Detection and treatment of metastasis founder cells

Abstract

The present invention is in the fields of diagnostics and treatment of cancer. The present invention provides, inter alia, means and methods for detecting and / or isolating metastasis founder cells based on the expression or expression level of the marker(s) c-KIT and / or MUC1, and, preferably, at least one additional disseminated cancer cell (DCC) marker such as EpCAM and / or (a) cytokeratin(s). Corresponding isolated cells or cell populations, and uses thereof, are provided as well. In one aspect, the invention relates to a method for detecting metastasis founder cells in a sample, comprising measuring in single cells in a sample the expression or expression level of (i) a first marker which is c-KIT and (ii) at least one disseminated cancer cell (DCC) marker, wherein expression of c-KIT and at least one DCC marker in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of c-KIT and at least one DCC marker in a cell above respective thresholds are indicative of the cell being a metastasis founder cell. Furthermore, the invention relates to methods for prognosing the development of metastases and / or the relapse, progression or outcome of a cancer, as well as to methods for measuring the success of an anti-cancer treatment and / or for stratifying subjects for treatment with (an) anti-cancer drug(s), based on the expression or expression level of said marker(s) in single cells. In addition, the present invention relates to one or more anti-cancer drugs for use in treating a patient having metastasis founder cells according to the invention. Further provided are therapeutics for targeting metastasis founder cells such as antibodies or fragments thereof binding both, c-KIT and EpCAM, or CAR-T cells targeting both, c-KIT and EpCAM.

Description

[0001] Detection and treatment of metastasis founder cells

[0002] The present invention is in the fields of diagnostics and treatment of cancer. The present invention provides, inter alia, means and methods for detecting and / or isolating metastasis founder cells based on the expression or expression level of the marker(s) c-KIT and / or MUC1, and, preferably, at least one additional disseminated cancer cell (DCC) marker such as EpCAM and / or (a) cytokeratin(s). Corresponding isolated cells or cell populations, and uses thereof, are provided as well. In one aspect, the invention relates to a method for detecting metastasis founder cells in a sample, comprising measuring in single cells in a sample the expression or expression level of (i) a first marker which is c-KIT and (ii) at least one disseminated cancer cell (DCC) marker, wherein expression of c-KIT and at least one DCC marker in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of c-KIT and at least one DCC marker in a cell above respective thresholds are indicative of the cell being a metastasis founder cell. Furthermore, the invention relates to methods for prognosing the development of metastases and / or the relapse, progression, or outcome of a cancer, as well as to methods for measuring the success of an anti-cancer treatment and / or for stratifying subjects for treatment with (an) anti-cancer drug(s), based on the expression or expression level of said marker(s) in single cells. In addition, the present invention relates to one or more anti-cancer drugs for use in treating a patient having metastasis founder cells according to the invention. Further provided are therapeutics for targeting metastasis founder cells such as antibodies or fragments thereof binding both, c-KIT and EpCAM, or CAR-T cells targeting both, c-KIT and EpCAM.

[0003] Cancer is a major lethal disease for humans and is caused by physiologically uncontrolled cell proliferation which affects normal physiological conditions of human body resulting in serious pathological reactions often leading to death. Although tremendous efforts on cancer studies and treatments have been made, presently, cancer is still the major cause of death to humans. There are multiple approaches to treat cancer patients including surgery, radiation therapy and chemotherapy.

[0004] Development of adjuvant therapies in the 21stcentury still goes along the classical path from treating first moribund patients, then perform controlled clinical trials enrolling stage IV metastatic patients and finally determine in large trials of neo-adjuvant and adjuvant settings progression-free and overall survival as therapy endpoints. Recent examples of adjuvant therapies include both targeted and immunotherapies, such as BRAF inhibition or ERBB2 targeting or immune checkpoint blockade. This process does not only consume substantial resources but slows clinical progress. In 1987, Gert Riethmuller and colleagues published a landmark study (G. Schlimok et al., 1987, Proc Natl Acad Sci U S A 84, 8672-8676), demonstrating that disseminated cancer cells (DCC; at that time called 'micrometastatic cells') can be detected by antibodies directed against epithelial intracellular cytokeratins (CK) and the epithelial surface marker EpCAM (then called '17-1A antigen') in bone marrow months to decades before clinical metastasis. Since then, it is the hope that DCC may not only be the harbingers of future metastasis, but also the actual and most important target cells of (neo)-adjuvant therapies and may therefore be developed into a novel therapy endpoint. Countless studies explored the clinical value of DCC detection in bone marrow and lymph nodes for almost all epithelial cancers (reviewed in (C. A. Klein, 2003, Adv Cancer Res 89, 35-67; S. Riethdorf, et al., 2008, Int J Cancer 123, 1991-2006) and applied the concept to blood samples where the two markers have, for example, been utilized in the FDA-approved CELLSEARCH® assay for the detection of circulating tumor cells (CTC) (see, e.g., M. Cristofanilli et aL, 2004, N Engl J Med 351, 781-791; L. Keller, K. Pantel, 2019, Nat Rev Cancer 19, 553-567; Cristofanilli et al., 2005, J Clin Oncol. 23(7): 1420-1430; de Bono et aL, 2008, Clin Cancer Res. 14:6302-6309; Cohen et al., 2008, J Clin Oncol. 26(19):3213- 3221). After initial enthusiasm and confirmation, results became complex as these studies included findings that detection by cytokeratin antibodies may also depend on specific intermediate filaments recognized (A. D. Hartkopf et al., 2021, Eur J Cancer 154, 128-137), and that enrichment methods and details in the staining protocols may have an impact on detection frequency and hence outcome prediction (B. Naume et al., 1998, Int J Cancer 78, 556-560; F. C. Cackowski et aL, 2019, Prostate 79, 1715-1727). Despite all these differences, there is wide-spread consensus and abundant data that for most, if not all epithelial cancer types, bone marrow-derived DCC impose a risk for progression and death. However, the size of effect of DCC detection has varied considerably and all studies so far concur in that it has been disappointingly low (A. D. Hartkopf et aL, 2021, Eur J Cancer 154, 128-137), for example, too low to be used as therapy endpoint.

[0005] Therefore, substantial effort has been invested into further characterization of DCC. To prove their malignant origin, genomic characterization was particularly relevant and significant insight into the biology and evolution of systemic cancer has been obtained from these studies (reviewed in C.A. Klein, 2009, Nat Rev Cancer, 9:302-312; C.A. Klein, 2013, Nature 501:365-372; C.A. Klein, 2020, Nat Rev Cancer, 20(ll):681-694).

[0006] A landmark study from the TRACERX consortium (Jamal-Hanjani (2017), N Engl J Med ;376(22) analyzed intratumor heterogeneity and cancer genome evolution of non-small cell lung cancer (NSCLC). It was found that driver mutations in EGFR, MET, BRAF, and TP53 formed the group of early initiating events in the formation of NSCLC. Moreover, KRAS mutations were found to be among the initiating early events in lung adenocarcinomas (LUAD).

[0007] To date, the prognosis of patients with NSCLC remains poor (R. Siegel et aL, 2021, CA Cancer J Clin, 71(l):7-33). Even after diagnosis at an early stage of disease, followed by curative tumor resection and adjuvant chemotherapy and / or radiotherapy, up to 50% of patients relapse within 5 years ( M.D. Taylor et aL, 2012, Ann Thorac Surg., 93(6): 1813-20; H. Uramoto and F. Tanaka, 2014, Transl Lung Cancer Res., 3(4):242-9). Available data suggest that the dissemination of lung cancer cells begins years before a lung tumor reaches stage T1 ( C.A. Klein, 2020, Nat Rev Cancer, 20(ll):681-694), thus targeting early DCC is very important for the prevention of metachronous metastasis. However, the detection of DCC and in particular their molecular characterization is hampered by insufficient knowledge of markers to identify early metastatic cells before clinical manifestation. Two reagents for the detection of DCC in the bone marrow that have been studied in detail are antibodies against epithelial intracellular cytokeratins (CK) and against EpCAM (S. Riethdorf, et al., 2008, Int J Cancer 123, 1991-2006), an epithelial surface marker.

[0008] Yet, not all patients having DCC or CTC as determined by prior art methods are doomed to develop metastases. In fact, the detection of DCC or CTC using EpCAM and / or CK does not provide enough prognostic value with regards to the development of metastases and, hence, patient survival. Therefore, detection of DCC or CTC using EpCAM and / or CK can currently not be used as surrogate endpoint for the typical study endpoints, progression-free, distant-disease free survival, cancer specific survival or overall survival. As such endpoints are achieved usually after several, significant numbers of patients are overtreated, i.e., receive toxic treatments that are not needed for the individual patient. Since it is still not possible to reliably predict the progression of a cancer (e.g., the development of metastases), many patients currently receive toxic treatments such as chemotherapies or checkpoint inhibitors, although they were never in need of such a therapy.

[0009] Accordingly, there is still a need for improved means and methods for the diagnosis of cancer, in particular for the prediction of metastases and / or the prognosis of patient survival.

[0010] The above technical problem is solved by the embodiments as characterized in the claims and as described herein below.

[0011] Specifically, the present invention provides two novel markers, namely c-KTT and mucin-1 (MUC1), demarcating a particularly aggressive subset of disseminated cancer cells (DCC) called "metastasis founder cells".

[0012] Accordingly, the present invention relates, inter alia, to a method for detecting metastasis founder cells in a sample, comprising measuring in single cells in a sample the expression or expression level of (i) a first marker which is c-KTT and (ii) at least one disseminated cancer cell (DCC) marker, wherein expression of c-KIT and at least one DCC marker in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of c-KIT and at least one DCC marker in a cell above respective thresholds are indicative of the cell being a metastasis founder cell.

[0013] The present invention also relates to a method for detecting metastasis founder cells in a sample, comprising measuring in single cells in a sample the expression or expression level of a first marker which is MUC1, wherein expression of MUC1 is indicative of the cell being a metastasis founder cell, or wherein the expression level of MUC1 in a cell above a threshold is indicative of the cell being a metastasis founder cell.

[0014] The invention is, inter alia, based on the surprising finding that patients having disseminated cancer cells (DCC) expressing c-KIT in addition to the classical DCC markers "EpCAM" and / or "cytokeratins (CK)", showed poor survival as compared to cancer patients having only c-KIT-negative DCCs, as also illustrated in the enclosed non-limiting examples (see, e.g., Examples 5 to 7). A "DCC", as used herein, refers to a cancer cell that breaks away from an original tumor and invades another tissue such as bone marrow. Unexpectedly, expression of c-KIT in DCCs dramatically changed hazard ratios for patients harboring DCCs from an epithelial cancer, specifically a non-small cell lung cancer (NSCLC); see Example 6 and Figure 10C. In fact, NSCLC patients having at least one c-KIT-positive DCC had an 2.1- 11.9-fold higher probability to die than patients having only c-KIT-negative DCCs. Moreover, the c-KIT expression or expression level had a very high negative prognostic value, regardless of whether c-KIT protein expression on the cell surface or c-KIT mRNA was measured; see, e.g., Figure 6E and Figures 9 and 10. In addition, it has been surprisingly found by the inventors that MUC1 mRNA expression in EpCAM+DCC was indicative of poor patient survival, as illustrated in Example 5 and Figure 6F, G. In contrast, mutations in TP53 and KRAS were very rare in DCC of NSCLC patients, and not predictive of patient survival (see Example 8 and Figures 12 and 13). This was an unexpected finding in view of reports in the prior art that TP53 and KRAS mutations are early events in NSCLC evolution. Hence, it has been surprisingly found in context of the present invention that c-KIT and MUC1 are negative prognostic markers in context of DCC (whereas TP53 or KRAS mutations are not). Poor survival of a cancer patient is also indicative of metastasis formation because it is well known in the art that the vast majority of cancer patients dies from metastases (see, e.g., H. DillekSs, 2019, Cancer Med., 8(12)). Since the cancer patients in the studies described in the enclosed non-limiting examples were free of metastases at day 0, the presence of c-KTT (or MUC1) positive DCCs is not only indicative of poor survival but further predicts the development of metastases. Hence, DCCs expressing c-KTT or MUC1 (in addition to at least one classical DCC marker such as EpCAM and / or CK) can be considered as "metastasis founder cells" (MFC), i.e., cells which have a high potential of forming metastases. In contrast, TP53 or KRAS mutations in DCCs are not indicative of MFCs, as there was no link to patient survival (Example 8 and Figure 13).

[0015] Detection of metastasis founder cells (MFC) after the removal of the primary tumor is particularly relevant as this is the time where currently no robust prediction on the risk of relapse and / or the development of metastases may be made, although it is very important for the overall treatment success and patient survival. Therefore, the detection of MFCs, as provided by the present invention, allows a reliable prognosis about the likelihood of metastases forming after the removal of the primary tumor. This information allows to assess the risk of relapse and take appropriate measures to prevent or control potential spread of cancer cells. Furthermore, the early detection of MFCs allows a close monitoring of the patient for the development of potential metastases. The detection of metastasis founder cells according to the present invention may become a standard test to stratify patients for further anti-cancer treatment, for example, after the removal of a primary tumor.

[0016] While the present inventors initially analyzed DCC from bone marrow as illustrated in the non-limiting examples, it is foreshadowed that the surprising findings described herein are also applicable to other non-epithelial tissue or body fluid samples such as blood (which is substantially a descendant from bone marrow). The presence of DCC in the blood, where they are also called "circulating tumor cells (CTC)", is the basis of tests in the prior art for determining the prognosis of cancer patients.

[0017] The current gold standard method for detecting / quantifiying CTC is the FDA-approved CELLSEARCH® Circulating Tumor Cell (CTC) Test by Menarini silicon biosystems (see, e.g., Cristofanilli et al., 2005, J Clin Oncol. 23(7): 1420- 1430; de Bono et aL, 2008, Clin Cancer Res. 14:6302-6309; Cohen et al., 2008, J Clin Oncol. 26(19):3213-3221). The CELLSEARCH® CTC Test detects and enumerates CTC in blood samples essentially by measuring the expression or the expression level of EpCAM and cytokeratins (CK) (in particular, CK8, CK18 and CK19).

[0018] As illustrated in the appended non-limited examples, various prior art DCC detection methods relying solely on EpCAM and / or CK expression using immunocytology and / or immunofluorescent staining (like the CELLSEARCH® CTC Test) could, however, not predict the survival of NSCLC patients; see Example 4, Table 6 and Figures 2 and 3. Yet, when these protocols were refined by further assessing the expression or expression level of c-KTT by immunostaining according to the present invention, it, surprisingly, became possible to predict patient survival; see, e.g., Example 6, Figures 9 and 10 and Table 7. Specifically, patients harboring c-KTT+DCC in bone marrow showed a significantly reduced probability of survival, as compared to patients having only c-KTT-negative DCC (i.e., DCC that were EpCAM+and / or CK+but did not show surface expression of c-KTT); see, e.g., Figure 10 and Example 6. Completely unexpectedly, already a single c-KTT-positive DCC in bone marrow outcompeted all clinical variables investigated as independent clinical prognostic variable (while the gold-standard CELLSEARCH® CTC Test did not achieve approval for NSCLC and uses five CTC per sample in breast cancer as threshold). Thus, the additional measurement of the c-KIT surface expression or expression level had a strong negative predictive value.

[0019] Therefore, assessing c-KIT expression by additionally employing an anti-c-KIT antibody in available standard immunostaining-based DCC detection assays positively impacts on assay reliability, improves outcome prediction, and increases effect size in patients having an epithelial cancer such as NSCLC.

[0020] Based on the surprising findings described herein above and as illustrated in the non-limiting Examples, a detection assay for metastasis founder cells is provided that is based on routine procedures, is cost effective, and can be standardized for operator-independent, quantitative evaluation. Accordingly, the present invention provides means and methods to reliable detect metastasis founder cells allowing the identification of patients with poor survival prognosis. In particular, the present invention further provides a method for analysing cells (especially for detecting metastasis founder cells), comprising a step of contacting a sample comprising cells with (i) an antigen-binding molecule binding c-KIT protein, or a probe or primer pair binding c-KIT RNA, and (ii) at least one antigen-binding molecule binding at least one DCC marker protein (such as EpCAM and / or CK) or at least one probe or primer pair binding at least one DCC marker RNA. In addition, the present invention also provides a kit-of-parts that may be used for detecting metastasis founder cells, wherein said kit-of-parts comprises (i) an antigen-binding molecule, e.g., an antibody or fragment thereof, binding c-KIT protein, and (ii) at least one antigen-binding molecule binding at least one DCC marker protein, e.g., an antibody or fragment thereof, binding EpCAM protein and / or an antibody or fragment thereof, binding at least one cytokeratin protein.

[0021] Based on the detection of c-KIT (or MUC1) expressing DCC, i.e., metastasis founder cells, the present invention further allows an early prognosis on the development of metastases, early prognosis of relapse, progression, and / or outcome of a cancer as well as measuring the success of a treatment, and / or stratification of patients for treatment. This is, inter alia, also useful for an improved study selection of patients. For example, for clinical trials with patients having a stage I-III cancer (no clinically detectable metastases), only patients that will likely develop metastases, i.e., have metastasis founder cells as determined according to the present invention, may be included. This will improve study planning and may, due to reduced study duration, yield faster and more reliable results. Furthermore, the present invention significantly reduces the proportion of (prophylactically) overtreated patients, thereby reducing the treatment associated side-affects for patients that would not have been in need of such a prophylactic treatment, for example, an adjuvant therapy using a chemotherapy, a checkpoint inhibitor and / or a targeted therapy etc. This will also reduce the associated costs entailed by such overtreatment associated side-effects on the health system and the society. The present invention also allows for the development of therapy algorithms by monitoring the presence of metastasis founder cells (MFCs), identified by the methods of the present invention, and predicting therapy success by detecting a reduction or disappearance of said MFCs.

[0022] Thus, the present invention provides a diagnostic test that is very useful for stratifying cancer patients to avoid unnecessary overtreatment and specifically select patients that benefit from a further anti-cancer therapy, e.g., an anti-cancer immunotherapy, a chemotherapy and / or a targeted therapy that may be tailored to target DCC expressing c-KIT on the cell surface. Hence, the present invention further provides surface markers and marker combinations such as c-KTT in combination with EpCAM that may also serve as therapy targets. Accordingly, the detection of MFCs according to the present invention may be also useful as companion diagnostics to treat individual patients at risk of developing metastases in a tailored manner. Hence, the present invention may further allow for an early, directed, and specific targeting of MFCs to prevent the development of metastases.

[0023] As indicated herein above, in one aspect, the present invention provides a method for detecting metastasis founder cells in a sample, comprising measuring in single cells in a sample the expression or expression level of (i) a first marker which is c-KTT and (ii) at least one DCC marker, wherein expression of c-KTT and at least one DCC marker in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of c-KTT and at least one DCC marker in a cell above respective thresholds are indicative of the cell being a metastasis founder cell.

[0024] The method of the present invention is, in particular, an exwVoor in vitro method, more specifically an ex vivo method, i.e., a method performed in vitro with a sample from a subject, e.g., a cancer patient.

[0025] As indicated herein above, the term "disseminated cancer cell" or "DCC", as used herein, describes a cancer cell that breaks away from a tumor (e.g. a primary tumor) and invades another tissue, such as bone marrow, lymph node or any other organ. DCCs may spread lymphatically or hematogenously and may reside in other organs and / or tissues in the body, such as bone marrow, lymph node, or central nervous system. DCCs may be obtained from a variety of organs and / or tissues (e.g., bone marrow) using methods well-known in the art (R. Lammers et al., 2002, Exp Hematol 30, 537-545; T. Fehm et aL, 2006, Cancer 107, 885-892; M. Werner-Klein et al., 2008, Nat Commun 9, 595; M. Guzvic et aL, 2014, Cancer Res 74, 7383-7394; which are all incorporated herein by reference) and as exemplified in nonlimiting Example l.C-E. With approximately 1-2 DCC / million mononucleated bone marrow cells DCCs are extremely rare in the bone marrow. DCCs sometimes are also referred to as "micrometastatic cells" in the art. In context of the invention, DCCs may be found, in particular, in cancer patients which have no metastases diagnosed and / or which have no clinically detectable metastases, as described herein. Thus, the DCCs described herein may also refer to "M0- DCC". In context of the present invention, DCC are, preferably, present in a non-epithelial tissue or body fluid as individual cells (i.e., not comprised in a cancer cell aggregate) and / or within small cancer cell aggregates, as described herein, e.g., in context of metastasis founder cells.

[0026] The term "circulating tumor cell" or "CTC" as used herein describes a cancer cell that circulates in the blood system. CTCs are obtainable from blood (e.g., from leukapheresis products) (Fischer et al., 2013, PNAS 110:16580-16585) using methods well-known in the art, in particular FDA-cleared CELLSEARCH® system (Veridex LLC / Menarini, silicon biosystems), DEPArray™ technology or flowcytometry, but CTC-isolation is not limited to these systems (Polzer et al., 2014, EMBO Mol Med. 6:1371-138) and may comprise a variety of other devices such as CellSieve or Parsortix®. In w / oCTC-capturing-devices may also be used. Examples for such devices are Gilupi, CellCollector (Gorges et al., 2015, ClinCancer Res. 1416.2015), cancer cell binding nanoparticles (Galanzha et aL, 2011, Cytometry 79:814-824) or medical wires (Saucedo-Zeni et al., 2012, Int J OncoL 41:1241-1250) but are not limited to these. Such devices or particles may be implanted or infused into a subject / patient to capture and enrich CTCs in the body of the subject / patient. The CTCs can then be retrieved from the CTC-capturing-device or -particles ex vivo or in vivo. In context of the present invention, the term "DCC" encompasses CTC. In other words, a CTC maybe also considered herein and in context of the invention as a DCC that is located in the blood of a subject. DCC in general, and CTC specifically, may be derived from a primary tumor, a site of metastasis, a metastasis, or a pre-stage of a manifest (esp. clinically detectable) metastasis.

[0027] The term "metastasis founder cell" or "MFC" as used herein describes specific cancers cells, especially DCC and / or CTC, with a potential to form metastases, in particular, clinically detectable metastases. In particular, MFCs may adapt to an organ other than the organ of origin of the primary tumor in a way that they can survive and use the local microenvironment of the distinct organ to grow again in an uncontrolled manner, i.e., as a metastasis, in particular a clinically detectable metastasis.

[0028] In the context of the present invention the metastasis founder cells is, preferably, from an epithelial cancer. An epithelial cancer in the context of the present invention may be, for example, a lung carcinoma, a prostate carcinoma (i.e., prostate cancer), a breast cancer, a head and neck carcinoma, a colorectal carcinoma, an endometrial carcinoma, a cervical carcinoma, a bladder carcinoma, a pancreatic carcinoma, an ovarian carcinoma, a hepatocellular carcinoma, a renal carcinoma, a thyroid carcinoma, a bile duct carcinoma, a gallbladder carcinoma, a laryngeal carcinoma or a parotid carcinoma. In preferred embodiments, the MFCs are from, a lung carcinoma, a breast cancer, or a prostate carcinoma, more preferably from a lung carcinoma. In even more preferred embodiments, the MFCs are from a non- small-cell lung cancer (NSCLC).

[0029] In the context of the present invention, the metastasis founder cells are, preferably, present (in particular in a non- epithelial tissue or body fluid) as individual cells (i.e. not comprised in a cancer cell aggregate) and / or within small cancer cell aggregates, i.e., a cancer cell aggregate comprising at most about 100000, at most about 50000, at most about 20000, at most about 10000, at most about 5000, at most about 2000, at most about 1000, at most about 500, at most about 400, at most about 300, at most about 200, at most about 100, at most about 50, at most about 20, at most about 10 or at most about 5 cancer cells In particular, such small cancer cell aggregates are usually not clinically detectable. In preferred embodiments, the small cancer cell aggregates described herein comprise at most about 100 cancer cells, more preferably at most about 50 cancer cells. Preferably, the metastasis founder cells described herein are predominantly not contained in clinically detectable cancer cell aggregates, i.e., a cancer cell aggregate such as a primary tumor or a metastasis, comprising more than about 1 million, more than about 5 million, more than about 10 million, more than about 50 million, more than about 100 million, more than about 500 million, more than about 1 billion, more than about 5 billion or more than about 10 billion cancer cells. Preferably, a clinically detectable cancer cell aggregate (e.g., a primary tumor or a metastasis) comprises more than about 100 million cancer cells. Furthermore, a clinically detectable cancer cell aggregate has, preferably, a volume of more than about 0.1 cm3, more than about 0.2 cm3, more than about 0.5 cm3or more than about 1 cm3, preferably more than about 0.5 cm3.

[0030] In the context of the present invention the metastasis founder cells have, in particular, a potential of forming metastases, especially clinically detectable metastases. Preferably, the metastasis founder cells have a higher potential of forming metastases than the bulk of cells from a corresponding primary tumor, in particular the primary tumor the MFCs are derived from. The term "metastasis" (sg.) as used herein describes a cancer cell aggregate, especially a cancer colony, that has formed at a site remote from the tissue of origin. In particular, in context of the present invention, a metastasis is a clinically detectable cancer cell aggregate, as described herein. Thus, a metastasis comprises, preferably, more than about 1 million, preferably more than about 100 million cancer cells and / or a volume of more than about 0.1 cm3, preferably more than about 0.5 cm3. A metastasis may also be referred to as secondary tumor. Metastases (pl.) may originate from MFCs that have spread to the respective tissues or organs. A metastasis is the same type of cancer as the primary tumor.

[0031] The term "marker" as used herein refers to a protein or polynucleotide, the expression or presence of which in or on a mammalian tissue or cell can be detected by any suitable standard methods, in particular, by the methods disclosed herein. For example, a marker may be detected by the use of (an) antigen-binding molecule(s) comprising (a) detectable label(s) such as, e.g., a fluorescent dye or that is / are bound by (a) molecule(s) comprising such (a) detectable label(s). More specific examples of detecting a marker are disclosed herein below. Herein, a marker refers, preferably, to the human version thereof, in particular, a human protein or a human mRNA. A particularly relevant marker in context of the present invention is c-KIT, especially human c-KIT.

[0032] The term "c-KIT" as used herein refers to a receptor tyrosine kinase protein also known as "CD117", "stem cell growth factor receptor (SCFR)" or "KIT" (or corresponding (m)RNA). Specifically, the wild type of c-KIT encodes a 145 kDa receptor tyrosine kinase (RTK) protein of 976 amino acids and is often referred to as stem cell factor (SCF) receptor due to its association with its ligand SCF (C.D. Mol et al., 2003, J Biol Chem, 278(34):31461-4). Preferably, "c-KIT", as used herein, refers to human c-KIT protein (preferably having a sequence as set forth in any one of SEQ ID NO. 15 to 22) and / or human c-KIT mRNA (preferably having a sequence as set forth in any one of SEQ ID NO. 7 to 14).

[0033] For reference, the human genomic locus of c-KIT may have a sequence as shown in SEQ ID NO. 5 or 6.

[0034] In humans, the 58 RTKs described to date are divided into 20 subfamilies or classes based on the structure of their amino (N)-terminus. Class III RTKs, which are characterized by the presence of five Ig-like EC domains, include platelet-derived growth factor a and p receptors (PDGFR o / P), colony-stimulating factor 1 receptor, fms-like RTK 3 and c-KIT (K. Verstraete and S. Swides, 2012, Nat Rev Cancer, 12(ll):753-66). c-KIT is expressed by various cells in the body, and signaling pathways stimulated by its activation by SCF under physiological conditions are involved in the regulation of cellular processes such as cell proliferation, survival, and migration (J. Lennartsson and Ronnstrand, 2012, Physiol Rev, 92(4): 1619-49). Sequencing of nearly 19,000 samples detected c-KIT alterations in 2.86% of the 59 cancer types studied, including very common and clinically impactful mutations (Consortium, 2017, Cancer Discov 7, 818- 831).

[0035] Although most cancer-associated c-KIT alterations are gain-of-function mutations that lead to constitutive activation of c-KIT in an SCF-independent manner, others involve amplification or loss-of-function mutations (M. Abbaspour Babaei et al., 2016, Drug Des Devel Ther, 1: 10:2443-59). "Gain-of-function mutations of c-KIT have been shown to drive the development of a variety of cancers / proliferative diseases, including GIST (A. Duensing et al., 2004, Cancer Invest, 22(1): 106-16), some subtypes of melanoma (Curtin et al., 2006, J Clin Oncol 24, 4340-4346), mastocytosis (C. Bodemer et al., 2010, J Invest Dermatol, 130(3):804-15), acute myeloid leukemia (AML) (A. Beghini et al., 2004, Haematologica 89, 920-925), and seminoma (Y. Sakuma et al., 2003, Cancer Sci, 94(6):486-91).

[0036] The expression of c-KTT in NSCLC primary tumors (PT) is rare (in contrast to SCLC) and has only been observed in individual foci in 9-17% of cases (KJ. Butnor et al., 2004, Arch Pathol Lab Med, 128(5):538-43). No survival data related to the expression of c-KIT protein in NSCLC-PT are available to date. However, the inventors' analyses of gene expression data from The Cancer Genome Atlas Program (TCGA) showed no association between high RNA expression in NSCLC-PT and a poor prognosis. In fact, while in most primary tumors such as squamous cell lung cancer, breast cancer or prostate cancer, c-KIT expression was not linked to patient survival, in primary tumors of lung adenocarcinoma, high c-KIT expression was even linked to a higher probability of survival, according to TCGA data- based analyses. Notably, squamous cell lung cancer and lung adenocarcinoma are, both, non-small cell lung cancers (NSCLC). It was, thus, highly unexpected that c-KIT positive DCC derived from NSCLC, are predictive of poor patient survival, and that c-KIT is a negative prognostic marker in context of DCC, as described herein and as illustrated in the appended non-limiting examples.

[0037] In other words, while c-KIT mutations were known to play a role in cancer, it was not expected that c-KIT expression demarcates a DCC subset that has a high potential of forming metastases and predicts poor patient survival, as described herein in context of the present invention. c-KIT is expressed on the cell surface, further facilitating staining procedures for diagnostic purposes. As a surface marker, c-KIT may also serve as a potential target, e.g., as target for CAR T cells or antigen-binding molecules, such as antibodies. For example, c-KIT is the target of the tyrosine kinase inhibitor imatinib that is used for the treatment of gastrointestinal stromal tumors (GIST) tumors that are characterized by mutations in c-KIT.

[0038] The term "DCC marker" as used herein refers to a marker that detects DCCs, thereby differentiating them from normal tissue cells, in particular, the cells of the tissue in which the DCC reside (especially the non-epithelial tissues or body fluids such as bone marrow or blood as described herein). As CTCs may be considered as a subset of DCCs that are located in the blood system, as described herein, they may also be detected by the same DCC marker(s).

[0039] In the context of the present invention the DCC marker(s) may comprise(s) at least one cytokeratin, EpCAM and / or MUC1, preferably at least EpCAM.

[0040] In preferred embodiments of the present invention, the DCC marker(s) comprise(s) at least EpCAM and optionally further at least one cytokeratin or MUC1. In context of the present invention, the cytokeratin(s) may comprise(s) CK8, CK18, and / or CK19.

[0041] The term "Epithelial cell adhesion molecule", "EpCAM" or "EPCAM" as used herein refers to a transmembrane glycoprotein (or a corresponding (m)RNA) also known as "CD326" or "17-1A antigen". Preferably, "EpCAM", as used herein, refers to human EpCAM protein (preferably having a sequence as set forth in SEQ ID NO. 4) and / or human EpCAM mRNA (preferably having a sequence as set forth in SEQ ID NO. 3). For reference, the human genomic locus of EpCAM may have a sequence as shown in any one of SEQ ID NO. 1 or 2.

[0042] EpCAM has been described as a DCC marker; see e.g., C.A. Klein, 2003, Adv Cancer Res 89, 35-67 and S. Riethdorf, et al., 2008, Int J Cancer 123, 1991-2006.

[0043] The terms "cytokeratin", "(a) cytokeratin(s)" or "CK" as used herein refer to at least one member selected from the group of keratin proteins found in the intracytoplasmic cytoskeleton of epithelial tissue (or corresponding (m)RNA), also referred to as epithelial intracellular cytokeratin(s). Preferably, in context of the invention, the cytokeratin(s) comprise(s) at least one CK selected from CK 1 to 8, 10, 14 to 20, more preferably CK8, CK18 and / or CK19.

[0044] Preferably, "CK", as used herein refers to at least one human CK protein and / or at least one human CK mRNA, in particular human CK1 protein (preferably having a sequence as set forth in SEQ ID NO. 26), human CK2 protein (preferably having a sequence as set forth in SEQ ID NO. 30), human CK3 protein (preferably having a sequence as set forth in SEQ ID NO. 35 or 36), human CK4 protein (preferably having a sequence as set forth in SEQ ID NO. 40), human CK5 protein (preferably having a sequence as set forth in SEQ ID NO. 44), human CK6A protein (preferably having a sequence as set forth in SEQ ID NO. 48), human CK6B protein (preferably having a sequence as set forth in SEQ ID NO. 52), human CK7 protein (preferably having a sequence as set forth in any one of SEQ ID NO. 64 to 72), human CK8 protein (preferably having a sequence as set forth in any one of SEQ ID NO. 80 to 83), human CK10 protein (preferably having a sequence as set forth in SEQ ID NO. 92 or 93), human CK14 protein (preferably having a sequence as set forth in SEQ ID NO. 108), human CK15 protein (preferably having a sequence as set forth in any one of SEQ ID NO. 117 to 122), human CK16 protein (preferably having a sequence as set forth in any one of SEQ ID NO. 126), human CK17 protein (preferably having a sequence as set forth in SEQ ID NO.130), human CK18 protein (preferably having a sequence as set forth in SEQ ID NO. 135 or 136), human CK19 protein (preferably having a sequence as set forth in any one of SEQ ID NO. 140), and / or human CK20 protein (preferably having a sequence as set forth in SEQ ID NO. 145), and / or human CK1 mRNA (preferably having a sequence as set forth in SEQ ID NO. 25), human CK2 mRNA (preferably having a sequence as set forth in SEQ ID NO. 29), human CK3 mRNA (preferably having a sequence as set forth in SEQ ID NO. 33 or 34), human CK4 mRNA (preferably having a sequence as set forth in SEQ ID NO. 39), human CK5 mRNA (preferably having a sequence as set forth in SEQ ID NO. 43), human CK6A mRNA (preferably having a sequence as set forth of SEQ ID NO. 47), human CK6B mRNA (preferably having a sequence as set forth in SEQ ID NO. 51), human CK7 mRNA (preferably having a sequence as set forth in any one of SEQ ID NO. 55 to 63), human CK8 mRNA (preferably having a sequence as set forth in any one of SEQ ID NO. 75 to 79), human CK10 mRNA (preferably having a sequence as set forth in SEQ ID NO. 90 or 91), human CK14 mRNA (preferably having a sequence as set forth in SEQ ID NO. 107), human CK15 mRNA (preferably having a sequence as set forth in any one of SEQ ID NO. Ill to 116), human CK16 mRNA (preferably having a sequence as set forth in SEQ ID NO. 125), human CK17 mRNA (preferably having a sequence as set forth in SEQ ID NO. 129), human CK18 mRNA (preferably having a sequence as set forth in SEQ ID NO. 133 or 134), human CK19 mRNA (preferably having a sequence as set forth in SEQ ID NO. 139), human CK20 mRNA (preferably having a sequence as set forth in SEQ ID NO. 144).

[0045] Furthermore, human CK9 protein may have a sequence as set forth in SEQ ID NO. 87, human CK9 mRNA may have a sequence as set forth in SEQ ID NO. 86, human CK12 protein may have a sequence as set forth in SEQ ID NO. 98, human CK12 mRNA have a sequence be as set forth in SEQ ID NO. 97, human CK13 protein may have a sequence as set forth in SEQ ID NO. 103 or 104, and / or human CK13 mRNA may have a sequence as set forth in SEQ ID NO. 101 or 102.

[0046] For reference, the human genomic locus of CK1 may have a sequence as shown in SEQ ID NO. 23 or 24, the human genomic locus of CK2 may have a sequence as shown in SEQ ID NO. T1 or 28, the human genomic locus of CK3 may have a sequence as shown in SEQ ID NO. 31 or 32, the human genomic locus of CK4 may have a sequence as shown in SEQ ID NO. 37 or 38, the human genomic locus of CK5 may have a sequence as shown in SEQ ID NO. 41 or 42, the human genomic locus of CK6A may have a sequence as shown in SEQ ID NO. 45 or 46, the human genomic locus of CK6B may have a sequence as shown in SEQ ID NO. 49 or 50, the human genomic locus of CK7 may have a sequence as shown in SEQ ID NO. 53 or 54, the human genomic locus of CK8 may have a sequence as shown in SEQ ID NO. 73 or 74, the human genomic locus of CK9 may have a sequence as shown in SEQ ID NO. 84 or 85, the human genomic locus of CK10 may have a sequence as shown in SEQ ID NO. 88 or 89, the human genomic locus of CK12 may have a sequence as shown in any one of SEQ ID NO. 94 to 96, the human genomic locus of CK13 may have a sequence as shown SEQ ID NO. 99 or 100, the human genomic locus of CK14 may have a sequence as shown in SEQ ID NO. 105 or 106, the human genomic locus of CK15 may have a sequence as shown in SEQ ID NO. 109 or 110, the human genomic locus of CK16 may have a sequence as shown in SEQ ID NO. 123 or 124, the human genomic locus of CK17 may have a sequence as shown in SEQ ID NO. 127 or 128, the human genomic locus of CK18 may have a sequence as shown in SEQ ID NO. 131 or 132, the human genomic locus of CK19 may have a sequence as shown in SEQ ID NO. 137 or 138, and / or the human genomic locus of CK20 may have a sequence as shown in any one of SEQ ID NO. 141 to 143.

[0047] Cytokeratins have been described as DCC markers; see e.g., C.A. Klein, 2003, Adv Cancer Res 89, 35-67 and S. Riethdorf, et al., 2008, Int J Cancer 123, 1991-2006.

[0048] The term "Mucin 1" or "MUC1" as used herein describes a glycoprotein (or corresponding (m)RNA) lining the apical surface of epithelial cells of for example lungs, stomach, intestine, eyes, and several other organs. MUC1 is also referred to as "CD227", "polymorphic epithelial mucin (PEM)" or "epithelial membrane antigen (EMA)". Preferably, "MUC1", as used herein, refers to human MUC1 protein (preferably having a sequence as set forth in any one of SEQ ID NO. 169 to 189) and / or human MUC1 mRNA (preferably having a sequence as set forth in any one of SEQ ID NO. 148 to 168).

[0049] For reference, the human genomic locus of MUC1 may have a sequence as shown in any one of SEQ ID NO. 146 to 147.

[0050] MUC1 has been also described as a DCC marker; see e.g., C.A. Klein, 2003, Adv Cancer Res 89, 35-67, S. Riethdorf, et al., 2008, Int J Cancer 123, 1991-2006 and S. Braun et aL, 2003, Breast., 12(6): 397-404.

[0051] The term "measuring the expression or expression level of a marker" in a single cell as used herein refers to experimentally determining the presence or amount of a marker in a single cell, respectively, employing appropriate methods of measuring expression of expression level as described elsewhere herein. Such a method of measuring expression or expression level is encompassed by the enclosed claims and is further illustrated in the enclosed nonlimiting Example 1. The term "in a cell" as used herein encompasses "on a cell". Accordingly, the term "expression in a cell" as used herein also encompasses "expression on the cell", in particular "expression on the cell surface".

[0052] In context of the present invention, the expression or expression level of a marker may correspond to the protein and / or RNA expression or expression level of the marker, respectively, preferably to the protein expression or expression level.

[0053] As used herein, a protein may be a polypeptide or a complex of polypeptides.

[0054] As used herein, the term "RNA" refers, in particular, to mRNA, and may also encompass mRNA precursors such as pre- mRNA.

[0055] In some embodiments, the expression or expression level of a marker may correspond to the RNA expression or expression level of the marker. In a preferred embodiment, the expression or expression level of a marker may correspond to the protein expression or expression level of the marker.

[0056] For example, the expression or expression level of c-KTT protein and EpCAM protein may be measured, and optionally the expression or expression level of CK protein may be further measured. Alternatively, the expression or expression level of c-KTT RNA and EpCAM protein may be measured. In another example, the expression or expression level of c-KIT protein and (a) cytokeratin(s) protein(s) may be measured, and optionally the expression or expression level of EpCAM protein may be further measured. In context of the measurement of EpCAM the measurement of the expression level may be preferred. In context of c-KIT, EpCAM, and CK measurements, the protein level may be preferred.

[0057] In some embodiments, the expression or expression level of c-KIT and at least one cytokeratin such as CK8, CK18 and / or CK19 may be measured in single cells, in particular, wherein (a) expression of c-KIT and at least one cytokeratin in a cell is indicative of the cell being a metastasis founder cell, or (b) expression levels of c-KIT and at least one cytokeratin in a cell above respective thresholds are indicative of the cell being a metastasis founder cell.

[0058] In some embodiments, the expression or expression level of c-KIT and EpCAM may be measured in single cells, in particular, wherein (a) expression of c-KIT and EpCAM in a cell is indicative of the cell being a metastasis founder cell, or (b) expression levels of c-KIT and EpCAM in a cell above respective thresholds are indicative of the cell being a metastasis founder cell.

[0059] In further embodiments, the expression level of EpCAM may be measured in single cells, in particular, wherein in a cell the combination of (i) expression of c-KIT and EpCAM, and (ii) a significantly higher expression level of EpCAM than in hematopoietic cells and / or blood cells, is indicative of the cell being a metastasis founder cell.

[0060] In some embodiments, e.g., in context of blood samples, the expression level of c-KIT and / or EpCAM may be measured in single cells, in particular, wherein in a cell the combination of (i) expression of c-KIT and EpCAM, and (ii) a significantly higher expression level of c-KTT and / or EpCAM than in blood cells, is indicative of the cell being a metastasis founder cell.

[0061] In further embodiments the expression level of c-KTT and / or EpCAM may be measured in single cells, in particular, wherein in a cell the combination of (i) expression of c-KTT and EpCAM, and (ii) a significantly higher expression level of c-KTT and / or EpCAM than in differentiated hematopoietic cells and / or blood cells, is indicative of the cell being a metastasis founder cell.

[0062] As used herein, the term "differentiated hematopoietic cells", refers, in particular, to the bulk of bone marrow and / or blood cells, and / or specifically excludes hematopoietic stem cells.

[0063] In some embodiments, the expression level of c-KTT, EpCAM and / or at least one cytokeratin such as CK8, CK18 and / or CK19 may be measured in single cells, in particular, wherein expression of c-KTT, EpCAM and at least one cytokeratin such as CK8, CK18 and / or CK19 in a cell is indicative of the cell being a metastasis founder cell, preferably, wherein in a cell the combination of (i) expression of c-KTT, EpCAM and at least one cytokeratin such as CK8, CK18 and / or CK19, and (ii) a significantly higher expression level of EpCAM than in differentiated hematopoietic cells and / or blood cells, is indicative of the cell being a metastasis founder cell.

[0064] In further embodiments the expression level of c-KTT, EpCAM and / or at least one cytokeratin such as CK8, CK18 and / or CK19 may be measured in single cells, in particular, wherein expression of c-KTT, EpCAM and at least one cytokeratin such as CK8, CK18 and / or CK19 in a cell is indicative of the cell being a metastasis founder cell, preferably, wherein in a cell the combination of (i) expression of c-KTT, EpCAM and at least one cytokeratin such as CK8, CK18 and / or CK19, and (ii) a significantly higher expression level of c-KTT and / or EpCAM than in differentiated hematopoietic cells and / or blood cells, is indicative of the cell being a metastasis founder cell.

[0065] In the context of the present invention the cells in the sample may be contacted with (i) an antigen-binding molecule binding c-KTT protein, or a probe or primer pair binding c-KTT RNA; and / or (ii) at least one antigen-binding molecule binding at least one DCC marker protein, or at least one probe or primer pair binding at least one DCC marker RNA.

[0066] Herein and in context of the present invention, an antigen-binding molecule is a molecule that specifically binds to a target antigen, in particular, an epitope on a peptide, polypeptide, or protein. An antigen-binding molecule may be, inter alia, an antibody or an antigen-binding fragment thereof, an aptamer, an affimer, or a darpin, all of which are well known in the art and are readily available or can be produced by routine methods. An antigen-binding molecule may be used for immunostaining to detect or quantify a target protein in single cells, as commonly performed in the art. In context of the present invention, an antigen-binding molecule is, preferably, an antibody or an antigen-binding fragment thereof.

[0067] In context of the present invention, an antibody or antigen-binding fragment thereof may be, inter alia, a full antibody (immunoglobulin), a F(ab)-fragment, a F(ab)2-fragment, a disulfide-linked Fvs (sdFv), an anti-idiotypic (anti-Id) antibody, an epitope-binding fragment, a single chain antibody such as a single-chain variable fragment (scFv), or a nanobody / single-domain antibody. Furthermore, the antibody or fragment thereof may be, inter alia, a monoclonal antibody, a polyclonal antibody, a recombinantly produced antibody, a chimeric antibody, a humanized antibody, a human antibody, a fully human antibody, or a CDR-grafted antibody, or an antigen-binding fragment of any one of said antibodies. In some embodiments, e.g., in context of medical uses, the antibody or fragment thereof may be a bivalent antibody-construct, a diabody, a triabody, a tetrabody (e.g., bispecific versions of the di-, tria-, or tetrabody), a multispecific antibody such as a bispecific single chain antibody, etc., or an antigen-binding fragment of any one of said antibodies. In some embodiments, in particular in context of medical uses, the antibody or fragment thereof may be multifunctional, i.e., exert its effects via more than one mode of action, such as for example by activating ADCC or CDC pathways.

[0068] In context of the present invention, a probe refers to a complementary nucleic acid molecule (e.g., a DNA or RNA) that specifically binds to a target RNA (esp. an mRNA) in a cell. In particular, a probe can be produced by routine methods and may be used for in-situ hybridization to detect or quantify a target RNA in single cells, as commonly performed in the art; see, e.g., S. Jamalzadeh et al., 2022, Lab Invest, 102(7):753-761. Furthermore, the probe may be labelled by a fluorescent tag and be used for fluorescence in-situ hybridization (FISH).

[0069] In context of the present invention, a primer pair refers, in particular, to a pair of oligonucleotides that specifically bind to a target RNA (esp. an mRNA) or, preferably, a corresponding cDNA in a cell or cell lysate. In particular, a primer pair can be produced by routine methods and may be used for reverse-transcriptase PCR (RT-PCT) to detect or quantify a target RNA in single cells, as commonly performed in the art. A particularly preferred primer pair for detecting c-KIT cDNA or RNA (e.g. for use in an RT-PCR) has the sequences SEQ ID NO: 190 and SEQ ID NO: 191, as shown in the following Table 1:

[0070] Table 1 - Particularly preferred primer pair for detecting c-KIT cDNA or RNA

[0071] The cells in the sample according to the present invention may be contacted with an antigen-binding molecule binding c-KIT protein, or a probe or primer pair binding c-KIT RNA, as described herein.

[0072] Furthermore, the cells in the sample may be contacted with at least one antigen-binding molecule binding at least one DCC marker protein (e.g., EpCAM and / or CK), or at least one probe or primer pair binding at least one DCC marker RNA, as described herein.

[0073] Thus, the cells in the sample may be contacted with (i) an antigen-binding molecule binding c-KIT protein, or a probe or primer pair binding c-KIT RNA; and (ii) at least one antigen-binding molecule binding at least one DCC marker protein, or at least one probe or primer pair binding at least one DCC marker RNA. In preferred embodiments of the present invention the cells in the sample may be contacted with (i) an antigen-binding molecule binding c-KTT protein; and / or (ii) at least one antigen-binding molecule binding at least one DCC marker protein (e.g., EpCAM and / or CK).

[0074] In a further aspect, the present invention relates to a method for analyzing cells, comprising a step of contacting a sample comprising cells with

[0075] (i) an antigen-binding molecule binding c-KTT protein, or a probe or primer pair binding c-KTT RNA; and

[0076] (ii) at least one antigen-binding molecule binding at least one DCC marker protein or at least one probe or primer pair binding at least one DCC marker RNA.

[0077] In preferred embodiments, the sample is contacted with an antigen-binding molecule binding c-KTT protein.

[0078] By contacting a sample with (i) an antigen-binding molecule binding c-KTT protein, or a probe or primer pair binding c-KTT RNA; and (ii) at least one antigen-binding molecule binding at least one DCC marker protein or at least one probe or primer pair binding at least one DCC marker RNA, the expression or expression level of c-KTT and at least one DCC marker can be measured in single cells in the sample. As described herein, the expression of c-KTT and at least one DCC marker in a cell may be indicative of the cell being a metastasis founder cell, or expression levels of c-KTT and at least one DCC marker in a cell above respective thresholds may be indicative of the cell being a metastasis founder cell.

[0079] In context of the present invention, an antigen-binding molecule (e.g., an antibody) or a probe may comprise a detectable label or may be bound by a molecule (e.g., a secondary antibody) comprising a detectable label. The detectable label may be, for example, a fluorescent tag (e.g., a fluorescent tag), a chromogenic enzyme, a luminogenic enzyme, a magnetic tag (e.g., a magnetic bead or a ferrofluid nanoparticle), a radioactive tag, or a DNA-tag. In preferred embodiments, the detectable label is a fluorescent tag. Furthermore, the detectable label according to the invention may produce a detectable signal, as described herein.

[0080] In context of the present invention the expression level of a marker may correspond to the level of a detectable signal that is associated with the marker. A detectable signal may be, e.g., a fluorescent, immunocytochemical, magnetic or radioactive detection signal (e.g., produced by an antibody or fragment thereof against a marker, coupled to a fluorescent tag, a chromo- or luminogenic enzyme, a magnetic tag, or radioactive tag, respectively) or a signal derived from a DNA-tag coupled, e.g., to an antibody against a marker, using PCR or sequencing as readout. In some preferred embodiments, the detectable signal is a fluorescent signal, e.g., generated by a fluorescent tag coupled to an antigenbinding molecule, preferably an antibody, binding a marker protein (e.g., c-KTT, EpCAM, CK, or MUC1), or binding another antigen-binding molecule, preferably an antibody, bound to the marker protein.

[0081] In some embodiments, the antigen-binding molecules comprises (i) an antigen-binding molecule, preferably an antibody or fragment, thereof binding c-KTT protein, and (ii) at least one antigen-binding molecule selected from the group consisting of: an antigen-binding molecule, preferably an antibody or fragment thereof, binding at least one cytokeratin protein, and an antigen-binding molecule, preferably an antibody or fragment thereof, binding EPCAM protein. In context of the invention, an antigen-binding molecule binding at least one cytokeratin protein may be a pan-anti- cytokeratin antibody or an antibody binding CK8, CK18 and CK19.

[0082] Exemplary antibodies (or fragments thereof) binding at least one cytokeratin (i.e., anti-CK) that may be used in context of the present invention include, inter alia, anti-CK8 / 18 antibodies such as LP34 (C. Thomas et al., 2009, Epub 2008, BJU Int., 104(l):29-34) or 5D3 (L.L. Volmer, et al., 2023, Int J Mol ScL, 24(18): 13930), anti- CK1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 10 / 14 / 15 / 16 / 19 antibodies such as AE1 and / or AE3 (A. Berg et al., 2007, Int J Cancer., 120(8): 1603-9; A. Berg et aL, 2008, Prostate, 68(15): 1607-14; W. Lilleby et aL, 2013, Int J Cancer., 133(l):149-55; L.L. Volmer et aL, 2023, Breast Cancer Res Treat., 202(l):67-72), anti-CK8 / 18 / 19 antibodies such as A45-B / B3 (J. Kdllermann et al., 2008, J Clin Oncol., 26(30) :4928-33; T. Todenhofer et aL, 2015, Prostate, 75(6):637-45; D. Weckermann et aL, 2001, J UroL, 166(2):699-703; P. Wimberger et aL, 2023, Breast Cancer Res., 25(1):32), anti- CK18 antibodies such as CK 2 (D. Weckermann et al., 1999, Urol Res., 27(5):285-90; D. Weckermann et aL, 1999, J Clin Oncol., 17(ll):3438-43), and anti-CK7 / 8 / 18 / 19 antibodies such as the pan-CK antibody #130-090-462 by Miltenyi biotec (T. Konig et al., 2023, Cancers (Basel). 15(3):568).

[0083] Exemplary antibodies (or fragments thereof) binding EpCAM (i.e., anti-EpCAM) that may be used in context of the present invention include, inter alia, HEA-125 (F.C. Cackowski et aL, 2019, Prostate, 79(14):1715-1727; MJ.M. Magbanua et aL, 2023, Breast Cancer Res Treat., 198(2):383-390), Ber-EP4 (WJ. Ellis et al., 2003, Urology, 61(2):277- 81; J. Pfitzenmaier et aL, 2006, BJU Int., 97(6): 1309-13; LN. Holcomb, 2008, Cancer Res., 68(14):5599-608; T.M. Morgan et aL, 2009, Clin Cancer Res., 15(2):677-83; CJ. Welty et aL, 2013, BMC Mol Biol., 14:6; L. Chery et aL, 2014, Oncotarget, 5(20):9939-51; Y. Wu et aL, 2016, J Mol Diagn., 18(l):131-43), and EBA-1 (F.C. Cackowski et al., 2019, Prostate, 79(14): 1715-1727; MJ.M. Magbanua et aL, 2023, Breast Cancer Res Treat,. 198(2):383-390).

[0084] In some embodiments, the antigen-binding molecule binding c-KIT protein is the anti-c-KIT antibody clone 104D2, the antigen-binding molecule binding EPCAM is the anti-EPCAM antibody clone HEA-125, and / or the antigen-binding molecule(s) binding at least one cytokeratin is the antibody clone A45-B / B3.

[0085] In context of the present invention, the expression or expression level of c-KIT and at least one DCC marker may be measured, for example, by imaging (in particular, by microscopy analysis), flow cytometry, mass cytometry (e.g., CyTOF or CyTOF imaging), PCR and / or sequencing. While imaging is particularly suitable for detecting or quantifying a fluorescent, immunocytochemical or radioactive detection signal, PCR and / or sequencing is particularly suitable for detecting or quantifying a signal derived from a DNA-tag or for directly detecting or quantifying the mRNA of a marker, as described herein. Flow cytometry is particularly suitable for detecting or quantifying a fluorescent detection signal, as described herein.

[0086] Furthermore, imaging (in particular, microscopy analysis) may be preferred for immunostaining of (a) protein(s) of (a) marker(s) such as c-KIT, EpCAM and / or CK, or for in-situ hybridization of an RNA of a marker such as MUC1.

[0087] Thus, in some preferred embodiments of the invention, the expression or expression level of at least one marker, e.g., c-KIT, MUC1, at least one cytokeratin and / or EPCAM, is measured by imaging. Preferably, the imaging may be performed by fully or semi-automated systems. Similarly, the subsequent data analysis of the images and identification of the metastasis founder cells may be performed fully or semi-automatically by computer-assisted (e.g., using artificial intelligence such as machine learning) methods.

[0088] Furthermore, fluorescent tags and fluorescent signals may be preferred in context of the present invention due to their advantages including their compatibility with automated imaging and / or flow cytometry platforms, a good safety profile, good availability, easy applicability, and suitability for multiplexing allowing the measurement of several markers in single cells.

[0089] Antigen-binding molecules, preferably antibodies or fragments thereof, coupled to a magnetic tag (e.g., a magnetic bead or a ferrofluid nanoparticle) may be used, for example, for enriching cells expressing a corresponding antigen (e.g., EpCAM) before analyzing the expression or expression level of a further marker (e.g., c-KTT) as described herein.

[0090] In addition to (preferably, prior to) measuring the expression or expression level of c-KTT and the DCC marker(s) according to the invention and / or in addition to (preferably, prior to) contacting the sample (e.g., a bone marrow or blood sample) with an antigen-binding molecule binding c-KIT protein, at least one antigen-binding molecule binding at least one DCC marker protein, a probe or primer pair binding c-KTT RNA, and / or at least one probe or primer pair binding at least one DCC marker RNA prior to the invention, according to the invention, erythrocytes and / or leukocytes such as granulocytes may be removed from the sample, (e.g., by using a density gradient centrifugation), and / or cells expressing CD45, CD33, CD11, CD325a, GlyA, CD27 and / or CD319 may be removed from the sample (e.g., by using a cell sorting method such as FACS, MACS or the CELLSEARCH® sytem). For example, cells expressing CD45, CD33, CD11 and GlyA may be removed from the sample, or cells expressing CD45, CD33, CD11, CD325a, and optionally CD27 and / or CD319, may be removed from the sample.

[0091] Such a cell removal (i.e., depletion) step as described above may increase the efficiency and / or accuracy of the method of the invention because the sample is enriched for DCC / CTS by removing potential contaminating cells in the sample (e.g., hematopoietic cell and / or blood cells).

[0092] In some embodiments of the invention, the sample is enriched for EpCAM -positive cells by sorting / purifying cells expressing EpCAM protein or expressing EpCAM protein above a respective threshold prior to measuring the expression or expression level of a further marker such as c-KTT, MUC1 and / or CK in single cells (e.g., by imaging or sequencing such as RNA sequencing, or by CELLSEARCH®), as described herein. The enrichment may comprise contacting the sample with an antigen-binding molecule (e.g., an antibody or fragment thereof) binding EpCAM protein, wherein said antigen-binding molecule is coupled to a fluorescent or magnetic tag or bound by a molecule (e.g., a secondary antibody) coupled to a fluorescent or magnetic tag, as described herein. Flow cytometry or other cell sorting techniques such as MACS® or the CELLTRACKS® AUTOPREP® System may then be used for sorting or purifying the cells bound by said antigen-binding molecule.

[0093] The detection or quantification of the negative prognostic markers identified in context of the present invention (in particular, c-KTT and / or MUC1) can be easily incorporated into existing approaches for detecting and / or quantifying DCC or CTC. The current gold standard method for detecting / quantifiying CTC is the FDA-approved CELLSEARCH® Circulating Tumor Cell (CTC) Test by Menarini silicon biosystems (Cristofanilli et al., 2005, J Clin Oncol. 23(7):1420-1430; de Bono et aL, 2008, Clin Cancer Res. 14:6302-6309; Cohen et al., 2008, J Clin Oncol. 26(19):3213-3221). The CELLSEARCH® Circulating Tumor Cell (CTC) Test detects and enumerates CTC in blood samples by measuring the expression or the expression level of EpCAM and cytokeratins (CK) (in particular, CK8, CK18 and CK19). Furthermore, CD45 is used as a negative marker to exclude contaminating blood cells.

[0094] Specifically, the CELLSEARCH® Circulating Tumor Cell (CTC) Test works as follows: solid blood components are separated from the plasma of a blood sample. By using ferrofluid nanoparticles comprising antibodies binding to EpCAM, EpCAM+CTCs are separated from the bulk of the other cells in the sample. The enriched cells are then stained with monoclonal antibodies against cytokeratins to further distinguish CTCs from the other cells in the sample. In addition, the cells are stained with a monoclonal antibody against CD45 to identify leukocytes that may have contaminated the sample. A DNA stain (in particular, DAPI) is added to visualize the nuclei of the cells. Then cells are brought into a single focal depth (in particular, by a magnetic cartridge). The CELLTRACKS ANALYZER II® System is used for scanning the cells, and the system automatically displays candidate cells that are positive for cytokeratin and DAPI, and which the operator can further review.

[0095] Thus, the CELLSEARCH® approach detects CTCs which are CD45-, EpCAM+, and cytokeratins 8+, 18+, and / or 19+ in a standardized and largely automated manner.

[0096] As surprisingly found in context of the present invention, and as detailed herein, the addition of the negative prognostic markers c-KIT and / or MUC1 to the classical DCC / CTC markers EpCAM and / or CK has a very strong negative predictive value. Thus, the CELLSEARCH® Circulating Tumor Cell (CTC) Test can be modified to stain for c-KIT and / or MUC1 (preferably c-KIT) in addition to or instead of cytokeratins 8, 18, and / or 19, which allows to identify metastasis founder cells, i.e., the DCC / CTC subset with a particularly strong negative predictive value. This should greatly enhance the sensitivity and / or specificity of the test and provide a more accurate diagnostics / prognostics for cancer patients.

[0097] Instead of the CELLSEARCH® system, other known platforms / technologies for marker-dependent or markerindependent enrichment of rare cell populations (e.g., down to a single cell) which are used for detecting DCC / CTC such as, inter alia, FACS or Parsortix® can be refined according to the invention, mutatis mutandis, by measuring c- KTT expression or expression level as additional marker.

[0098] In general, the skilled person can easily determine suitable thresholds for the expression level(s) of the employed marker(s) according to the invention, e.g., c-KIT, MUC1, EpCAM, and / or CK, based on controls and determine whether the expression level of a marker is above the respective threshold.

[0099] In particular, in context of the present invention the expression level of a marker in a cell may be considered above the respective threshold when the expression level of the marker in said cell is significantly higher than the background in the sample or significantly higher than the average expression level of the marker in the cells in the sample, preferably higher than the background in the sample. In context of protein expression, antigen-binding molecules and immunostainings, the background level in the sample may be determined by contacting a control sample (esp. a sample from the same tissue as the test sample) with an isotype control antibody, in particular, an antibody of the same isotype and comprising the same detectable label as the antibody used for measuring a maker protein such as c-KTT, EpCAM, CK or MUC1, which does not specifically bind to any antigen in the sample, and which may be, for example, directed against mineral oil.

[0100] For example, when the expression level of a marker is measured by imaging, the expression level of a marker in a cell may be considered above the respective threshold when the level of a detectable signal associated with the marker in the cell is higher than the mean signal in 10 neighboring cells plus three standard deviations of the signal in said 10 neighboring cells. This is also illustrated, for example, in the appended Examples l.G and l.H. Here, after fluorescently staining the cells and subsequent image acquisition, potentially positive cells and 10 neighboring negative cells may be manually labeled in an image handler program, and their respective mean gray values (MGVs) may be quantified. The MGV of a cell of interest should surpass the mean of the neighboring 10 cells plus three standard deviations of the MGVs of these cells for the respective fluorescent channel to be considered positive.

[0101] In addition, the present invention provides means (e.g., kits) for detecting metastasis founder cells, which may be used in the methods according to the invention.

[0102] Thus, in a one aspect, the present invention relates to a kit for use in detecting metastasis founder cells, said kit comprising

[0103] (a) an antigen-binding molecule binding c-KIT protein, an antigen-binding molecule binding EPCAM protein and / or an antigen-binding molecule binding at least one cytokeratin protein, as described herein; and / or

[0104] (b) a probe or primer pair binding c-KIT RNA, a probe or primer pair binding EPCAM RNA and / or a probe or primer pair binding at least one cytokeratin RNA, as described herein.

[0105] In some embodiments, the kit comprises an antigen-binding molecule, preferably an antibody or fragment thereof, binding c-KIT protein, as described herein. The kit may be preferably further combined with an antigen-binding molecule, preferably an antibody or fragment thereof, binding EPCAM protein, as described herein. Furthermore, the kit may be preferably further combined with an antigen-binding molecule, preferably an antibody or fragment thereof, binding at least one cytokeratin, as described herein. The kit may be also further combined with an antigen-binding molecule, preferably an antibody or fragment thereof, binding MUC1, as described herein, and / or a probe or primer pair binding MUC1 RNA.

[0106] Furthermore, present invention relates to the use of a kit according to the invention for detecting metastasis founder cells. In particular, said use is ex vivo or in vitro.

[0107] The present invention further relates to a combination of antigen-binding molecules comprising (i) an antigen-binding molecule binding c-KIT protein, and (ii) at least one antigen-binding molecule binding at least one DCC marker protein (e.g., EpCAM, at least one cytokeratin and / or MUC1), as described herein. Additionally, the present invention relates to a kit-of-parts comprising (i) an antigen-binding molecule binding c-KTT protein, and (ii) at least one antigen-binding molecule binding at least one DCC marker protein (e.g., EpCAM, at least one cytokeratin and / or MUC1), as defined herein.

[0108] In one aspect, the present invention further relates to a composition comprising (i) an antigen-binding molecule binding c-KTT protein, and (ii) at least one antigen-binding molecule binding at least one DCC marker protein, as described herein.

[0109] In context of the present invention, the kit or kit-of-parts may further comprise a brochure or leaflet with instructions for its use, for example, in a method according to the present invention.

[0110] The combination of the invention, the kit-of-parts of the invention, or the composition of the invention may be used for detecting metastasis founder cells as described herein, in particular, according to a method of the present invention.

[0111] The above-described combination, kit, kit-of-parts, or composition may comprise an antigen-binding molecule binding MUC1 protein and an antigen-binding molecule binding EPCAM protein, as described herein.

[0112] In a further aspect, the present invention relates to a method of isolating metastasis founder cells from a sample, comprising the steps of:

[0113] (a) contacting cells in a sample with an antigen-binding molecule binding c-KTT protein, and at least one antigenbinding molecule binding at least one DCC marker protein, as described herein, and

[0114] (b) isolating cells from the sample which express c-KTT and at least one DCC marker, wherein expression of c-KTT and at least one DCC marker in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of c-KTT and at least one DCC marker in a cell above respective thresholds are indicative of the cell being a metastasis founder cell, as described herein.

[0115] The metastasis founder cells may be isolated from the sample by using a micromanipulator, a cell sorting method employing FACS, MACS and / or ferrofluid nanoparticles (e.g., according to CELLSEARCH®), and / or a microfluidic device. Preferably, the cells are isolated in an automated and / or high-throughput manner, preferably by using FACS or a suitable microfluidic device (that may be coupled to imaging).

[0116] Preferably, the DCC marker(s) comprise(s) or consist(s) of EpCAM. Since c-KTT and EpCAM are surface markers, the cells can be kept alive during the isolation process. Therefore, the cells are, preferably, alive after isolating from the sample.

[0117] In some embodiments of the above isolation method of the invention, the sample is further contacted with an antigenbinding molecule binding MUC1 protein and EPCAM protein, wherein cells expressing c-KTT, MUC1 and EPCAM are isolated from the sample.

[0118] The kit, combination, composition, or kit-of-parts according to the invention may be also used for isolating metastasis founder cells from a sample, as described herein, in particular in a method according to the invention. In the context of the present invention the term "sample" may refer to a tissue sample. Furthermore, all or part of the cells in the sample may be connected to each other, or all or part of the cells may be separate from each other.

[0119] In the context of the present invention a sample may be obtained from tissues, body fluids, and / or organs of a subject / patient. As illustrated in the appended non-limiting examples, the sample may be a bone marrow (BM) aspirate. Furthermore, the sample may be a blood sample, as is used, e.g., in the CELLSEARCH® Circulating Tumor Cell (CTC) Test. "Blood" may be considered herein as a tissue or a body fluid. Samples of body fluids (i.e., liquid biopsies) can be obtained by well-known techniques and include samples of blood, lymphatic fluid, cerebrospinal fluid, or derivative thereof that may contain DCC and / or CTC. The cells of a sample may be subjected to a variety of well-known postcollection preparative and storage techniques prior to assessing the level of the markers in the sample.

[0120] Preferably herein, the tissue sample is from bone marrow, blood, lymph nodes or cerebrospinal fluid. In context of the present invention, it is particularly preferred that the sample is a bone marrow sample or a blood sample.

[0121] The bone marrow as an indicator organ for the detection of hematogenous systemic cancer spread is attractive not only because there are no autochthonous epithelial cells, but also because recent experimental data suggest that DCCs receive important instructional signals in the bone marrow that facilitate metastasis elsewhere (W. Zhang et aL, 2021, Cell 187, 2471-2486).

[0122] As bone marrow cells and blood cells both share a common lineage and originate from the same precursor cells, i.e., hematopoietic stem cells and progenitors, located in the bone marrow, detecting MFCs in blood is envisaged by the present invention. It is foreshadowed and plausible, especially in view of the ample experimental evidence provided herein with bone marrow samples, that the invention will also work well with blood samples.

[0123] In context of the present invention, the sample may comprise (i) at least about lxlO4, at least about 5xl04, at least about lxlO5or at least about 5xl05cells or (ii) from about lxlO4to about lxlO7or from about 5xl05to about 5xl05cells. In some preferred embodiments the sample may comprise (i) at least about lxlO5cells or (ii) from about lxlO5to about 2xl05or from about 1.5xl05to about 3xl05cells. In some more preferred embodiments, the sample may comprise (i) at least about 2xl05cells or (ii) about 2xl05cells.

[0124] In some preferred embodiments, the sample may be a bone marrow sample comprising (i) at least about lxlO4, at least about 5xl04, at least about lxlO5or at least about 5xl05mononucleated cells, or (ii) from about lxlO4to about lxlO7or from about 5xl05to about 5xl05mononucleated cells. In some preferred embodiment, the sample may be a bone marrow sample comprising (i) at least about lxlO5mononucleated cells, or (ii) from about lxlO5to about 2xl05or from about 1.5xl05to about 3xl05mononucleated cells. In some more preferred embodiments, the sample may be a bone marrow sample comprising (i) at least about 2xl05mononucleated cells, or (ii) about 2xl05mononucleated cells.

[0125] In some embodiments, the method for detecting metastasis founder cells in a sample comprising measuring in single cells in a sample the expression or expression level of a first marker which is c-KIT, further comprises measuring in single cells in the sample the expression or expression level of MUC1 and EPCAM, wherein expression of (i) c-KTT and / or MUC1 in combination with (ii) EPCAM in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of c-KTT and / or MUC1 above respective thresholds in combination with (ii) an expression level of EPCAM above a threshold in a cell are indicative of the cell being a metastasis founder cell.

[0126] In some embodiments, the method for analysing cells comprising a step of contacting a sample comprising cells with an antigen-binding molecule binding c-KTT protein, or a probe or primer pair binding c-KTT RNA according to the invention, further comprises contacting the cells in the sample (i) with an antigen-binding molecule binding MUC1 protein, or a probe or primer pair binding MUC1 RNA, preferably a probe or primer pair binding MUC1 RNA, and / or (ii) with an antigen-binding molecule binding EPCAM protein, or a probe or primer pair binding EPCAM RNA, preferably an antigen-binding molecule binding EPCAM protein.

[0127] The present invention further relates to a method for detecting metastasis founder cells in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) c-KTT and (ii) at least one DCC marker, wherein expression of c-KTT and at least one DCC marker in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of c-KTT and at least one DCC marker in a cell above respective thresholds are indicative of the cell being a metastasis founder cell, as described herein.

[0128] In the context of the present invention the subject may be a mammal such as a human, a horse, a cow, a monkey, a tiger, a mouse, a rat, a rabbit, or a guinea pig etc. Preferably herein, the subject is a patient, preferably a human patient, in particular, a patient that has, previously had, or is suspected of having a cancer. Furthermore, the patient may have previously had a metastasis and / or may be suspected of having and / or developing a metastasis.

[0129] Furthermore, the subject or patient according to the invention is, preferably, free of metastases (esp. full-blown metastases). While, in some embodiments, the subject or patient may be suspected of having a metastasis, in preferred embodiments the subject or patient has been diagnosed to currently not have metastases (although the subject or patient may have previously had a metastasis and / or may be suspected of developing a metastasis).

[0130] Furthermore, the subject according to the invention may be or may have been treated by surgery, anti-cancer radiation, and / or with at least one anti-cancer drug. In particular, the surgery may comprise or may have comprised full or partial resection of a tumor, e.g., a primary tumor. The anti-cancer drug may comprise or may have comprised a chemotherapy, a targeted therapy and / or an immunotherapy such as a checkpoint inhibitor.

[0131] In context of the present invention, the cancer is preferably an epithelial cancer. Herein and in context of the present invention, an epithelial cancer may be selected from the group consisting of: a lung carcinoma, a prostate carcinoma (i.e., prostate cancer), a breast cancer, a head and neck carcinoma, a colorectal carcinoma, an endometrial carcinoma, a cervical carcinoma, a bladder carcinoma, a pancreatic carcinoma, an ovarian carcinoma, a hepatocellular carcinoma, a renal carcinoma, a thyroid carcinoma, a bile duct carcinoma, a gallbladder carcinoma, a laryngeal carcinoma and a parotid carcinoma. In preferred embodiments, the epithelial cancer is a lung carcinoma, a breast cancer or a prostate carcinoma. In more preferred embodiments, the cancer is a lung carcinoma, more preferably a non-small-cell lung cancer (NSCLC). The presence of metastasis founder cells in a subject may indicate that the subject is likely to develop a metastasis, and / or the absence of metastasis founder cells in the subject as described herein may indicate that the subject is not likely to develop a metastasis.

[0132] Furthermore, the presence of metastasis founder cells in a subject may indicate that the cancer is likely to relapse or progress, and / or the absence of metastasis founder cells in the subject may indicate that the cancer is not likely to relapse or progress.

[0133] In addition, the presence of metastasis founder cells in a subject may indicate a negative outcome of the cancer, and / or the absence of metastasis founder cells in the subject may indicate a positive outcome of the cancer. In particular, a positive outcome may comprise survival of the subject, and / or a negative outcome may comprise death of the subject. The time of survival / the time until death may depend on the type of cancer, patient characteristics such as the age and / or the treatment of the patient. Thus, a positive outcome (e.g., survival) of a subject who is free of metastasis founder cells is relative to the corresponding outcome of a subject that has metastasis founder cells. Similarly, a negative outcome (e.g., death) of a subject who has metastasis founder cells is relative to the corresponding outcome of a subject that is free of metastasis founder cells.

[0134] Furthermore, the presence of metastasis founder cells in a subject allows to monitor and / or predict the success of an anti-cancer therapy such as a surgery (e.g., resection of a primary tumor), anti-cancer radiation and / or treatment with anti-cancer drug(s) (e.g., chemotherapy, a targeted therapy and / or an immunotherapy).

[0135] In particular, the presence of metastasis founder cells in a subject may indicate that an anti-cancer treatment is, will be or was not successful, and / or wherein the absence of metastasis founder cells in the subject may indicate that the anti-cancer treatment is, will be or was successful.

[0136] In addition, the presence of metastasis founder cells in a subject may indicate that at least one anti-cancer drug as described herein is to be administered to the subject, and / or the absence of metastasis founder cells in the subject may indicate that said anti-cancer drug(s) is / are not to be administered to the subject.

[0137] In a further aspect, the present invention relates to a method of prognosing the development of metastases in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) c- KTT and (ii) at least one DCC marker, as described herein,

[0138] (a) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing c-KTT and at least one DCC marker in the sample indicates that the subject is likely to develop a metastasis, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing c- KTT and at least one DCC marker in the sample indicates that the subject will likely not develop a metastasis, or

[0139] (b) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing c-KIT and at least one DCC marker at levels above respective thresholds in the sample indicates that the subject is likely to develop a metastasis, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the subject will likely not develop a metastasis.

[0140] The present invention also relates to a method of prognosing the relapse of a cancer in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) c-KTT and (ii) at least one DCC marker, as described herein,

[0141] (a) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing c-KIT and at least one DCC marker in the sample indicates that the cancer is likely to relapse, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing c-KIT and at least one DCC marker in the sample indicates that the cancer is not likely to relapse, or

[0142] (b) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing c-KIT and at least one DCC marker at levels above respective thresholds in the sample indicates that the cancer is likely to relapse, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing c-KIT and at least one DCC marker at levels above respective thresholds in the sample indicates that the cancer is not likely to relapse.

[0143] In another aspect the present invention relates to a method of prognosing the progression of a cancer in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) c-KIT and (ii) at least one DCC marker, as described herein,

[0144] (a) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing c-KIT and at least one DCC marker in the sample indicates that the cancer is likely to progress, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing c-KIT and at least one DCC marker in the sample indicates that the cancer is not likely to progress, or

[0145] (b) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing c-KIT and at least one DCC marker at levels above respective thresholds in the sample indicates that the cancer is likely to progress, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing c-KIT and at least one DCC marker at levels above respective thresholds in the sample indicates that the cancer is not likely to progress.

[0146] The present invention further relates to a method for prognosing the outcome of a cancer in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) c-KIT and (ii) at least one DCC marker, as described herein,

[0147] (a) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing c-KIT and at least one DCC marker in the sample is indicative of a negative outcome, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing c-KIT and at least one DCC marker in the sample is indicative of a positive outcome, or

[0148] (b) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing c-KIT and at least one DCC marker at levels above respective thresholds in the sample is indicative of a negative outcome, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing c-KIT and at least one DCC marker at levels above respective thresholds in the sample is indicative of a positive outcome. In particular, in context of the present invention a positive outcome may comprise survival of the subject for more than about 18 months or for more than about 24 months, and / or a negative outcome may comprise death of the subject within about 18 months or within about 24 months, respectively.

[0149] In a further aspect, the present invention relates to a method of measuring the success of a treatment of a subject that is or had been treated with at least one anti-cancer drug, said method comprising measuring in single cells in a sample from the subject the expression or expression level of (i) c-KTT and (ii) at least one DCC marker, as described herein,

[0150] (a) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing c-KTT and at least one DCC marker in the sample indicates that the treatment with the anti-cancer drug(s) is or was not successful, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing c-KTT and at least one DCC marker in the sample indicates that the treatment with the anti-cancer drug(s) is or was successful, or

[0151] (b) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the treatment with the anti-cancer drug(s) is or was not successful, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the treatment with the anti-cancer drug(s) is or was successful.

[0152] Furthermore, the present invention relates to a method of stratifying subjects for treatment with at least one anticancer drug, comprising measuring in single cells in a sample from a subject the expression or expression level of (i) c-KTT and (ii) at least one DCC marker, as described herein,

[0153] (a) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing c-KTT and at least one DCC marker in the sample indicates that the anti-cancer drug(s) are to be administered to the subject, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing c-KTT and at least one DCC marker in the sample indicates that the anti-cancer drug(s) are not to be administered to the subject, or

[0154] (b) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the anti-cancer drug(s) are to be administered to the subject, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the anti-cancer drug(s) are not to be administered to the subject.

[0155] In preferred embodiments of the present invention, the presence of at least one cell expressing c-KTT and at least one DCC marker, or expressing c-KTT and at least one DCC marker at levels above respective thresholds, as described herein, in the sample indicates that the subject is likely to develop a metastasis, indicates that the cancer is likely to relapse or progress, indicates a negative outcome, indicates that the treatment with the anti-cancer drug(s) is or was not successful, and / or indicates that the anti-cancer drug(s) are to be administered to the subject, and the absence of a cell expressing c-KTT and at least one DCC marker, or expressing c-KTT and at least one DCC marker at levels above respective thresholds, in the sample indicates that the subject will likely not develop a metastasis, indicates that the cancer is not likely to relapse or progress, indicates a positive outcome, indicates that the treatment with the anti- cancer drug(s) is or was successful, and / or indicates that the anti-cancer drug(s) are not to be administered to the subject, respectively.

[0156] In some embodiments of the present invention, the presence of at least one cell expressing c-KTT and at least one DCC marker, or expressing c-KTT and at least one DCC marker at levels above respective thresholds, as described herein, out of about lxlO4, about 5xl04, about lxlO5, about 5xl05, about lxlO5, about 2xl05, about 5xl05or about lxlO7cells in the sample indicates that the subject is likely to develop a metastasis, indicates that the cancer is likely to relapse or progress, indicates a negative outcome, indicates that the treatment with the anti-cancer drug(s) is or was not successful, and / or indicates that the anti-cancer drug(s) are to be administered to the subject, and the absence of a cell expressing c-KTT and at least one DCC marker, or expressing c-KTT and at least one DCC marker at levels above respective thresholds, out of about lxlO4, about 5xl04, about lxlO5, about 5xl05, about lxlO5, about 2xl05, about 5xl05or about lxlO7cells in the sample, respectively, indicates that the subject will likely not develop a metastasis, indicates that the cancer is not likely to relapse or progress, indicates a positive outcome, indicates that the treatment with the anti-cancer drug(s) is or was successful, and / or indicates that the anti-cancer drug(s) are not to be administered to the subject, respectively.

[0157] In some embodiments, the above methods of the invention may further comprise measuring in single cells in the sample, the expression or expression level of MUC1 and EPCAM, wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing (i) c-KTT and / or MUC1 and (ii) EPCAM in the sample, or the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing (i) c-KTT and / or MUC1 and (ii) EPCAM at levels above respective thresholds in the sample indicates that the subject is likely to develop a metastasis, the cancer is likely to relapse or progress, indicates a negative outcome, indicates that the treatment with the anti-cancer drug(s) is or was not successful or that the anti-cancer drug(s) are to be administered to the subject, respectively; and / or wherein the absence of at least one cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing (i) c-KTT and / or MUC1 and (ii) EPCAM in the sample, or the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing (i) c-KTT and / or MUC1 and (ii) EPCAM at levels above respective thresholds in the sample indicates that the subject will likely not develop a metastasis, that the cancer is not likely to relapse or progress, indicates a positive outcome, indicates that the treatment with the anti -cancer drug(s) is or was successful or that the anti-cancer drug(s) are not to be administered to the subject, respectively.

[0158] As described herein, the subject may be a patient that has, previously had, or is suspected of having a cancer. Furthermore, the patient may have previously had a metastasis and / or may be suspected of having and / or developing a metastasis.

[0159] As also described herein, the cancer is preferably an epithelial cancer such as a lung carcinoma, breast cancer, or prostate carcinoma. In preferred embodiments, the cancer is a lung carcinoma, preferably a non-small-cell lung cancer (NSCLC). In addition to c-KTT and MUC1, further markers were expressed on EpCAM -positive cells from patients with poor prognosis; see, e.g., Example 5 and Figures 6 and 7. These further markers include, in particular, KCNK5, SPN, TMEM63A, ENG, LRP5, USP5, ERH, AHCY, SNRPD1, LETM1, SULT1A3, C19orf48, NOL6, MEPCE, MCM3AP, CYB5RL, RABEPK, SCO1, PPRC1, KCNH2, CHD1, LMNA, NACA, WDR92, CHD8, CLUH, SSBP1, SAMD4B, FKBR4, RPL19, RPS10, SF1, RPS18, and SRRT.

[0160] Therefore, in a further aspect, the present invention relates to a method for detecting metastasis founder cells in a sample, comprising measuring in single cells in a sample the expression or expression level of (i) a first marker or marker combination selected from the group consisting of c-KIT, MUC1, KCNK5, SPN, TMEM63A, ENG, LRP5, USP5, ERH, AHCY, SNRPD1, LETM1, SULT1A3, C19orf48, NOL6, MEPCE, MCM3AP, CYB5RL, RABEPK, SCO1, PPRC1, KCNH2, CHD1, LMNA, NACA, WDR92, CHD8, CLUH, SSBP1, SAMD4B, FKBR4, RPL19, RPS10, SF1, RPS18, SRRT, and combinations thereof and (ii) at least one DCC marker, wherein expression of the first marker or marker combination and at least one DCC marker in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of the first marker or marker combination and at least one DCC marker in a cell above respective thresholds are indicative of the cell being a metastasis founder cell.

[0161] In some embodiments of the present invention, the first marker may be selected from the group consisting of c-KIT, MUC1, KCNK5, SPN, TMEM63A, ENG, LRP5, and combinations thereof.

[0162] In some further embodiments of the present invention, the first marker may be selected from the group consisting of USP5, ERH, AHCY, SNRPD1, LETM1, SULT1A3, C19orf48, NOL6, MEPCE, MCM3AP, CYB5RL, RABEPK, SCO1, PPRC1, KCNH2, CHD1, LMNA, NACA, WDR92, CHD8, CLUH, SSBP1, SAMD4B, FKBR4, RPL19, RPS10, SF1, RPS18, SRRT, and combinations thereof.

[0163] In preferred embodiments of the present invention the first marker is c-KIT. In some embodiments c-KIT may be replaced by MUC1 as a first marker or combined with MUC1 as a further marker.

[0164] The surprising finding in context of the present invention that c-KIT and / or MUC1 mark(s) a subset of DCC / CTC, i.e., metastasis founder cells, is not only of high relevance for the diagnostics of cancer but also provides a new target for anti-cancer therapies. While c-KIT has been already targeted in the treatment of cancer in different contexts by various drugs, it was not expected that c-KIT marks a subset of DCC / CTC, i.e., metastasis founder cells. Therefore, c-KIT may be targeted specifically in patients having metastasis founder cells (e.g., as identified according to the inventive methods provided herein), preferably in combination with an antigen binding molecule binding EpCAM that may be used for delivering a compound targeting c-KIT specifically to DCC or CTC.

[0165] Therefore, the present invention further provides one or more anti-cancer drugs for treating a patient having metastasis founder cells, as described herein.

[0166] As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated. Desirable effects of treatment include, but are not limited to, prophylaxis, preventing occurrence or recurrence of disease or symptoms associated with disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, improved prognosis and cure.

[0167] Accordingly, the present invention further relates to one or more anti-cancer drugs for use in treating a patient having metastasis founder cells, wherein said anti-cancer drug(s) comprise(s)

[0168] (a) a c-KTT inhibitor such as Imatinib, Sorafenib, Dasatinib, Sunitinib, Nilotinib, Amuvatinib, Regorafenib, Avapritinib, Ripretinib, Tivozanib, or any of compound 1 to 37 according to Table 2,

[0169] (b) an antigen-binding molecule, preferably an antibody of fragment thereof, binding c-KTT.

[0170] (c) an antigen-binding molecule, preferably an antibody of fragment thereof, binding EPCAM

[0171] (d) an antigen-binding molecule, preferably an antibody of fragment thereof, binding c-KIT and EPCAM, preferably, and / or

[0172] (e) CAR-T cells binding c-KIT and / or EPCAM, preferably, wherein said CAR T cells comprise

[0173] (i) a CAR-T cell comprising a CAR binding c-KIT and additionally a CAR binding EPCAM,

[0174] (ii) a CAR-T cell comprising a CAR binding c-KIT, and additionally a CAR-T cell comprising a CAR binding EPCAM, and / or

[0175] (iii) a CAR-T cell comprising a CAR binding c-KIT and EPCAM.

[0176] In particular, said metastasis founder cells express c-KIT and at least one DCC marker, or have expression levels of c- KTT and at least one DCC marker above respective thresholds, as described herein.

[0177] Preferably, the antigen-binding molecule binding c-KIT according to (b) blocks c-KIT signaling and / or is conjugated to a drug, preferably a cytostatic and / or cytotoxic drug such as Vedotin, Mafodotin, Deruxtecan, Govitecan, Tesirine, Ozagamyzin, Pasudotox, Soravtansine or Emtansine.

[0178] As used herein, Vedotin may also refer to Monomethyl auristatin E (MMAE), Mafodotin may also refer to maleimidocaproyl monomethyl auristatin F (mcMMAF), and Tesirine may also refer to pyrrolobenzodiazepine (PBD).

[0179] Preferably, the antigen-binding molecule binding EpCAM according to (c), is conjugated to a c-KIT inhibitor selected from the group consisting of Imatinib, Sorafenib, Dasatinib, Sunitinib, Nilotinib, Amuvatinib, Regorafenib, Avapritinib, Ripretinib, Tivozanib, or any of compound 1 to 37 according to Table 2, preferably Imatinib.

[0180] Preferably, the antigen-binding molecule binding c-KIT and EPCAM according to (d) blocks c-KIT signalling and / or is conjugated to a drug, preferably a cytostatic and / or cytotoxic drug such as Vedotin, Mafodotin, Deruxtecan, Govitecan, Tesirine, Ozagamyzin, Pasudotox, Soravtansine or Emtansine, and / or a c-KIT inhibitor selected from the group consisting of Imatinib, Sorafenib, Dasatinib, Sunitinib, Nilotinib, Amuvatinib, Regorafenib, Avapritinib, Ripretinib, Tivozanib, or any of compound 1 to 37 according to Table 2, preferably Imatinib. Preferably, the antigen-binding molecule binding c-KIT and EPCAM according to (d) is a bi-specific antibody or fragment thereof as described herein.

[0181] In particular, the antigen-binding molecule binding c-KIT and / or EpCAM may be used for delivering a cytostatic and / or cytotoxic drug to the metastasis founder cells, thereby eliminating these cells. This may prevent the development of metastases, prevent a relapse and / or progression of the cancer and / or prolong the survival of the patient. The treatment of a patient according to the present invention may comprise, in particular, preventing the development of metastases, preventing the relapse of a cancer, preventing progression (in particular, systemic progression) of a cancer and / or prolonging the survival of the patient.

[0182] It is further foreshadowed that patients having metastasis founder cells particularly benefit from the treatment with the drug(s) described herein as compared to other cancer patients, e.g., as compared to patients having a cancer not expressing corresponding marker or marker combinations (e.g., c-KTT and EpCAM, MUC1 and EpCAM, or c-KTT and MUC1), patients having only a primary tumor expressing said marker or marker combinations (but no metastasis founder cells), or patients already having metastases, esp. full-blown metastases.

[0183] In context of the present invention, an inhibitor of c-KTT may be selected from the group consisting of Imatinib, Sorafenib, Dasatinib, Sunitinib, Nilotinib, Amuvatinib, Regorafenib, Avapritinib, Ripretinib, Tivozanib, and compounds 1 to 37 according to Table 2. Preferably, the inhibitor of c-KIT is selected from the group consisting of Imatinib, Sorafenib, Dasatinib, Sunitinib, Nilotinib, Amuvatinib, Regorafenib, Avapritinib, Ripretinib, and Tivozanib. In some particularly preferred embodiments, the inhibitor of c-KIT is Imatinib.

[0184] Table 2: List of further c-KIT inhibitors as described in Pathania et al., 2021, BBA-Reviews on Cancer,

[0185] 1876(2):188631)

[0186]

[0187]

[0188] In some preferred embodiments of the invention, the c-KTT inhibitor is Imatinib.

[0189] Furthermore, the present invention provides novel means that may be used for treating a patient having metastasis founder cells. Accordingly, the present invention further relates to an antibody or fragment thereof binding c-KTT and EpCAM. In particular, said antibody or fragment thereof is bispecific. For example, said antibody or fragment thereof may be a bispecific antibody, a bispecific single chain antibody such as a bispecific scFv or a bispecific diabody.

[0190] The generation and validation of bispecific antibodies or fragments thereof, as described herein, can be done by employing well established routine methods.

[0191] Furthermore, the antibody or fragment thereof binding c-KIT and EpCAM according to the invention may block c-KIT signalling (e.g., by blocking the interaction of SCF and / or METRNL with c-KIT).

[0192] The antibody or fragment thereof binding c-KIT and EpCAM according to the invention may be conjugated to a drug, preferably a cytostatic and / or cytotoxic drug such as Vedotin, Mafodotin, Deruxtecan, Govitecan, Tesirine, Ozagamyzin, Pasudotox, Soravtansine or Emtansine, and / or a c-KIT inhibitor as described herein. An exemplary antibody-drug conjugate targeting c-KIT is described, e.g., in Kim et al., 2022, Int. J. Mol. Sci., 23:2264 which is incorporated herein by reference in its entirety.

[0193] The inventive antibody or fragment thereof binding c-KIT and EpCAM targets metastasis founder cells as described herein in context of the present invention, in particular DCC / CTC expressing c-KIT protein and EpCAM protein (especially on the cell surface). It is envisaged that the inventive antibody or fragment thereof binding c-KIT and EpCAM kills metastasis founder cells and / or prevents metastasis founder cells from forming a metastasis.

[0194] In addition, the present invention relates to a T cell comprising

[0195] (i) a chimeric antigen receptor (CAR) binding c-KIT and additionally a CAR binding EPCAM, and / or

[0196] (ii) a CAR binding c-KIT and EPCAM.

[0197] Chimeric antigen receptors (CAR) (incl. bispecific CARs) and T cells having such CARs (i.e., CART cells) are well known in the art or can be produced and validated by routine methods. Exemplary CAR T-cells targeting c-KIT are described, e.g., in Myburgh et al., 2020, Immunotherapy, 34:2688-2703, which is incorporated herein by reference in its entirety.

[0198] It is also possible to combine different types of CAR-T cells, each type having a CAR against a different antigen.

[0199] Therefore, the present invention also relates to a population of CAR-T cells comprising a CAR-T cell binding c-KIT and further a CAR-T cell binding EPCAM.

[0200] The inventive CAR-T cell or CAR-T cell population may target metastasis founder cells as described herein in context of the present invention, in particular DCC / CTC expressing c-KIT protein and EpCAM protein (especially also on the cell surface). It is envisaged that the inventive CAR-T cell or CAR-T cell population kills metastasis founder cells.

[0201] The inventive antibody or fragment thereof (binding e.g., c-KIT and EpCAM) or the inventive CAR-T cell or CAR-T cell population (targeting, e.g., c-KIT and EpCAM) may be used for treating a patient having metastasis founder cells, as described herein. A patient that is treated by one or more drugs according to the invention may have been previously diagnosed to have metastasis founder cells according to an inventive method provides herein.

[0202] Therefore, the present invention further relates to a method of treating a subject in need thereof comprising the steps of

[0203] (a) performing a method of detecting metastasis founder cells in a subject according to a method of present invention, and

[0204] (b) administering an effective amount of one or more anti-cancer drugs as described herein, when the subject has metastasis founder cells.

[0205] As already indicated herein above and as illustrated in Example 5 and Fig. 6F and G, next to c-KTT, Mucin 1 (MUC1) has been surprisingly found in context of the present invention as a negative predictive marker which has a strong negative predictive value in context of DCC, in particular, as regards the survival of patients having a cancer such as a NSCLC or a prostate cancer. Specifically, patients having EpCAM-positive DCC expressing a high level of MUC1 RNA had a higher risk to die than patients having only EpCAM-positive DCC expressing a low level of MUC1 RNA (P=0.0036 for NSCLC and 0.0023 for prostate cancer).

[0206] Therefore, MUC1 is, unexpectedly, a further marker of metastasis founder cells, as described herein. This is particularly surprising as mucins have been tested for many years in the field as DCC markers and were found not to be prognostic in most studies (S. Braun et al., 2003, Breast., 12(6): 397-404; C.A. Klein, 2003, Adv Cancer Res., 89:35-67). It is thought that the specificity of mucin (e.g., MUC1) protein detection in bone marrow greatly depends on the antibody clone chosen and may be related to the type of glycosylation. In contrast, in the present study, MUC1 mRNA has been tested and found to have a negative predictive value that was highly significant. Since the use of MUC1 RNA avoids the difficulties in reliably measuring MUC1 protein by an immunostaining (which may depend, e.g., on the glycosylation pattern), preferably MUC1 RNA is measured context of the present invention, for example by using a probe (and preferably in situ hybridization) or a primer pair (and preferably PCR and / or sequencing), as described herein.

[0207] While MUC1 had a negative predictive value in particular in the context of EpCAM protein-positive DCCs as demonstrated in the appended Examples, it is thought that EpCAM may be omitted as a marker. It was surprisingly found in context of the present invention that a MUC1 antibody (antibody clone 16A) did not detect a single MUC1- positive cell among 2 x 105bone marrow cells in a bone marrow sample of a non-cancer patient. Thus, MUC1 may be specific for DCC / CTC in non-epithelial tissues or body fluids, e.g., in the bone marrow, and further DCC marker such as EpCAM and / or CK may thus not be required. Hence, MUC1 (e.g., MUC1 mRNA) may be also used as a single marker for detecting metastasis founder cells, as described herein.

[0208] Accordingly, the present invention relates, in a further aspect, to a method for detecting metastasis founder cells in a sample, comprising measuring in single cells in a sample the expression or expression level of a first marker which is MUC1, wherein expression of MUC1 is indicative of the cell being a metastasis founder cell, or wherein the expression level of MUC1 in a cell above a threshold is indicative of the cell being a metastasis founder cell. MUC1 may be combined with c-KTT as a further marker, and / or at least one additional DCC marker such as EpCAM and / or at least one cytokeratin, as described herein.

[0209] In some embodiments, the method further comprises measuring in single cells in the sample the expression or expression level of a second marker which is c-KTT, wherein expression of MUC1 and / or c-KTT is indicative of the cell being a metastasis founder cell, or wherein expression levels of MUC1 and / or c-KTT in a cell above respective thresholds are indicative of the cell being a metastasis founder cell.

[0210] In further embodiments (which may be combined, e.g., with the embodiments directly above), the method further comprises measuring in single cells in the sample the expression or expression level of at least one additional disseminated cancer cell (DCC) marker such as EPCAM, wherein expression of (i) MUC1 and / or c-KTT in combination with (ii) expression of the additional DCC marker(s) in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of MUC1 and / or c-KTT above respective thresholds in combination with expression level(s) of the additional DCC marker(s) above respective thresholds in a cell are indicative of the cell being a metastasis founder cell.

[0211] As described herein, the expression or expression level of a marker may correspond to the protein and / or RNA expression or expression level of the marker. In context of the present invention, the expression or expression level of MUC1 is, preferably, the RNA expression or expression level of MUC1, respectively.

[0212] The cells in the sample may be contacted with an antigen-binding molecule binding MUC1 protein or a probe or primer pair binding MUC1 RNA, preferably a probe or primer pair binding MUC1 RNA, as described herein. Optionally, the cells in the sample may be contacted with an antigen-binding molecule binding c-KTT protein or a probe or primer pair binding c-KTT RNA, preferably an antigen-binding molecule binding c-KTT protein. Furthermore, the cells in the sample may be contacted with at least one antigen-binding molecule binding at least one additional DCC marker protein or at least one probe or primer pair binding at least one additional DCC marker RNA, as described herein.

[0213] Furthermore, the present invention relates to a method for analysing cells, comprising a step of contacting a sample comprising cells with

[0214] (i) an antigen-binding molecule binding MUC1 protein or a probe or primer pair binding MUC1 RNA, preferably a probe or primer pair binding MUC1 RNA; and

[0215] (ii) an antigen-binding molecule binding c-KTT protein a probe or primer pair binding c-KTT RNA, preferably an antigen-binding molecule binding c-KTT protein.

[0216] Optionally, the cells in the sample may be further contacted with at least one additional antigen-binding molecule binding at least one DCC marker protein or at least one additional probe or primer pair binding at least one DCC marker RNA, as described herein.

[0217] In addition, the expression or expression level of MUC1 and c-KTT, and optionally at least one additional DCC marker, may be measured in single cells in the sample, as described herein. In particular, the expression of MUC1 and / or c- KTT, and optionally at least additional one DCC marker such as EPCAM, in a cell is indicative of the cell being a metastasis founder cell, or expression levels of MUC1 and / or c-KTT, and optionally at least one additional DCC marker such as EPCAM, above respective thresholds are indicative of the cell being a metastasis founder cell, as described herein.

[0218] The present invention also relates to a method for analysing cells, comprising a step of contacting a sample comprising cells with

[0219] (i) an antigen-binding molecule binding MUC1 protein or a probe or primer pair binding MUC1 RNA, preferably a probe or primer pair binding MUC1 RNA; and

[0220] (ii) at least one antigen-binding molecule binding at least one additional DCC marker protein or at least one probe or primer pair binding at least one additional DCC marker RNA, preferably at least an antigen-binding molecule binding EPCAM.

[0221] Optionally, the cells in the sample may be further contacted with an antigen-binding molecule binding c-KTT protein a probe or primer pair binding c-KTT RNA, preferably an antigen-binding molecule binding c-KIT protein, as described herein.

[0222] In addition, the expression or expression level of MUC1 and at least one additional DCC marker, and optionally c-KIT, may be measured in single cells in the sample, as described herein.

[0223] In particular, expression of MUC1 and at least one additional DCC marker, and optionally c-KIT, in a cell is indicative of the cell being a metastasis founder cell, or expression levels of MUC1 and at least one additional DCC marker, and optionally c-KIT, above respective thresholds are indicative of the cell being a metastasis founder cell, as described herein.

[0224] Preferably, the additional DCC marker(s) (in addition to MUC1 which may be also considered as a DCC marker) comprise(s) at least one cytokeratin and / or EPCAM, more preferably at least EPCAM. In preferred embodiments, the cytokeratin(s) comprise(s) CK8, CK18 and / or CK19.

[0225] In a particularly preferred embodiment, the additional DCC marker(s) comprise(s) or consist(s) of EPCAM.

[0226] In some preferred embodiments, the expression or expression level of MUC1 and EPCAM, and optionally c-KIT, is measured in single cells, wherein (a) expression of MUC1 and EPCAM, and optionally c-KIT, in a cell is indicative of the cell being a metastasis founder cell, or (b) expression levels of MUC1 and EPCAM, and optionally c-KIT, in a cell above respective thresholds are indicative of the cell being a metastasis founder cell.

[0227] In some embodiments, the expression level of MUC1 and EPCAM is measured in single cells, wherein in a cell the combination of (i) expression of MUC1 and EPCAM, and (ii) a significantly higher expression level of EPCAM than in hematopoietic cells and / or blood cells, is indicative of the cell being a metastasis founder cell.

[0228] The method may further comprise a step of removing erythrocytes and / or leukocytes and / or cells expressing CD45, CD33, CD11, CD325a, GlyA, CD27 and / or CD319, as described herein. Furthermore, the present invention relates to a method for detecting metastasis founder cells in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) MUC1 and, optionally (ii) c-KTT and / or (iii) at least one additional DCC marker, wherein expression of MUC1 and / or c-KTT and optionally at least one additional DCC marker, in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of MUC1 and / or c-KTT and optionally at least one additional DCC marker in a cell above respective thresholds are indicative of the cell being a metastasis founder cell, as described herein.

[0229] In particular, the presence of metastasis founder cells in the subject may indicate that the subject is likely to develop a metastasis, and / or the absence of metastasis founder cells in the subject may indicate that the subject is not likely to develop a metastasis.

[0230] Furthermore, the presence of metastasis founder cells in the subject may indicate that the cancer is likely to relapse or progress, and / or the absence of metastasis founder cells in the subject may indicate that the cancer is not likely to relapse or progress.

[0231] In addition, the presence of metastasis founder cells in the subject may indicate a negative outcome of the cancer as described herein, and / or the absence of metastasis founder cells in the subject may indicate a positive outcome of the cancer, as described herein.

[0232] Furthermore, the presence of metastasis founder cells in the subject (in particular a subject that is or has been treated with at least one anti-cancer drug) may indicate that the treatment with the anti-cancer drug(s) is or was not successful, and / or wherein the absence of metastasis founder cells in the subject indicates that the treatment with the anti-cancer drug(s) is or was successful.

[0233] The presence of metastasis founder cells in the subject may further indicate that at least one anti-cancer drug as described herein is to be administered to the subject, and / or that the absence of metastasis founder cells in the subject indicates that said anti-cancer drug(s) is / are not to be administered to the subject.

[0234] In a further aspect, the present invention relates to a method of prognosing the development of metastases in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) MUC1 and / or c-KTT and optionally (ii) at least one additional DCC marker, as described herein,

[0235] (a) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, in the sample indicates that the subject is likely to develop a metastasis, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, in the sample indicates that the subject will likely not develop a metastasis, or

[0236] (b) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample indicates that the subject is likely to develop a metastasis, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample indicates that the subject will likely not develop a metastasis. The present invention also relates to a method of prognosing the relapse of a cancer in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) MUC1 and / or c-KTT and optionally (ii) at least one additional DCC marker, as described herein,

[0237] (a) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, in the sample indicates that the cancer is likely to relapse, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing MUC1 and / or c-KIT and optionally at least one additional DCC marker, in the sample indicates that the cancer is not likely to relapse, or

[0238] (b) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing MUC1 and / or c-KIT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample indicates that the cancer is likely to relapse, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing MUC1 and / or c-KIT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample indicates that the cancer is not likely to relapse.

[0239] Furthermore, the present invention relates to a method of prognosing the progression of a cancer in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) MUC1 and / or c-KIT and optionally (ii) at least one DCC marker, as described herein,

[0240] (a) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing MUC1 and / or c-KIT and optionally at least one additional DCC marker, in the sample indicates that the cancer is likely to progress, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing MUC1 and / or c-KIT and optionally at least one additional DCC marker, in the sample indicates that the cancer is not likely to progress, or

[0241] (b) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing MUC1 and / or c-KIT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample indicates that the cancer is likely to progress, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing MUC1 and / or c-KIT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample indicates that the cancer is not likely to progress.

[0242] In a further aspect, the present invention relates to a method for prognosing the outcome of a cancer in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) MUC1 and / or c-KIT and optionally (ii) at least one additional DCC marker, as described herein,

[0243] (a) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing MUC1 and / or c-KIT and optionally at least one additional DCC marker, in the sample is indicative of a negative outcome as described herein, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing MUC1 and / or c-KIT and optionally at least one additional DCC marker, in the sample is indicative of a positive outcome as described herein, or

[0244] (b) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing MUC1 and / or c-KIT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample is indicative of a negative outcome as described herein, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample is indicative of a positive outcome as described herein.

[0245] The invention further relates to a method of measuring the success of a treatment of a subject that is or had been treated with at least one anti-cancer drug, said method comprising measuring in single cells in a sample from the subject the expression or expression level of (i) MUC1 and / or c-KTT and optionally (ii) at least one additional DCC marker, as described herein,

[0246] (a) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing MUC1 and / or c-KIT and optionally at least one additional DCC marker, in the sample indicates that the treatment with the anti-cancer drug(s) is or was not successful, and / or wherein the absence of a cell cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing MUC1 and / or c-KIT and optionally at least one additional DCC marker, in the sample indicates that the treatment with the anti-cancer drug(s) is or was successful, or

[0247] (b) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing MUC1 and / orc- KTT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample indicates that the treatment with the anti-cancer drug(s) is or was not successful, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing MUC1 and / or c-KIT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample indicates that the treatment with the anti-cancer drug(s) is or was successful.

[0248] Furthermore, the present invention relates to a method of stratifying subjects for treatment with at least one anticancer drug, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) MUC1 and / or c-KIT and optionally (ii) at least one additional DCC marker, as described herein,

[0249] (a) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing MUC1 and / or c-KIT and optionally at least one additional DCC marker, in the sample indicates that the anti-cancer drug(s) are to be administered to the subject, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing MUC1 and / or c-KIT and optionally at least one additional DCC marker, in the sample indicates that the anti-cancer drug(s) are not to be administered to the subject, or

[0250] (b) wherein the presence of at least one cell (or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells) expressing MUC1 and / or c-KIT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample indicates that the anti-cancer drug(s) are to be administered to the subject, and / or wherein the absence of a cell (or the presence of less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells, respectively) expressing MUC1 and / or optionally c-KIT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample indicates that the anti-cancer drug(s) are not to be administered to the subject.

[0251] Furthermore, the present invention relates to a kit for use in detecting metastasis founder cells, said kit comprising

[0252] (a) an antigen-binding molecule binding MUC1 protein, an antigen-binding molecule binding c-KIT protein, and / or an antigen-binding molecule binding EPCAM protein, as described herein; and / or (b) a probe or primer pair binding MUC1 RNA, a probe or primer pair binding c-KTT RNA, and / or a probe or primer pair binding EPCAM RNA.

[0253] In some embodiments, the kit comprises a probe or primer pair binding MUC1 RNA and additionally an antigen-binding molecule binding c-KTT protein and / or an antigen-binding molecule binding EPCAM protein.

[0254] The invention also relates to the use of the kit of the invention for detecting metastasis founder cells.

[0255] Furthermore, the present invention relates to a combination of antigen-binding molecules comprising (i) an antigenbinding molecule binding MUC1 protein, and (ii) an antigen-binding molecule binding c-KTT protein, and optionally (iii) an antigen-binding molecule binding EPCAM protein, as described herein.

[0256] The present invention further relates to a combination of antigen-binding molecules comprising (i) an antigen-binding molecule binding MUC1 protein, and (ii) an antigen-binding molecule binding EPCAM protein, and optionally (iii) an antigen-binding molecule binding c-KTT protein, as described herein.

[0257] Furthermore, the present invention relates to a kit-of-parts of antigen-binding molecules comprising (i) an antigenbinding molecule binding MUC1 protein, and (ii) an antigen-binding molecule binding c-KTT protein, and optionally (iii) an antigen-binding molecule binding EPCAM protein, as described herein.

[0258] The present invention also relates to a kit-of-parts of antigen-binding molecules comprising (i) an antigen-binding molecule binding MUC1 protein, and (ii) an antigen-binding molecule binding EPCAM protein, and optionally (iii) an antigen-binding molecule binding c-KTT protein, as described herein.

[0259] As described herein, the kit or kit-of-parts may further comprise a brochure or leaflet with instructions for the use, preferably for the use in a method according to the present invention.

[0260] The present invention further relates to a composition comprising (i) an antigen-binding molecule binding MUC1 protein, and (ii) an antigen-binding molecule binding c-KTT protein, and optionally (iii) an antigen-binding molecule binding EPCAM protein, as described herein.

[0261] Furthermore, the present invention relates to a composition comprising (i) an antigen-binding molecule binding MUC1 protein, and (ii) an antigen-binding molecule binding EPCAM protein, and optionally (iii) an antigen-binding molecule binding c-KTT protein, as described herein.

[0262] The present invention also relates to the use of the inventive combination provided herein, the kit-of-parts provided herein, or the composition provided herein for detecting metastasis founder cells. In particular, said metastasis founder cells may express MUC1, and optionally c-KTT and / or at least one additional DCC marker, or have expression levels of MUC1, and optionally c-KTT and at least one additional DCC marker, above respective thresholds, as described herein. Furthermore, the present invention relates to a method of isolating metastasis founder cells from a sample, comprising the steps of:

[0263] (a) contacting cells in a sample with an antigen-binding molecule binding MUC1 protein, and optionally, an antigen-binding molecule binding c-KTT and / or at least one additional antigen-binding molecule binding at least one DCC marker protein, as described herein, and

[0264] (b) isolating cells from the sample which express MUC1, and optionally c-KTT and / or at least one additional DCC marker, wherein expression of MUC1, and optionally c-KTT and / or at least one additional DCC marker, in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of MUC1, and optionally c- KTT and / or at least one additional DCC marker, in a cell above respective thresholds are indicative of the cell being a metastasis founder cell, as described herein.

[0265] In preferred embodiments, said additional DCC marker(s) comprise(s) or consist(s) of EPCAM.

[0266] Furthermore, the cells are, preferably, alive after isolating from the sample, as described herein.

[0267] The metastasis founder cells may be isolated from the sample by using a micromanipulator, a cell sorting method employing FACS, MACS and / or ferrofluid nanoparticles (e.g., according to CELLSEARCH®), and / or a microfluidic device. Preferably, the cells are isolated in an automated and / or high-throughput manner, preferably by using FACS or a suitable microfluidic device (that may be coupled to imaging), as described herein.

[0268] Metastasis founder cells marked by MUC1 (and optionally c-KTT and / or EpCAM) may be also promising target in anticancer therapies.

[0269] Accordingly, the present invention relates, in a further aspect, to one or more anti-cancer drugs for use in treating a patient having metastasis founder cells, wherein said anti-cancer drug(s) comprise(s)

[0270] (a) an antigen-binding molecule, preferably an antibody of fragment thereof, binding MUC1, and, preferably, wherein said antigen-binding molecule is conjugated to a drug, preferably

[0271] (i) a c-KTT inhibitor as described herein and / or

[0272] (ii) a cytostatic and / or cytotoxic drug, such as Vedotin, Mafodotin, Deruxtecan, Govitecan, Tesirine, Ozagamyzin, Pasudotox, Soravtansine or Emtansine;

[0273] (b) an antigen-binding molecule, preferably an antibody of fragment thereof, binding MUC1 and c-KTT, wherein said antigen-binding molecule blocks c-KTT signalling and / or is conjugated to a c-KTT inhibitor and / or a cytostatic and / or cytotoxic drug as described herein;

[0274] (c) an antigen-binding molecule, preferably an antibody of fragment thereof, binding MUC1 and EPCAM, wherein said antigen-binding molecule is conjugated to a cytostatic and / or cytotoxic drug as described herein; and / or

[0275] (d) CAR-T cells binding MUC1, and optionally c-KTT and / or EPCAM, preferably, wherein said CART cells comprise

[0276] (i) a CAR-T cell comprising a CAR binding MUC1, and wherein said CAR-T cell optionally comprises a CAR binding c-KTT and / or a CAR binding EPCAM,

[0277] (ii) a CAR-T cell comprising a CAR binding c-KTT, and optionally, a CAR-T cell comprising a CAR binding c-KTT and / or a CAR-T cell comprising a CAR binding EPCAM, (iii) a CAR-T cell comprising a CAR binding MUC1 and c-KTT, and / or

[0278] (iv) a CAR-T cell comprising a CAR binding MUC1 and EPCAM.

[0279] In particular, in context of the above aspect, said metastasis founder cells express MUC1, and optionally c-KTT and / or at least one additional DCC marker, or have expression levels of MUC1, and optionally c-KIT and at least one additional DCC marker, above respective thresholds, as described herein.

[0280] Furthermore, the present invention relates to an antibody or fragment thereof, binding MUC1 and c-KIT. In particular, said antibody or fragment thereof may be a bispecific antibody or fragment thereof, as described herein. Furthermore, said antibody or fragment thereof may block c-KIT signalling. In addition, said antibody or fragment thereof may be conjugated to a c-KIT inhibitor and / or a cytostatic and / or cytotoxic drug as described herein.

[0281] The inventive antibody or fragment thereof binding MUC1 and c-KIT targets metastasis founder cells as described herein in context of the present invention, in particular DCC / CTC expressing c-MUCl protein and c-KIT protein (especially on the cell surface). It is envisaged that the inventive antibody or fragment thereof binding MUC1 and c- KTT kills metastasis founder cells and / or prevents metastasis founder cells from forming a metastasis.

[0282] The present invention also relates to an antibody or fragment thereof, binding MUC1 and EPCAM. In particular, said antibody or fragment thereof may be a bispecific antibody or fragment thereof, as described herein. In addition, said antibody or fragment thereof may be conjugated to a cytotoxic drug, as described herein.

[0283] The inventive antibody or fragment thereof binding MUC1 and EPCAM targets metastasis founder cells as described herein in context of the present invention, in particular DCC / CTC expressing MUC1 protein and EpCAM protein (especially on the cell surface). It is envisaged that the inventive antibody or fragment thereof binding MUC1 and EPCAM kills metastasis founder cells and / or prevents metastasis founder cells from forming a metastasis.

[0284] The present invention also relates to an antibody or fragment thereof, binding MUC1, c-KIT and EpCAM. In particular, said antibody or fragment thereof may be a trispecific antibody or fragment thereof (e.g., a trispecific triabody or a trispecific scFv), as described herein. Furthermore, said antibody or fragment thereof may block c-KIT signalling. In addition, said antibody or fragment thereof may be conjugated to a c-KIT inhibitor and / or a cytostatic and / or cytotoxic drug as described herein.

[0285] The inventive antibody or fragment thereof binding MUC1, c-KIT and EPCAM targets metastasis founder cells as described herein in context of the present invention, in particular DCC / CTC expressing MUC1 protein, c-KIT protein and EpCAM protein (especially on the cell surface). It is envisaged that the inventive antibody or fragment thereof binding MUC1, c-KIT and EPCAM kills metastasis founder cells and / or prevents metastasis founder cells from forming a metastasis.

[0286] In a further aspect, the present invention relates to a T cell comprising

[0287] (i) a chimeric antigen receptor (CAR) binding MUC1 and additionally a CAR binding c-KIT, and / or

[0288] (ii) a CAR binding MUC1 and c-KIT. The present invention also relates to a T cell comprising

[0289] (i) a chimeric antigen receptor (CAR) binding MUC1 and additionally a CAR binding EPCAM, and / or

[0290] (ii) a CAR binding MUC1 and EPCAM.

[0291] Further present invention further relates to a T cell comprising

[0292] (i) a chimeric antigen receptor (CAR) binding MUC1, a CAR binding c-KTT, and additionally a CAR binding EPCAM, and / or

[0293] (ii) a CAR binding MUC1, c-KTT and EPCAM.

[0294] Furthermore, the present invention relates to a population of CAR-T cells comprising (i) a CAR-T cell binding MUC1, and additionally (ii) a CAR-T cell binding c-KTT and / or a CAR-T cell binding EPCAM.

[0295] The inventive CAR-T cell or CAR-T cell population may target metastasis founder cells as described herein in context of the present invention, e.g., DCC / CTC expressing MUC1 protein, and optionally c-KTT protein and / or EpCAM protein (especially also on the cell surface). It is envisaged that the inventive CAR-T cell or CAR-T cell population kills metastasis founder cells.

[0296] The inventive antibody or fragment thereof (binding e.g., MUC1 and c-KTT and / or MUC1 and EpCAM) or the inventive CAR-T cell or CAR-T cell population (targeting, e.g., MUC1 and c-KTT and / or MUC1 and EpCAM) may be used for treating a patient having metastasis founder cells, as described herein.

[0297] In a further aspect the invention relates to an isolated cell expressing (i) a first marker which is c-KTT and (ii) at least one disseminated cancer cell (DCC) marker, as described herein. In particular, said cell is a metastasis founder cell, as described herein. Furthermore, said cell has been, preferably, isolated from a non -epithelial tissue or body fluid, as described herein. Preferably, said cell is obtainable or obtained by a method of isolating metastasis founder cells from a sample according to the present invention, wherein said method comprises contacting cells in a sample with an antigen-binding molecule binding c-KTT protein, and (ii) at least one antigen-binding molecule binding at least one DCC marker protein, as described herein.

[0298] The term "isolated", as used herein, refers to the isolation of a substance or biological material (e.g., a cell) from its natural environment in a subject. Therefore, an isolated substance or biological material (e.g., a cell) is in a non-natural environment, e.g., in a vial, a dish or a well of a cell culture plate, preferably, together with a suitable buffer. In particular, an isolated cell, as used herein, refers to a cell that has been isolated from a tissue or body fluid of a subject. Therefore, an isolated cell, as used herein, is not part of a natural tissue or body fluid. An isolated cell may be contained in a vial, a dish or a well of a cell culture plate, preferably further containing a suitable buffer or culture medium.

[0299] The present invention also relates to a population of isolated cells, wherein at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or all cells in the population express (i) a first marker which is c-KTT and (ii) at least one disseminated cancer cell (DCC) marker, as described herein.

[0300] Preferably, said cells are obtainable or obtained by a method of isolating metastasis founder cells from a sample according to the present invention, wherein said method comprises contacting cells in a sample with an antigen-binding molecule binding c-KTT protein, and (ii) at least one antigen-binding molecule binding at least one DCC marker protein, as described herein.

[0301] Preferably, the isolated cell or population of isolated cells expressing c-KTT express EpCAM and / or MUC1, preferably at least EpCAM.

[0302] In addition, the present invention relates to a population of isolated cells, wherein at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or all cells in the population express a first marker which is MUC1. Preferably, the cells expressing MUC1 further express a second marker which is c-KTT. It is also preferred that the cells expressing MUC1 further express at least one additional disseminated cancer cell (DCC) marker, preferably EpCAM, as described herein.

[0303] Preferably, said cells are obtainable or obtained by a method of isolating metastasis founder cells from a sample according to the present invention, wherein said method comprises contacting cells in a sample with an antigen-binding molecule binding MUC1, as described herein.

[0304] Furthermore, the isolated cell(s) expressing the first marker (i.e., c-KTT or MUC1) according to the invention, preferably, express EpCAM at a level that is significantly higher than the EpCAM expression level in hematopoietic cells and / or blood cells.

[0305] Furthermore, the isolated cell(s) expressing c-KTT according to the invention may express c-KTT at a level that is significantly higher than the c-KTT expression level in hematopoietic cells and / or blood cells.

[0306] Preferably, the invention population of isolated cells provided comprises at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1000, at least 2000, at least 5000 or at least 10000 cells.

[0307] In particular, the isolated cell(s) according to the present invention are metastasis founder cells, as described herein.

[0308] In a further aspect, the present invention relates to a cell culture comprising an isolated cell according to the invention or a population of isolated cells according to the invention, and, additionally, a medium for maintaining and / or proliferating the cell(s). Optionally, the cell culture further comprises stromal cells. In particular, the medium and / or the stromal cells promote the maintenance and / or proliferation of the isolated cell(s) according to the invention. The medium may comprise or may be supplemented with at least one ligand of c-KTT. Furthermore, the stromal cells may express (e.g., on the cell surface) and / or secrete at least one ligand of c-KTT.

[0309] Herein and in context of the present invention, the ligands of c-KTT may comprise, in particular, stem cell factor (SCF, also known as KL) and / or meteorin-like (METRNL).

[0310] Furthermore, the present invention relates to a method of culturing cells, comprising culturing an isolated cell of the invention or a population of isolated cells of the invention in a medium for maintaining and / or proliferating the cell(s). Optionally, said cell(s) are cultured on stromal cells, and preferably, wherein the medium comprises at least one ligand of c-KTT, and / or the stromal cells express or secrete at least one ligand of c-KTT of c-KTT, as described herein.

[0311] The isolated cell of the invention, the population of isolated cells according to the invention, or the cell culture or method of culturing cells according to the invention may be used for drug screening.

[0312] In particular, the isolated cell(s) of the invention may be used for identifying and / or validating drugs that target metastasis founder cells. Such drugs may be used for treating a patient having metastasis founder cells, as described herein.

[0313] Furthermore, the isolated cell of the invention, the population of isolated cells according to the invention, or the cell culture or method of culturing cells according to the invention may be used for identifying drug targets. This may comprise detecting one or more mutations in the genomic DNA of the isolated cell(s), preferably in one or more open reading frames (ORF). A protein encoded by an ORF harbouring one or more mutations may be a target for the treatment of a patient having metastasis founder cells.

[0314] Furthermore, the present invention relates to the following items:

[0315] 1. A method for detecting metastasis founder cells in a sample, comprising measuring in single cells in a sample the expression or expression level of (i) a first marker which is c-KIT and (ii) at least one disseminated cancer cell (DCC) marker, wherein expression of c-KIT and at least one DCC marker in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of c-KIT and at least one DCC marker in a cell above respective thresholds are indicative of the cell being a metastasis founder cell.

[0316] 2. The method of item 1, wherein the expression or expression level of a marker corresponds to the protein and / or RNA expression or expression level of the marker, respectively, preferably to the protein expression or expression level.

[0317] 3. The method of item 1 or 2, wherein the cells in the sample are contacted with

[0318] (i) an antigen-binding molecule binding c-KIT protein, or a probe or primer pair binding c-KIT RNA, preferably an antigen-binding molecule binding c-KIT protein; and / or

[0319] (ii) at least one antigen-binding molecule binding at least one DCC marker protein, or at least one probe or primer pair binding at least one DCC marker RNA.

[0320] 4. A method for analysing cells, comprising a step of contacting a sample comprising cells with

[0321] (i) an antigen-binding molecule binding c-KIT protein, or a probe or primer pair binding c-KIT RNA, preferably an antigen-binding molecule binding c-KIT protein; and

[0322] (ii) at least one antigen-binding molecule binding at least one DCC marker protein or at least one probe or primer pair binding at least one DCC marker RNA.

[0323] 5. The method of item 4, wherein the expression or expression level of c-KIT and at least one DCC marker is measured in single cells in the sample. The method of item 5, wherein expression of c-KTT and at least one DCC marker in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of c-KIT and at least one DCC marker in a cell above respective thresholds are indicative of the cell being a metastasis founder cell. The method of any one of items 1 to 6, wherein the DCC marker(s) comprise(s) EPCAM, at least one cytokeratin, and / or MUC1; preferably wherein the DCC marker(s) comprise(s) at least EPCAM, and optionally further at least one cytokeratin or MUC1; and, preferably, wherein the cytokeratin(s) comprise(s) CK8, CK18 and / or CK19. The method of any one of items 1 to 7, wherein the expression or expression level of c-KTT and at least one cytokeratin such as CK8, CK18 and / or CK19 is measured in single cells, and wherein (a) expression of c-KTT and at least one cytokeratin in a cell is indicative of the cell being a metastasis founder cell, or (b) expression levels of c-KTT and at least cytokeratin in a cell above respective thresholds are indicative of the cell being a metastasis founder cell. The method of any one of items 1 to 8, wherein the expression or expression level of c-KTT and EPCAM is measured in single cells, and wherein (a) expression of c-KTT and EPCAM in a cell is indicative of the cell being a metastasis founder cell, or (b) expression levels of c-KTT and EPCAM in a cell above respective thresholds are indicative of the cell being a metastasis founder cell. The method of any one of items 1 to 9, wherein the expression level of c-KTT and / or EPCAM is measured in single cells, and wherein in a cell the combination of (i) expression of c-KTT and EPCAM, and (ii) a significantly higher expression level of EPCAM than in hematopoietic cells and / or blood cells, is indicative of the cell being a metastasis founder cell. The method of any one of items 1 to 10, wherein the expression level of c-KTT, at least one cytokeratin and / or EPCAM such as CK8, CK18 and / or CK19 is measured in single cells, and wherein expression of c-KTT, at least one cytokeratin and EPCAM in a cell is indicative of the cell being a metastasis founder cell; preferably, wherein in a cell the combination of (i) expression of c-KTT, at least one cytokeratin and EPCAM, and (ii) a significantly higher expression level EPCAM than in hematopoietic cells and / or blood cells, is indicative of the cell being a metastasis founder cell. The method of any one of items 3 to 11, wherein said antigen-binding molecule(s) is / are antibody / ies or antigen-binding fragment(s) thereof. The method of any one of items 3 to 12, wherein said antigen-binding molecule(s) comprise(s) (a) detectable label(s) or is / are bound by (a) molecule(s) comprising (a) detectable label(s). The method of item 13, wherein the detectable label is a fluorescent tag, a chromogenic enzyme, a luminogenic enzyme, a radioactive tag or a DNA-tag, preferably a fluorescent dye. The method of any one of items 3 to 13, wherein said antigen-binding molecules comprise) (i) an antigenbinding molecule, preferably an antibody or fragment, thereof binding c-KTT protein, and (ii) at least one antigen-binding molecule selected from the group consisting of: an antigen-binding molecule, preferably an antibody or fragment thereof, binding at least one cytokeratin protein, and an antigen-binding molecule, preferably an antibody or fragment thereof, binding EPCAM protein. 16. The method of item 15, wherein the antigen-binding molecule binding at least one cytokeratin protein is a pan-anti-cytokeratin antibody or an antibody binding CK8, CK18 and CK19.

[0324] 17. The method of any one of items 3 to 16, wherein the antigen-binding molecule binding c-KTT protein is the anti-c-KTT antibody clone 104D2, the antigen-binding molecule binding EPCAM is the anti-EPCAM antibody clone HEA-125, and / or wherein the antigen-binding molecule(s) binding at least one cytokeratin comprises the antibody clone A45-B / B3.

[0325] 18. The method of any one of items 1 to 3 and 5 to 17, wherein the expression or expression level of c-KTT and at least one DCC marker is measured by imaging, flow cytometry, PCR, and / or sequencing; preferably wherein the expression or expression level of c-KTT, at least one cytokeratin and / or EPCAM is measured by imaging.

[0326] 19. The method of any one of items 1 to 3 and 5 to 18, wherein the expression level of a marker corresponds to the level of a detectable signal, preferably a fluorescent signal, that is associated with the marker.

[0327] 20. The method of any one of items 1 to 3 and 6 to 19, wherein the expression level of a marker in a cell is above the respective threshold when the expression level of the marker in said cell is significantly higher than the background in the sample or significantly higher than the average expression level of the marker in the cells in the sample.

[0328] 21. The method of any one of items 1 to 3 and 6 to 19, wherein the expression level of a marker is measured by imaging, and wherein the expression level of a marker in a cell is above the respective threshold when the level of a detectable signal associated with the marker in the cell is higher than the mean signal in 10 neighbouring cells plus three standard deviations of the signal in said 10 neighbouring cells.

[0329] 22. The method of any one of items 1 to 21, wherein the sample is a tissue sample.

[0330] 23. The method of any one of items 1 to 22, wherein all or part of the cells in the sample are connected to each other, or wherein all or part of the cells are separate from each other.

[0331] 24. The method of any one of items 1 to 23, wherein the sample is from a non -epithelial tissue or a body fluid, preferably from bone marrow, blood, lymph nodes or cerebrospinal fluid, and more preferably, wherein the sample is a bone marrow sample or a blood sample.

[0332] 25. The method of any one of items 1 to 24, wherein

[0333] (i) the sample comprises at least about lxlO4, at least about 5xl04, at least about lxlO5or at least about 5xl05, preferably at least about lxlO5, more preferably at least about 2xl05cells; and, preferably, wherein the sample is a bone marrow sample comprising at least about lxlO4, at least about 5xl04, at least about lxlO5or at least about 5xl05, preferably at least about lxlO5, more preferably at least about 2xl05mononucleated cells; and / or

[0334] (ii) the sample comprises from about lxlO4to about lxlO7or from about 5xl05to about 5xl05, preferably from about lxlO5to about 2xl05or from about 1.5xl05to about 3xl05, more preferably about 2xl05cells; and preferably, wherein the sample is a bone marrow sample comprising from about lxlO4to about lxlO7or from about 5xl05to about 5xl05, preferably from about lxlO5to about 2xl05or from about 1.5xl05to about 3xl05, more preferably about 2xl05mononucleated cells.

[0335] 26. The method of any one of items 1 to 25, wherein the method comprises prior to measuring the expression or expression level of c-KIT and the DCC marker(s) and / or prior to contacting the sample with an antigen-binding molecule binding c-KIT protein, at least one antigen-binding molecule binding at least one DCC marker protein, a probe or primer pair binding c-KIT RNA, and / or at least one probe or primer pair binding at least one DCC marker RNA, a step of

[0336] (i) removing erythrocytes and / or leukocytes such as granulocytes from the sample, preferably by using a density gradient centrifugation, and / or

[0337] (ii) removing cells expressing CD45, CD33, CD11, CD325a, GlyA, CD27 and / or CD319 from the sample, preferably by using a cell sorting method such as FACS, MACS or CELLSEARCH®.

[0338] Zl. The method of any one of items 1 to 3 and 6 to 26, wherein the metastasis founder cells are disseminated cancer cells (DCCs) or circulating tumor cells (CTCs).

[0339] 28. The method of any one of items 1 to 3 and 6 to Zl, wherein the metastasis founder cells are from an epithelial cancer such as a lung carcinoma, breast cancer or prostate carcinoma, preferably from a lung carcinoma, more preferably from a non-small-cell lung cancer (NSCLC).

[0340] 29. The method of any one of items 1 to 3 and 6 to 28, wherein the metastasis founder cells are present as individual cells and / or within small cancer cell aggregates each comprising at most about 100000, at most about 10000, at most about 1000, or at most about 100 cancer cells, preferably at most about 100 cancer cells.

[0341] 30. The method of any one of items 1 to 3 and 6 to 29, wherein the metastasis founder cells have a potential of forming metastases, preferably a clinically detectable metastasis, in particular, a metastasis having a volume of more than about 0.5 cm3and / or comprising more than about 100 million cells; and, preferably wherein the metastasis founder cells have a higher potential of forming metastases than the bulk of cells in a corresponding primary tumour.

[0342] 31. A method for detecting metastasis founder cells in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) c-KIT and (ii) at least one DCC marker, wherein expression of c-KIT and at least one DCC marker in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of c-KIT and at least one DCC marker in a cell above respective thresholds are indicative of the cell being a metastasis founder cell, as defined in any one of items 1 to 3 and 7 to 30.

[0343] 32. The method of item 31, wherein the subject is a patient that has, previously had or is suspected of having a cancer.

[0344] 33. The method of item 31 or 32, wherein the patient previously had a metastasis and / or is suspected of having and / or developing a metastasis. 34. The method of item 32 or 33, wherein the cancer is an epithelial cancer such as a lung carcinoma, breast cancer or prostate carcinoma, preferably a lung carcinoma, more preferably a non-small-cell lung cancer (NSCLC).

[0345] 35. The method of any one of items 31 to 34, wherein the presence of metastasis founder cells in the subject indicates that the subject is likely to develop a metastasis, and / or wherein the absence of metastasis founder cells in the subject indicates that the subject is not likely to develop a metastasis.

[0346] 36. The method of any one of items 31 to 35, wherein the presence of metastasis founder cells in the subject indicates that the cancer is likely to relapse or progress, and / or wherein the absence of metastasis founder cells in the subject indicates that the cancer is not likely to relapse or progress.

[0347] 37. The method of any one of items 31 to 36, wherein the presence of metastasis founder cells in the subject indicates a negative outcome of the cancer, and / or wherein the absence of metastasis founder cells in the subject indicates a positive outcome of the cancer.

[0348] 38. The method of item 37, wherein a positive outcome comprises survival of the subject, and / or wherein a negative outcome comprises death of the subject.

[0349] 39. The method of any one of items 31 to 38, wherein the subject is or has been treated with at least one anticancer drug.

[0350] 40. The method of item 39, wherein the presence of metastasis founder cells in the subject indicates that the treatment with the anti-cancer drug(s) is or was not successful, and / or wherein the absence of metastasis founder cells in the subject indicates that the treatment with the anti-cancer drug(s) is or was successful.

[0351] 41. The method of any one of items 31 to 40, wherein the presence of metastasis founder cells in the subject indicates that at least one anti-cancer drug is to be administered to the subject, and / or wherein the absence of metastasis founder cells in the subject indicates that said anti-cancer drug(s) is / are not to be administered to the subject, wherein said anti-cancer drug(s) is / are as defined in item 50.

[0352] 42. A method of prognosing the development of metastases in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) c-KTT and (ii) at least one DCC marker, as defined in any one of items 1 to 3 and 5 to 30,

[0353] (a) wherein the presence of at least one cell expressing c-KTT and at least one DCC marker in the sample indicates that the subject is likely to develop a metastasis, and / or wherein the absence of a cell expressing c-KTT and at least one DCC marker in the sample indicates that the subject will likely not develop a metastasis, or

[0354] (b) wherein the presence of at least one cell expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the subject is likely to develop a metastasis, and / or wherein the absence of a cell expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the subject will likely not develop a metastasis, and, preferably, wherein the expression level above (a) threshold(s) is as defined in item 20 or 21. A method of prognosing the relapse of a cancer in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) c-KTT and (ii) at least one DCC marker, as defined in any one of items 1 to 3 and 5 to 30,

[0355] (a) wherein the presence of at least one cell expressing c-KTT and at least one DCC marker in the sample indicates that the cancer is likely to relapse, and / or wherein the absence of a cell expressing c-KTT and at least one DCC marker in the sample indicates that the cancer is not likely to relapse, or

[0356] (b) wherein the presence of at least one cell expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the cancer is likely to relapse, and / or wherein the absence of a cell expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the cancer is not likely to relapse, and, preferably, wherein the expression level above (a) threshold(s) is as defined in item 20 or 21. A method of prognosing the progression of a cancer in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) c-KTT and (ii) at least one DCC marker, as defined in any one of items 1 to 3 and 5 to 30,

[0357] (a) wherein the presence of at least one cell expressing c-KTT and at least one DCC marker in the sample indicates that the cancer is likely to progress, and / or wherein the absence of a cell expressing c-KTT and at least one DCC marker in the sample indicates that the cancer is not likely to progress, or

[0358] (b) wherein the presence of at least one cell expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the cancer is likely to progress, and / or wherein the absence of a cell expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the cancer is not likely to progress, and, preferably, wherein the expression level above (a) threshold(s) is as defined in item 20 or 21. A method for prognosing the outcome of a cancer in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) c-KTT and (ii) at least one DCC marker, as defined in any one of items 1 to 3 and 5 to 30,

[0359] (a) wherein the presence of at least one cell expressing c-KTT and at least one DCC marker in the sample is indicative of a negative outcome, and / or wherein the absence of a cell expressing c-KTT and at least one DCC marker in the sample is indicative of a positive outcome, or

[0360] (b) wherein the presence of at least one cell expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample is indicative of a negative outcome, and / or wherein the absence of a cell expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample is indicative of a positive outcome, and, preferably, wherein the expression level above (a) threshold(s) is as defined in item 20 or 21. The method of item 45, wherein a positive outcome comprises survival of the subject for more than about 18 months or for more than about 24 months, and / or wherein a negative outcome comprises death of the subject within about 18 months or within about 24 months, respectively. 47. A method of measuring the success of a treatment of a subject that is or had been treated with at least one anti-cancer drug, said method comprising measuring in single cells in a sample from the subject the expression or expression level of (i) c-KTT and (ii) at least one DCC marker, as defined in any one of items 1 to 3 and 5 to 30,

[0361] (a) wherein the presence of at least one cell expressing c-KTT and at least one DCC marker in the sample indicates that the treatment with the anti-cancer drug(s) is or was not successful, and / or wherein the absence of a cell expressing c-KTT and at least one DCC marker in the sample indicates that the treatment with the anti-cancer drug(s) is or was successful, or

[0362] (b) wherein the presence of at least one cell expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the treatment with the anti-cancer drug(s) is or was not successful, and / or wherein the absence of a cell expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the treatment with the anti - cancer drug(s) is or was successful, and, preferably, wherein the expression level above (a) threshold(s) is as defined in item 20 or 21.

[0363] 48. A method of stratifying subjects for treatment with at least one anti-cancer drug, comprising measuring in single cells in a sample from a subject the expression or expression level of (i) c-KTT and (ii) at least one DCC marker, as defined in any one of items 1 to 3 and 5 to 30,

[0364] (a) wherein the presence of at least one cell expressing c-KTT and at least one DCC marker in the sample indicates that the anti-cancer drug(s) are to be administered to the subject, and / or wherein the absence of a cell expressing c-KTT and at least one DCC marker in the sample indicates that the anticancer drug(s) are not to be administered to the subject, or

[0365] (b) wherein the presence of at least one cell expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the anti-cancer drug(s) are to be administered to the subject, and / or wherein the absence of a cell expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the anti-cancer drug(s) are not to be administered to the subject, and, preferably, wherein the expression level above (a) threshold(s) is as defined in item 20 or 21.

[0366] 49. The method of any one of items 42 to 48, wherein the subject is as defined in any one of items 32 to 34.

[0367] 50. One or more anti-cancer drugs for use in treating a patient having metastasis founder cells, wherein said anticancer drug(s) comprise(s)

[0368] (a) a c-KTT inhibitor such as imatinib,

[0369] (b) an antigen-binding molecule, preferably an antibody of fragment thereof, binding c-KTT, and, preferably, wherein said antigen-binding molecule blocks c-KTT signaling and / or is conjugated to a drug, preferably a cytostatic and / or cytotoxic drug such as Vedotin, Mafodotin, Deruxtecan, Govitecan, Tesirine, Ozagamyzin, Pasudotox, Soravtansine or Emtansine. (c) an antigen-binding molecule, preferably an antibody of fragment thereof, binding EPCAM, preferably, wherein said antigen binding molecule is conjugated to a c-KTT inhibitor as defined in (a),

[0370] (d) an antigen-binding molecule, preferably an antibody of fragment thereof, binding c-KTT and EPCAM, preferably, wherein said antigen-binding molecule blocks c-KTT signalling and / or is conjugated to a drug as defined in (b) and / or a c-KTT inhibitor as defined in (a), and / or(e) CAR-T cells binding c-KTT and / or EPCAM, preferably, wherein said CAR T cells comprise

[0371] (i) a CAR-T cell comprising a CAR binding c-KTT and additionally a CAR binding EPCAM,

[0372] (ii) a CAR-T cell comprising a CAR binding c-KTT, and additionally a CAR-T cell comprising a CAR binding EPCAM, and / or

[0373] (iii) a CAR-T cell comprising a CAR binding c-KTT and EPCAM.

[0374] 51. The one or more anti-cancer drugs for use according to item 50, wherein the metastasis founder cells express c-KTT and at least one DCC marker, or have expression levels of c-KTT and at least one DCC marker above respective thresholds, as defined in any one of items 1 to 41.

[0375] 52. An antibody or fragment thereof binding c-KTT and EPCAM.

[0376] 53. The antibody or fragment thereof according to item 52, which blocks c-KTT signaling.

[0377] 54. The antibody or fragment thereof according to item 52 or 53, which is conjugated to a drug as defined in item 50(b) and / or a c-KTT inhibitor as defined in item 50(a).

[0378] 55. A T cell comprising

[0379] (i) a chimeric antigen receptor (CAR) binding c-KTT and additionally a CAR binding EPCAM, and / or

[0380] (ii) a CAR binding c-KTT and EPCAM.

[0381] 56. A population of CAR-T cells comprising a CAR-T cell binding c-KTT and a CAR-T cell binding EPCAM.

[0382] 57. A kit for use in detecting metastasis founder cells, said kit comprising

[0383] (a) an antigen-binding molecule binding c-KTT protein, an antigen-binding molecule binding EPCAM protein and / or an antigen-binding molecule binding at least one cytokeratin protein, as defined in any one of items 3 and 12 to 17; and / or

[0384] (b) a probe or primer pair binding c-KTT RNA, a probe or primer pair binding EPCAM RNA and / or a probe or primer pair binding at least one cytokeratin RNA.

[0385] 58. Use of a kit for detecting metastasis founder cells, said kit comprising

[0386] (a) an antigen-binding molecule binding c-KTT protein, an antigen-binding molecule binding EPCAM protein and / or an antigen-binding molecule binding at least one cytokeratin protein, as defined in any one of items 3 and 12 to 17; and / or (b) a probe or primer pair binding c-KTT RNA, a probe or primer pair binding EPCAM RNA and / or a probe or primer pair binding at least one cytokeratin RNA.

[0387] 59. A combination of antigen-binding molecules comprising (i) an antigen-binding molecule binding c-KTT protein, and (ii) at least one antigen-binding molecule binding at least one DCC marker protein, as defined in any one of items 3 and 12 to 17.

[0388] 60. A kit-of-parts comprising (i) an antigen-binding molecule binding c-KTT protein, and (ii) at least one antigenbinding molecule binding at least one DCC marker protein, as defined in any one of items 3 and 12 to 17.

[0389] 61. The kit of item 57, the use of item 58, or the kit-of-parts of item 60, wherein the kit further comprises a brochure or leaflet with instructions for the use.

[0390] 62. A composition comprising (i) an antigen-binding molecule binding c-KTT protein, and (ii) at least one antigenbinding molecule binding at least one DCC marker protein, as defined in any one of items 3 and 12 to 17.

[0391] 63. Use of the combination of item 58, the kit-of-parts of item 60 or the composition of item 62 for detecting metastasis founder cells.

[0392] 64. The kit of item 57 or 61 or the use of item 58 or 63, wherein the metastasis founder cells express c-KTT and at least one DCC marker, or have expression levels of c-KTT and at least one DCC marker above respective thresholds, as defined in any one of items 1 to 41.

[0393] 65. A method of isolating metastasis founder cells from a sample, comprising the steps of:

[0394] (a) contacting cells in a sample with an antigen-binding molecule binding c-KTT protein, and (ii) at least one antigen-binding molecule binding at least one DCC marker protein, as defined in any one of items 3 and 12 to 17, and

[0395] (b) isolating cells from the sample which express c-KTT and at least one DCC marker, wherein expression of c-KTT and at least one DCC marker in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of c-KTT and at least one DCC marker in a cell above respective thresholds are indicative of the cell being a metastasis founder cell, as defined in any one of items 1 to 3 and 7 to 30.

[0396] 66. The method of item 65, wherein the DCC marker(s) comprise(s) or consist(s) of EPCAM.

[0397] 67. The method of item 65 or 66, wherein the cells are alive after isolating from the sample.

[0398] 68. The method of any one of items 65 to 67, wherein the cells are isolated by using a micromanipulator, a cell sorting method employing FACS, MACS and / or ferrofluid nanoparticles, and / or a microfluidic device.

[0399] 69. A method for detecting metastasis founder cells in a sample, comprising measuring in single cells in a sample the expression or expression level of a first marker which is MUC1, wherein expression of MUC1 is indicative of the cell being a metastasis founder cell, or wherein the expression level of MUC1 in a cell above a threshold is indicative of the cell being a metastasis founder cell. 70. The method of item 69, further comprising measuring in single cells in the sample the expression or expression level of a second marker which is c-KIT, wherein expression of MUC1 and / or c-KTT is indicative of the cell being a metastasis founder cell, or wherein expression levels of MUC1 and / or c-KTT in a cell above respective thresholds are indicative of the cell being a metastasis founder cell.

[0400] 71. The method of item 69 or 70, further comprising measuring in single cells in the sample the expression or expression level of at least one additional disseminated cancer cell (DCC) marker such as EPCAM, wherein expression of (i) MUC1 and / or c-KTT in combination with (ii) expression of the additional DCC marker(s) in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of MUC1 and / or c- KTT above respective thresholds in combination with expression level(s) of the additional DCC marker(s) above respective thresholds in a cell are indicative of the cell being a metastasis founder cell.

[0401] 72. The method of any one of items 69 to 71, wherein the expression or expression level of a marker corresponds to the protein and / or RNA expression or expression level of the marker, respectively; preferably, wherein the expression or expression level of MUC1 is the RNA expression or expression level of MUC1, respectively.

[0402] 73. The method of any one of items 69 to 72, wherein the cells in the sample are contacted with

[0403] (i) an antigen-binding molecule binding MUC1 protein or a probe or primer pair binding MUC1 RNA, preferably a probe or primer pair binding MUC1 RNA, and optionally,

[0404] (ii) an antigen-binding molecule binding c-KTT protein or a probe or primer pair binding c-KTT RNA, preferably an antigen-binding molecule binding c-KTT protein, and / or

[0405] (iii) at least one antigen-binding molecule binding at least one additional DCC marker protein or at least one probe or primer pair binding at least one additional DCC marker RNA.

[0406] 74. A method for analysing cells, comprising a step of contacting a sample comprising cells with

[0407] (i) an antigen-binding molecule binding MUC1 protein or a probe or primer pair binding MUC1 RNA, preferably a probe or primer pair binding MUC1 RNA; and

[0408] (ii) an antigen-binding molecule binding c-KTT protein a probe or primer pair binding c-KTT RNA, preferably an antigen-binding molecule binding c-KTT protein; and optionally

[0409] (iii) at least one additional antigen-binding molecule binding at least one DCC marker protein or at least one additional probe or primer pair binding at least one DCC marker RNA.

[0410] 75. The method of item 74, wherein the expression or expression level of MUC1 and c-KTT, and optionally at least one additional DCC marker, is measured in single cells in the sample.

[0411] 76. The method of item 75, wherein expression of MUC1 and / or c-KTT, and optionally at least additional one DCC marker such as EPCAM, in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of MUC1 and / or c-KTT, and optionally at least one additional DCC marker such as EPCAM, above respective thresholds are indicative of the cell being a metastasis founder cell. A method for analysing cells, comprising a step of contacting a sample comprising cells with

[0412] (i) an antigen-binding molecule binding MUC1 protein or a probe or primer pair binding MUC1 RNA, preferably a probe or primer pair binding MUC1 RNA; and

[0413] (ii) at least one antigen-binding molecule binding at least one additional DCC marker protein or at least one probe or primer pair binding at least one additional DCC marker RNA, preferably at least an antigen-binding molecule binding EPCAM; and optionally

[0414] (iii) an antigen-binding molecule binding c-KTT protein a probe or primer pair binding c-KTT RNA, preferably an antigen-binding molecule binding c-KTT protein. The method of item 77, wherein the expression or expression level of MUC1 and at least one additional DCC marker, and optionally c-KTT, is measured in single cells in the sample. The method of item 78, wherein expression of MUC1 and at least one additional DCC marker, and optionally c-KTT, in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of MUC1 and at least one additional DCC marker, and optionally c-KTT, above respective thresholds are indicative of the cell being a metastasis founder cell. The method of any one of items 71 to 79, wherein the additional DCC marker(s) comprise(s) at least one cytokeratin and / or EPCAM, preferably at least EPCAM, and, preferably, wherein the cytokeratin(s) comprise(s) CK8, CK18 and / or CK19. The method of any one of items 71 to 80, wherein the DCC marker(s) comprise(s) or consist(s) of EPCAM. The method of any one of items 71 to 81, wherein the expression or expression level of MUC1 and EPCAM, and optionally c-KTT, is measured in single cells, and wherein (a) expression of MUC1 and EPCAM, and optionally c-KTT, in a cell is indicative of the cell being a metastasis founder cell, or (b) expression levels of MUC1 and EPCAM, and optionally c-KTT, in a cell above respective thresholds are indicative of the cell being a metastasis founder cell. The method of any one of items 71 to 82, wherein the expression level of MUC1 and EPCAM is measured in single cells, and wherein in a cell the combination of (i) expression of MUC1 and EPCAM, and (ii) a significantly higher expression level of EPCAM than in hematopoietic cells and / or blood cells, is indicative of the cell being a metastasis founder cell. The method of any one of items 73 to 83, wherein the antigen-binding molecule(s) is / are defined as in items 12 to 14. The method of any one of items 69 to 84, wherein the expression or expression level of a marker is measured as defined in item 18 or 21, the expression level of a marker is as defined in item 19 and / or the expression level of a marker above the respective threshold is as defined in item 20 or 21. 86. The method of any one of items 69 to 85, wherein the sample is as defined in any one of items 22 to 25.

[0415] 87. The method of any one of items 69 to 86, wherein the method further comprises a step of removing erythrocytes and / or leukocytes and / or cells expressing CD45, CD33, CD11, CD325a, GlyA, CD27 and / or CD319, as defined in item 26.

[0416] 88. The method of any one of items 69 to 87, wherein the metastasis founder cells are as defined in any one of items 27 to 30.

[0417] 89. A method for detecting metastasis founder cells in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) MUC1 and, optionally (ii) c-KTT and / or (iii) at least one additional DCC marker, wherein expression of MUC1 and / or c-KTT and optionally at least one additional DCC marker, in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of MUC1 and / or c-KTT and optionally at least one additional DCC marker in a cell above respective thresholds are indicative of the cell being a metastasis founder cell, as defined in any one of items 69 to 73, 76 and 79 to 88.

[0418] 90. The method of item 89, wherein the subject is as defined in any one of items 32 to 34.

[0419] 91. The method of item 89 or 90, wherein the presence of metastasis founder cells in the subject indicates that the subject is likely to develop a metastasis, and / or wherein the absence of metastasis founder cells in the subject indicates that the subject is not likely to develop a metastasis.

[0420] 92. The method of any one of items 89 to 91, wherein the presence of metastasis founder cells in the subject indicates that the cancer is likely to relapse or progress, and / or wherein the absence of metastasis founder cells in the subject indicates that the cancer is not likely to relapse or progress.

[0421] 93. The method of any one of items 89 to 92, wherein the presence of metastasis founder cells in the subject indicates a negative outcome of the cancer, and / or wherein the absence of metastasis founder cells in the subject indicates a positive outcome of the cancer; preferably wherein the positive outcome and / or negative outcome is as defined in item 38.

[0422] 94. The method of any one of items 89 to 93, wherein the subject is or has been treated with at least one anticancer drug.

[0423] 95. The method of item 94, wherein the presence of metastasis founder cells in the subject indicates that the treatment with the anti-cancer drug(s) is or was not successful, and / or wherein the absence of metastasis founder cells in the subject indicates that the treatment with the anti-cancer drug(s) is or was successful.

[0424] 96. The method of any one of items 89 to 95, wherein the presence of metastasis founder cells in the subject indicates that at least one anti-cancer drug is to be administered to the subject, and / or wherein the absence of metastasis founder cells in the subject indicates that said anti-cancer drug(s) is / are not to be administered to the subject, wherein said anti-cancer drug(s) is / are as defined in item 50.

[0425] 97. A method of prognosing the development of metastases in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) MUC1 and / or c-KTT and optionally (ii) at least one additional DCC marker, as defined in any one of items 69 to 73, 75, 76, and 78 to 96, (a) wherein the presence of at least one cell expressing MUC1 and / orc-KTT and optionally at least one additional DCC marker, in the sample indicates that the subject is likely to develop a metastasis, and / or wherein the absence of a cell expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, in the sample indicates that the subject will likely not develop a metastasis, or

[0426] (b) wherein the presence of at least one cell expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample indicates that the subject is likely to develop a metastasis, and / or wherein the absence of a cell expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample indicates that the subject will likely not develop a metastasis, and, preferably, wherein the expression level above (a) threshold(s) is as defined in item 20 or 21.

[0427] 98. A method of prognosing the relapse of a cancer in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) MUC1 and / or c-KTT and optionally (ii) at least one additional DCC marker, as defined in any one of items 69 to 73, 75, 76, and 78 to 96,

[0428] (a) wherein the presence of at least one cell expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, in the sample indicates that the cancer is likely to relapse, and / or wherein the absence of a cell expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, in the sample indicates that the cancer is not likely to relapse, or

[0429] (b) wherein the presence of at least one cell expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample indicates that the cancer is likely to relapse, and / or wherein the absence of a cell expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample indicates that the cancer is not likely to relapse, and, preferably, wherein the expression level above (a) threshold(s) is as defined in item 20 or 21.

[0430] 99. A method of prognosing the progression of a cancer in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) MUC1 and / or c-KTT and optionally (ii) at least one DCC marker, as defined in any one of items 69 to 73, 75, 76, and 78 to 96,

[0431] (a) wherein the presence of at least one cell expressing MUC1 and / orc-KTT and optionally at least one additional DCC marker, in the sample indicates that the cancer is likely to progress, and / or wherein the absence of a cell expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, in the sample indicates that the cancer is not likely to progress, or

[0432] (b) wherein the presence of at least one cell expressing MUC1 and / orc-KTT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample indicates that the cancer is likely to progress, and / or wherein the absence of a cell expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample indicates that the cancer is not likely to progress, and, preferably, wherein the expression level above (a) threshold(s) is as defined in item 20 or 21. A method for prognosing the outcome of a cancer in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) MUC1 and / or c-KTT and optionally (ii) at least one additional DCC marker, as defined in any one of items 69 to 73, 75, 76, and 78 to 96,

[0433] (a) wherein the presence of at least one cell expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, in the sample is indicative of a negative outcome, and / or wherein the absence of a cell expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, in the sample is indicative of a positive outcome, or

[0434] (b) wherein the presence of at least one cell expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample is indicative of a negative outcome, and / or wherein the absence of a cell expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample is indicative of a positive outcome, and, preferably, wherein the expression level above (a) threshold(s) is as defined in item 20 or 21. The method of item 100, wherein a positive outcome comprises survival of the subject, and / or wherein a negative outcome comprises death of the subject. A method of measuring the success of a treatment of a subject that is or had been treated with at least one anti-cancer drug, said method comprising measuring in single cells in a sample from the subject the expression or expression level of (i) MUC1 and / or c-KTT and optionally (ii) at least one additional DCC marker, as defined in any one of items 69 to 73, 75, 76, and 78 to 96,

[0435] (a) wherein the presence of at least one cell expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, in the sample indicates that the treatment with the anti-cancer drug(s) is or was not successful, and / or wherein the absence of a cell expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, in the sample indicates that the treatment with the anti-cancer drug(s) is or was successful, or

[0436] (b) wherein the presence of at least one cell expressing MUC1 and / orc-KTT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample indicates that the treatment with the anti-cancer drug(s) is or was not successful, and / or wherein the absence of a cell expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample indicates that the treatment with the anti-cancer drug(s) is or was successful, and, preferably, wherein the expression level above (a) threshold(s) is as defined in item 20 or 21. A method of stratifying subjects for treatment with at least one anti-cancer drug, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) MUC1 and / or c-KTT and optionally (ii) at least one additional DCC marker, as defined in any one of items 69 to 73, 75, 76, and 78 to 96,

[0437] (a) wherein the presence of at least one cell expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, in the sample indicates that the anti-cancer drug(s) are to be administered to the subject, and / or wherein the absence of a cell expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, in the sample indicates that the anti-cancer drug(s) are not to be administered to the subject, or

[0438] (b) wherein the presence of at least one cell expressing MUC1 and / or c-KTT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample indicates that the anticancer drug(s) are to be administered to the subject, and / or wherein the absence of a cell expressing MUC1 and / or optionally c-KTT and optionally at least one additional DCC marker, at levels above respective thresholds in the sample indicates that the anti-cancer drug(s) are not to be administered to the subject, and, preferably, wherein the expression level above (a) threshold(s) is as defined in item 20 or 21. The method of any one of items 97 to 103, wherein the subject is as defined in any one of items 32 to 34. One or more anti-cancer drugs for use in treating a patient having metastasis founder cells, wherein said anticancer drug(s) comprise(s)

[0439] (a) an antigen-binding molecule, preferably an antibody of fragment thereof, binding MUC1, and, preferably, wherein said antigen-binding molecule is conjugated to a drug, preferably

[0440] (i) a c-KTT inhibitor as defined in item 50 and / or

[0441] (ii) a cytostatic and / or cytotoxic drug, such as Vedotin, Mafodotin, Deruxtecan, Govitecan, Tesirine, Ozagamyzin, Pasudotox, Soravtansine or Emtansine;

[0442] (b) an antigen-binding molecule, preferably an antibody of fragment thereof, binding MUC1 and c-KTT, wherein said antigen-binding molecule blocks c-KTT signalling and / or is conjugated to a c-KTT inhibitor and / or a cytostatic and / or cytotoxic drug as defined in (a);

[0443] (c) an antigen-binding molecule, preferably an antibody of fragment thereof, binding MUC1 and EPCAM, wherein said antigen-binding molecule is conjugated to a cytostatic and / or cytotoxic drug as defined in (a); and / or

[0444] (d) CAR-T cells binding MUC1, and optionally c-KTT and / or EPCAM, preferably, wherein said CAR T cells comprise

[0445] (i) a CAR-T cell comprising a CAR binding MUC1, wherein said CAR-T cell optionally comprises a CAR binding c-KTT and / or a CAR binding EPCAM,

[0446] (ii) a CAR-T cell comprising a CAR binding c-KTT, and optionally, a CAR-T cell comprising a CAR binding c-KTT and / or a CAR-T cell comprising a CAR binding EPCAM,

[0447] (iii) a CAR-T cell comprising a CAR binding MUC1 and c-KTT, and / or

[0448] (iv) a CAR-T cell comprising a CAR binding MUC1 and EPCAM. The one or more anti-cancer drugs for use according to item 105, wherein the metastasis founder cells express MUC1, and optionally c-KTT and / or at least one additional DCC marker, or have expression levels of MUC1, and optionally c-KTT and at least one additional DCC marker, above respective thresholds, as defined in any one of items 69 to 88.

[0449] 107. An antibody or fragment thereof, binding MUC1 and c-KTT.

[0450] 108. The antibody or fragment thereof according to item 107, which blocks c-KTT signaling.

[0451] 109. The antibody or fragment thereof according to item 107 or 108, which is conjugated to a c-KTT inhibitor and / or a cytostatic and / or cytotoxic drug as defined in item 105.

[0452] 110. An antibody or fragment thereof, binding MUC1 and EPCAM.

[0453] 111. A T cell comprising

[0454] (i) a chimeric antigen receptor (CAR) binding MUC1 and additionally a CAR binding c-KTT, and / or

[0455] (ii) a CAR binding MUC1 and c-KTT.

[0456] 112. A T cell comprising

[0457] (i) a chimeric antigen receptor (CAR) binding MUC1 and additionally a CAR binding EPCAM, and / or

[0458] (ii) a CAR binding MUC1 and EPCAM.

[0459] 113. A population of CAR-T cells comprising (i) a CAR-T cell binding MUC1, and additionally (ii) a CAR-T cell binding c-KTT and / or a CAR-T cell binding EPCAM.

[0460] 114. A kit for use in detecting metastasis founder cells, said kit comprising

[0461] (a) an antigen-binding molecule binding MUC1 protein, an antigen-binding molecule binding c-KTT protein, and / or an antigen-binding molecule binding EPCAM protein, as defined in any one of items 73, 74 and 84; and / or

[0462] (b) a probe or primer pair binding MUC1 RNA, a probe or primer pair binding c-KTT RNA, and / or a probe or primer pair binding EPCAM RNA.

[0463] 115. Use of a kit for detecting metastasis founder cells, said kit comprising

[0464] (a) an antigen-binding molecule binding MUC1 protein, an antigen-binding molecule binding c-KTT protein, and / or an antigen-binding molecule binding EPCAM protein, as defined in any one of items 73, 74 and 84; and / or

[0465] (b) a probe or primer pair binding MUC1 RNA, a probe or primer pair binding c-KTT RNA, and / or a probe or primer pair binding EPCAM RNA.

[0466] 116. A combination of antigen-binding molecules comprising (i) an antigen-binding molecule binding MUC1 protein, and (ii) an antigen-binding molecule binding c-KTT protein, and optionally (iii) an antigen-binding molecule binding EPCAM protein, as defined in any one of items 73, 74 and 84. 117. A combination of antigen-binding molecules comprising (i) an antigen-binding molecule binding MUC1 protein, and (ii) an antigen-binding molecule binding EPCAM protein, and optionally (iii) an antigen-binding molecule binding c-KTT protein, as defined in any one of items 73, 74 and 84.

[0467] 118. A kit-of-parts of antigen-binding molecules comprising (i) an antigen-binding molecule binding MUC1 protein, and (ii) an antigen-binding molecule binding c-KTT protein, and optionally (iii) an antigen-binding molecule binding EPCAM protein, as defined in any one of items 73, 74 and 84.

[0468] 119. A kit-of-parts of antigen-binding molecules comprising (i) an antigen-binding molecule binding MUC1 protein, and (ii) an antigen-binding molecule binding EPCAM protein, and optionally (iii) an antigen-binding molecule binding c-KTT protein, as defined in any one of items 73, 74 and 84.

[0469] 120. The kit of item 114, the use of item 115, or the kit-of-parts of item 118 or 119, wherein the kit further comprises a brochure or leaflet with instructions for the use.

[0470] 121. A composition comprising (i) an antigen-binding molecule binding MUC1 protein, and (ii) an antigen-binding molecule binding c-KTT protein, and optionally (iii) an antigen-binding molecule binding EPCAM protein, as defined in any one of items 73, 74 and 84.

[0471] 122. A composition comprising (i) an antigen-binding molecule binding MUC1 protein, and (ii) an antigen-binding molecule binding EPCAM protein, and optionally (iii) an antigen-binding molecule binding c-KTT protein, as defined in any one of items 73, 74 and 84.

[0472] 123. Use of the combination of item 116 or 117, the kit-of-parts of item 118 or 119 or the composition of item 121 or 122 for detecting metastasis founder cells.

[0473] 124. The kit of item 114, or 123, or the use of item 115 or 123, wherein the metastasis founder cells express MUC1, and optionally c-KTT and / or at least one additional DCC marker, or have expression levels of MUC1, and optionally c-KTT and at least one additional DCC marker, above respective thresholds, as defined in any one of items 69 to 96.

[0474] 125. A method of isolating metastasis founder cells from a sample, comprising the steps of:

[0475] (a) contacting cells in a sample with an antigen-binding molecule binding MUC1 protein, and optionally, an antigen-binding molecule binding c-KTT and / or at least one additional antigen-binding molecule binding at least one DCC marker protein, as defined in any one of items 74, 77 and 84, and

[0476] (b) isolating cells from the sample which express MUC1, and optionally c-KTT and / or at least one additional DCC marker, wherein expression of MUC1, and optionally c-KTT and / or at least one additional DCC marker, in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of MUC1, and optionally c- KTT and / or at least one additional DCC marker, in a cell above respective thresholds are indicative of the cell being a metastasis founder cell, as defined in any one of items 69 to 73, 76 and 79 to 96.

[0477] 126. The method of item 125, wherein the additional DCC marker(s) comprise(s) or consist(s) of EPCAM.

[0478] 127. The method of item 125 or 126, wherein the cells are alive after isolating from the sample. 128. The method of any one of items 125 to 126, wherein the cells are isolated by using a micromanipulator, a cell sorting method employing FACS, MACS and / or ferrofluid nanoparticles, and / or a microfluidic device.

[0479] 129. The method of any one of items 1 to 3, and 5 to 41, comprising measuring in single cells in the sample the expression or expression level of MUC1 and EPCAM, wherein expression of (i) c-KTT and / or, MUC1 in combination with (ii) EPCAM in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of c-KTT and / or MUC1 above respective thresholds in combination with (ii) an expression level of EPCAM above a threshold in a cell are indicative of the cell being a metastasis founder cell.

[0480] 130. The method of any one of items 4 to 30, wherein the cells in the sample are contacted (i) with an antigenbinding molecule binding MUC1 protein, or a probe or primer pair binding MUC1 RNA, preferably a probe or primer pair binding MUC1 RNA, and / or (ii) with an antigen-binding molecule binding EPCAM protein, or a probe or primer pair binding EPCAM RNA, preferably an antigen-binding molecule binding EPCAM protein.

[0481] 131. The method of any one of items 42 to 49, comprising measuring in single cells in the sample, the expression or expression level of MUC1 and EPCAM, wherein the presence of at least one cell expressing (i) c-KTT and / or MUC1 and (ii) EPCAM in the sample, or the presence of at least one cell expressing (i) c-KTT and / or MUC1 and (ii) EPCAM at levels above respective thresholds in the sample indicates that the subject is likely to develop a metastasis, the cancer is likely to relapse or progress, indicates a negative outcome, indicates that the treatment with the anti-cancer drug(s) is or was not successful or that the anti-cancer drug(s) are to be administered to the subject, respectively; and / or wherein the absence of at least one cell expressing (i) c-KIT and / or MUC1 and (ii) EPCAM in the sample, or the absence of a cell expressing (i) c-KTT and / or MUC1 and (ii) EPCAM at levels above respective thresholds in the sample indicates that the subject will likely not develop a metastasis, that the cancer is not likely to relapse or progress, indicates a positive outcome, indicates that the treatment with the anti-cancer drug(s) is or was successful or that the anti-cancer drug(s) are not to be administered to the subject, respectively.

[0482] 132. The one of more drugs for use according to item 50 or 51, wherein said one or more drugs further comprise at least one drug as defined in item 105, preferably wherein the metastasis founder cells express MUC1 and EPCAM.

[0483] 133. The combination of item 58, the kit-of-parts of item 60 or 61, the composition of item 62, or the use of item 63, wherein the combination, kit-of-parts or composition comprises an antigen-binding molecule binding MUC1 protein and an antigen-binding molecule binding EPCAM protein.

[0484] 134. The method of any one of items 65 to 68, wherein the sample is contacted with an antigen-binding molecule binding MUC1 protein and EPCAM protein, and wherein cells expressing c-KTT, MUC1 and EPCAM are isolated from the sample.

[0485] 135. An isolated cell expressing (i) a first marker which is c-KTT and (ii) at least one disseminated cancer cell (DCC) marker.

[0486] 136. A population of isolated cells, wherein at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or all cells in the population express (i) a first marker which is c-KTT and (ii) at least one disseminated cancer cell (DCC) marker. 137. The cell of item 135 or the population of cells of item 136, wherein said cell(s) is / are obtainable by the method according to any one of items 65 to 68.

[0487] 138. A population of isolated cells, wherein at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or all cells in the population express a first marker which is MUC1.

[0488] 139. The population of cells of item 138, wherein the cells expressing MUC1 further express a second marker which is c-KTT.

[0489] 140. The population of cells of item 138 or 139, wherein the cells expressing MUC1 further express at least one additional disseminated cancer cell (DCC) marker, preferably EPCAM.

[0490] 141. The population of cells of any one of items 138 to 140, wherein said cell(s) is / are obtainable by the method according to any one of items 125 to 128.

[0491] 142. The cell of item 135 or the population of cells of item 136 or 137, wherein the DCC marker(s) comprise(s) or consist(s) of EPCAM and / or MUC1, preferably EPCAM.

[0492] 143. The cell of any one of items 135, 137 and 142 or the population of cells of any one of items 136 to 142, wherein the cell(s) expressing said first marker further express EPCAM at a level that is significantly higher than the EPCAM expression level in hematopoietic cells and / or blood cells.

[0493] 144. The cell of any one of items 135, 137, 142 and 143 or the population of cells of any one of items 136, 137 and 139 to 142, wherein the cells expressing c-KTT express c-KTT at a level that is significantly higher than the c-KTT expression level in differentiated hematopoietic cells and / or blood cells.

[0494] 145. The population of cells of any one of items 136 to 144, comprising at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1000, at least 2000, at least 5000 or at least 10000 cells.

[0495] 146. The cell of any one of items 135, 137 and 142 to 144 or the population of cells of any one of items 136 to 145, wherein said cell(s) are metastasis founder cells, preferably as defined in any one of items 27 to 30.

[0496] 147. A cell culture comprising the cell of any one of items 135, 137, 142 to 144 and 146 or the population of cells of any one of items 136 to 146, a medium for maintaining and / or proliferating the cell(s), and optionally, stromal cells; and preferably, wherein the medium comprises at least one ligand of c-KTT, and / or the stromal cells express or secrete at least one ligand of c-KTT; and, preferably, wherein the ligand(s) of c-KTT comprise(s) SCF and / or meteorin-like (METRNL).

[0497] 148. A method of culturing cells, comprising culturing the cell of any one of items 135, 137, 142 to 144 and 146 or the population of cells of any one of items 136 to 146 in a medium for maintaining and / or proliferating the cell(s); optionally, wherein said cells are cultured on stromal cells; and preferably, wherein the medium comprises at least one ligand of c-KTT, and / or the stromal cells express or secrete at least one ligand of c-KTT of c-KTT; and, preferably, wherein the ligand(s) of c-KTT comprise(s) SCF and / or meteorin-like (METRNL).

[0498] 149. Use of the cell of any one of items 135, 137, 142 to 144 and 146, the population of cells of any one of items 136 to 146 or the cell culture of item 147 for drug screening. 150. Use of the cell of any one of items 135, 137, 142 to 144 and 146, the population of cells of any one of items 136 to 146 for identifying drug targets.

[0499] 151. The use of item 150, comprising detecting one or more mutations in the genomic DNA of the cell(s), preferably in one or more open reading frames.

[0500] 152. A method of treating a subject in need thereof comprising the steps of

[0501] (a) performing a method of detecting metastasis founder cells in a subject according to any one of items 31 to 41 and 129, and

[0502] (b) administering an effective amount of one or more anti-cancer drugs as defined in item 50 to the subject, when the subject has metastasis founder cells.

[0503] 153. A method of treating a subject in need thereof comprising the steps of

[0504] (a) performing a method of detecting metastasis founder cells in a subject according to any one of items 89 to 96, and

[0505] (b) administering an effective amount of one or more anti-cancer drugs as defined in item 105 to the subject, when the subject has metastasis founder cells.

[0506] Sequence listina:

[0507] A sequence listing according to WIPO St. 26 is attached herewith. Further meta information regarding the sequences can be found in the following:

[0508] EPCAM epithelial cell adhesion molecule [ Homo sapiens (human) ]

[0509] Gene ID: 4072, updated on 10-Mar-2024

[0510] EpCAM, Gene sequence HGNC:HGNC: 11529

[0511] SEQ ID NO. 1:

[0512] >NC_000002.12:47369311-47387020 EPCAM [organism=Homo sapiens] [GeneID=4072] [chromosome=2]

[0513] SEQ ID NO. 2:

[0514] >NC_060926.1:47374345-47392054 EPCAM [organ ism = Homo sapiens] [GeneID=4072] [chromosome=2]

[0515] EpCAM, mRNA sequence

[0516] SEQ ID NO. 3:

[0517] >NM_002354.3 EPCAM [organ ism = Homo sapiens] [GeneID=4072]

[0518] EpCAM, Protein sequence

[0519] SEQ ID NO. 4:

[0520] >NP_002345.2 EPCAM [organism=Homo sapiens] [GeneID=4072]

[0521] KIT KIT proto-oncogene, receptor tyrosine kinase [ Homo sapiens (human) ]

[0522] Gene ID: 3815, updated on 17-Mar-2024 KTT, Gene sequence HGNC:HGNC:6342

[0523] SEQ ID NO. 5:

[0524] >NC_000004.12:54657957-54740715 KTT [organism=Homo sapiens] [GeneID=3815] [chromosome=4]

[0525] SEQ ID NO. 6:

[0526] >NC_060928.1:58146698-58229411 KTT [organism=Homo sapiens] [GeneID=3815] [chromosome=4]

[0527] KTT, mRNA sequence

[0528] SEQ ID NO. 7:

[0529] >NM_001385284.1 KTT [organism=Homo sapiens] [GeneID=3815] [transcript=3]

[0530] SEQ ID NO. 8:

[0531] >NM_001385290.1 KTT [organism=Homo sapiens] [GeneID=3815] [transcript=7]

[0532] SEQ ID NO. 9:

[0533] >NM_001385288.1 KTT [organism=Homo sapiens] [GeneID=3815] [transcript=6]

[0534] SEQ ID NO. 10:

[0535] >NM_001385292.1 KTT [organism=Homo sapiens] [GeneID=3815] [transcript=8]

[0536] SEQ ID NO. 11:

[0537] >NM_000222.3 KTT [organism=Homo sapiens] [GeneID=3815] [transcript=l]

[0538] SEQ ID NO. 12:

[0539] >NM_001385285.1 KTT [organism=Homo sapiens] [GeneID=3815] [transcript=4]

[0540] SEQ ID NO. 13:

[0541] >NM_001093772.2 KTT [organism=Homo sapiens] [GeneID=3815] [transcript=2]

[0542] SEQ ID NO. 14:

[0543] >NM_001385286.1 KTT [organism=Homo sapiens] [GeneID=3815] [transcript=5]

[0544] KTT, Protein sequence

[0545] SEQ ID NO. 15:

[0546] >NP_000213.1 KTT [organ ism = Homo sapiens] [GeneID=3815] [isoform=l precursor]

[0547] SEQ ID NO. 16:

[0548] >NP_001087241.1 KTT [organism=Homo sapiens] [GeneID=3815] [isoform=2 precursor]

[0549] SEQ ID NO. 17:

[0550] >NP_001372213.1 KTT [organism=Homo sapiens] [GeneID=3815] [isoform=3 precursor]

[0551] SEQ ID NO. 18:

[0552] >NP_001372214.1 KTT [organism=Homo sapiens] [GeneID=3815] [isoform=4 precursor]

[0553] SEQ ID NO. 19:

[0554] >NP_001372215.1 KTT [organism=Homo sapiens] [GeneID=3815] [isoform=5 precursor]

[0555] SEQ ID NO. 20:

[0556] >NP_001372217.1 KTT [organism=Homo sapiens] [GeneID=3815] [isoform=6 precursor]

[0557] SEQ ID NO. 21:

[0558] >NP_001372219.1 KTT [organism=Homo sapiens] [GeneID=3815] [isoform=7 precursor]

[0559] SEQ ID NO. 22:

[0560] >NP_001372221.1 KTT [organism=Homo sapiens] [GeneID=3815] [isoform=8 precursor] KRT1 keratin 1 [ Homo sapiens (human) ]

[0561] Gene ID: 3848, updated on 5-Mar-2024

[0562] KRT1, Gene sequence HGNC:HGNC:6412

[0563] SEQ ID NO. 23:

[0564] >NC_000012.12x52680407-52674736 KRT1 [organ ism = Homo sapiens] [GeneID=3848] [chromosome=12]

[0565] SEQ ID NO. 24:

[0566] >NC_060936.1x52644932-52639281 KRT1 [organism=Homo sapiens] [GeneID=3848] [chromosome =12]

[0567] KRT1, mRNA sequence

[0568] SEQ ID NO. 25:

[0569] >NM_006121.4 KRT1 [organ ism = Homo sapiens] [GeneID=3848]

[0570] KRT1, Protein sequence

[0571] SEQ ID NO. 26:

[0572] >NP_006112.3 KRT1 [organism=Homo sapiens] [GeneID=3848]

[0573] KRT2 keratin 2 [ Homo sapiens (human) ]

[0574] Gene ID: 3849, updated on 5-Mar-2024

[0575] KRT2, Gene sequence HGNC:HGNC:6439

[0576] SEQ ID NO. 27:

[0577] >NC_000012.12x52652211-52644558 KRT2 [organ ism = Homo sapiens] [GeneID=3849] [chromosome=12]

[0578] SEQ ID NO. 28:

[0579] >NC_060936.1x52616770-52609117 KRT2 [organism=Homo sapiens] [GeneID=3849] [chromosome =12]

[0580] KRT2, mRNA sequence

[0581] SEQ ID NO. 29:

[0582] >NM_000423.3 KRT2 [organ ism = Homo sapiens] [GeneID=3849]

[0583] KRT2, protein sequence

[0584] SEQ ID NO. 30:

[0585] >NP_000414.2 KRT2 [organism=Homo sapiens] [GeneID=3849]

[0586] KRT3 keratin 3 [ Homo sapiens (human) ]

[0587] Gene ID: 3850, updated on 5-Mar-2024

[0588] KRT3, Gene sequence HGNC:HGNC:6440

[0589] SEQ ID NO. 31:

[0590] >NC_000012.12x52796117-52789685 KRT3 [organ ism = Homo sapiens] [GeneID=3850] [chromosome=12]

[0591] SEQ ID NO. 32:

[0592] >NC_060936.1x52770193-52754139 KRT3 [organism=Homo sapiens] [GeneID=3850] [chromosome =12]

[0593] KRT3, mRNA sequence

[0594] SEQ ID NO. 33:

[0595] >NM_057088.3 KRT3 [organ ism = Homo sapiens] [GeneID=3850]

[0596] SEQ ID NO. 34: >XM_054372016.1 KRT3 [organ ism = Homo sapiens] [GeneID=3850] [transcript=Xl]

[0597] KRT3, Protein sequence

[0598] SEQ ID NO. 35:

[0599] >NP_476429.2 KRT3 [organism=Homo sapiens] [GeneID=3850]

[0600] SEQ ID NO. 36:

[0601] >XP_054227991.1 KRT3 [organ ism = Homo sapiens] [GeneID=3850] [isoform=Xl]

[0602] KRT4 keratin 4 [ Homo sapiens (human) ]

[0603] Gene ID: 3851, updated on 5-Mar-2024

[0604] KRT4, Gene sequence HGNC:HGNC:6441

[0605] SEQ ID NO. 37:

[0606] >NC_000012.12x52814116-52806549 KRT4 [organ ism = Homo sapiens] [GeneID=3851] [chromosome=12]

[0607] SEQ ID NO. 38:

[0608] >NC_060936.1x52778574-52771007 KRT4 [organism=Homo sapiens] [GeneID=3851] [chromosome =12]

[0609] KRT4, mRNA sequence

[0610] SEQ ID NO. 39:

[0611] >NM_002272.4 KRT4 [organ ism = Homo sapiens] [GeneID=3851]

[0612] KRT4, Protein sequence

[0613] SEQ ID NO. 40:

[0614] >NP_002263.3 KRT4 [organism=Homo sapiens] [GeneID=3851]

[0615] KRT5 keratin 5 [ Homo sapiens (human) ]

[0616] Gene ID: 3852, updated on 5-Mar-2024

[0617] KRT5, Gene sequence HGNC:HGNC:6442

[0618] SEQ ID NO. 41:

[0619] >NC_000012.12x52520394-52514575 KRT5 [organ ism = Homo sapiens] [GeneID=3852] [chromosome=12]

[0620] SEQ ID NO. 42:

[0621] >NC_060936.1x52484995-52479176 KRT5 [organism=Homo sapiens] [GeneID=3852] [chromosome =12]

[0622] KRT5, mRNA sequence

[0623] SEQ ID NO. 43:

[0624] >NM_000424.4 KRT5 [organ ism = Homo sapiens] [GeneID=3852]

[0625] KRT5, Protein sequence

[0626] SEQ ID NO. 44:

[0627] >NP_000415.2 KRT5 [organism=Homo sapiens] [GeneID=3852]

[0628] KRT6A keratin 6A [ Homo sapiens (human) ]

[0629] Gene ID: 3853, updated on 23-Mar-2024

[0630] KRT6A, Gene sequence HGNC:HGNC:6443

[0631] SEQ ID NO. 45:

[0632] >NC_000012.12x52493257-52487176 KRT6A [organism=Homo sapiens] [GeneID=3853] [chromosome=12] SEQ ID NO. 46:

[0633] >NC_060936.1x52457856-52451772 KRT6A [organ ism = Homo sapiens] [GeneID=3853] [chromosome=12]

[0634] KRT6A, mRNA sequence

[0635] SEQ ID NO. 47:

[0636] >NM_005554.4 KRT6A [organism=Homo sapiens] [GeneID=3853]

[0637] KRT6A, Protein sequence

[0638] SEQ ID NO. 48:

[0639] >NP_005545.1 KRT6A [organism=Homo sapiens] [GeneID=3853]

[0640] KRT6B keratin 6B [ Homo sapiens (human) ]

[0641] Gene ID: 3854, updated on 5-Mar-2024

[0642] KRT6B, Gene sequence HGNC:HGNC:6444

[0643] SEQ ID NO. 49:

[0644] >NC_000012.12x52452146-52446651 KRT6B [organ ism = Homo sapiens] [GeneID=3854] [chromosome=12]

[0645] SEQ ID NO. 50:

[0646] >NC_060936.1x52416753-52411258 KRT6B [organism=Homo sapiens] [GeneID=3854] [chromosome=12]

[0647] KRT6B, mRNA sequence

[0648] SEQ ID NO. 51:

[0649] >NM_005555.4 KRT6B [organism=Homo sapiens] [GeneID=3854]

[0650] KRT6B, Protein sequence

[0651] SEQ ID NO. 52:

[0652] >NP_005546.2 KRT6B [organ ism = Homo sapiens] [GeneID=3854]

[0653] KRT7 keratin 7 [ Homo sapiens (human) ]

[0654] Gene ID: 3855, updated on 3-Apr-2024

[0655] KRT7, Gene sequence HGNC:HGNC:6445

[0656] SEQ ID NO. 53:

[0657] >NC_000012.12:52233243-52255853 KRT7 [organism=Homo sapiens] [GeneID=3855] [chromosome =12]

[0658] SEQ ID NO. 54:

[0659] >NC_060936.1:52196307-52215830 KRT7 [organism=Homo sapiens] [GeneID=3855] [chromosome=12]

[0660] KRT7, mRNA sequence

[0661] SEQ ID NO. 55:

[0662] >XM_011538325.3 KRT7 [organ ism = Homo sapiens] [GeneID=3855] [transcript=Xl]

[0663] SEQ ID NO. 56:

[0664] >NM_005556.4 KRT7 [organ ism = Homo sapiens] [GeneID=3855]

[0665] SEQ ID NO. 57:

[0666] >XM_047428827.1 KRT7 [organ ism = Homo sapiens] [GeneID=3855] [transcript=X2]

[0667] SEQ ID NO. 58:

[0668] >XM_017019294.2 KRT7 [organ ism = Homo sapiens] [GeneID=3855] [transcript=X3] SEQ ID NO. 59:

[0669] >XM_054372019.1 KRT7 [organ ism = Homo sapiens] [GeneID=3855] [transcript=X6]

[0670] SEQ ID NO. 60:

[0671] >XM_054372018.1 KRT7 [organ ism = Homo sapiens] [GeneID=3855] [transcript=X5]

[0672] SEQ ID NO. 61:

[0673] >XM_054372020.1 KRT7 [organ ism = Homo sapiens] [GeneID=3855] [transcript=Xl]

[0674] SEQ ID NO. 62:

[0675] >XM_054372017.1 KRT7 [organ ism = Homo sapiens] [GeneID=3855] [transcript=X4]

[0676] SEQ ID NO. 63:

[0677] >XM_054372021.1 KRT7 [organ ism = Homo sapiens] [GeneID=3855] [transcript=X7]

[0678] KRT7, Protein sequence

[0679] SEQ ID NO. 64:

[0680] >NP_005547.3 KRT7 [organism=Homo sapiens] [GeneID=3855]

[0681] SEQ ID NO. 65:

[0682] >XP_011536627.1 KRT7 [organism=Homo sapiens] [GeneID=3855] [isoform=Xl]

[0683] SEQ ID NO. 66:

[0684] >XP_016874783.1 KRT7 [organ ism = Homo sapiens] [GeneID=3855] [isoform=X3]

[0685] SEQ ID NO. 67:

[0686] >XP_047284783.1 KRT7 [organ ism = Homo sapiens] [GeneID=3855] [isoform=X2]

[0687] SEQ ID NO. 68:

[0688] >XP_054227992.1 KRT7 [organ ism = Homo sapiens] [GeneID=3855] [isoform=X4]

[0689] SEQ ID NO. 69:

[0690] >XP_054227993.1 KRT7 [organ ism = Homo sapiens] [GeneID=3855] [isoform=X5]

[0691] SEQ ID NO. 70:

[0692] >XP_054227994.1 KRT7 [organ ism = Homo sapiens] [GeneID=3855] [isoform=X6]

[0693] SEQ ID NO. 71:

[0694] >XP_054227995.1 KRT7 [organ ism = Homo sapiens] [GeneID=3855] [isoform=Xl]

[0695] SEQ ID NO. 72:

[0696] >XP_054227996.1 KRT7 [organ ism = Homo sapiens] [GeneID=3855] [isoform=X7]

[0697] KRT8 keratin 8 [ Homo sapiens (human) ]

[0698] Gene ID: 3856, updated on 31-Mar-2024

[0699] KRT8, Gene sequence HGNC:HGNC:6446

[0700] SEQ ID NO. 73:

[0701] >NC_000012.12x52949860-52897191 KRT8 [organ ism = Homo sapiens] [GeneID=3856] [chromosome=12]

[0702] SEQ ID NO. 74:

[0703] >NC_060936.1x52914419-52861778 KRT8 [organism=Homo sapiens] [GeneID=3856] [chromosome =12]

[0704] KRT8, mRNA sequence

[0705] SEQ ID NO. 75:

[0706] >NR_045962.2 KRT8 [organism=Homo sapiens] [GeneID=3856] [transcript=4] SEQ ID NO. 76:

[0707] >NM_001256293.2 KRT8 [organism=Homo sapiens] [GeneID=3856] [transcript=3]

[0708] SEQ ID NO. 77:

[0709] >NM_001256282.2 KRT8 [organism=Homo sapiens] [GeneID=3856] [transcript=l]

[0710] SEQ ID NO. 78:

[0711] >NM_002273.4 KRT8 [organ ism = Homo sapiens] [GeneID=3856] [transcript=2]

[0712] SEQ ID NO. 79:

[0713] >XM_054372022.1 KRT8 [organ ism = Homo sapiens] [GeneID=3856] [transcript=Xl]

[0714] KRT8, Protein sequence

[0715] SEQ ID NO. 80:

[0716] >NP_001243211.1 KRT8 [organism=Homo sapiens] [GeneID=3856] [isoform=l]

[0717] SEQ ID NO. 81:

[0718] >NP_001243222.1 KRT8 [organism=Homo sapiens] [GeneID=3856] [isoform=2]

[0719] SEQ ID NO. 82:

[0720] >NP_002264.1 KRT8 [organism=Homo sapiens] [GeneID=3856] [isoform=2]

[0721] SEQ ID NO. 83:

[0722] >XP_054227997.1 KRT8 [organ ism = Homo sapiens] [GeneID=3856] [isoform=Xl]

[0723] KRT9 keratin 9 [ Homo sapiens (human) ]

[0724] Gene ID: 3857, updated on 5-Mar-2024

[0725] KRT9, Gene sequence HGNC:HGNC:6447

[0726] SEQ ID NO. 84:

[0727] >NC_000017.11x41572059-41565836 KRT9 [organ ism = Homo sapiens] [GeneID=3857] [chromosome=17]

[0728] SEQ ID NO. 85:

[0729] >NC_060941.1x42427646-42421421 KRT9 [organism=Homo sapiens] [GeneID=3857] [chromosome =17]

[0730] KRT9, mRNA sequence

[0731] SEQ ID NO. 86:

[0732] >NM_000226.4 KRT9 [organ ism = Homo sapiens] [GeneID=3857]

[0733] KRT9, Protein sequence

[0734] SEQ ID NO. 87:

[0735] >NP_000217.2 KRT9 [organism=Homo sapiens] [GeneID=3857]

[0736] KRT10 keratin 10 [ Homo sapiens (human) ]

[0737] Gene ID: 3858, updated on 5-Mar-2024

[0738] KRT10, Gene sequence HGNC:HGNC:6413

[0739] SEQ ID NO. 88:

[0740] >NC_000017.11x40822614-40818117 KRT10 [organism=Homo sapiens] [GeneID=3858] [chromosome=17]

[0741] SEQ ID NO. 89:

[0742] >NC_060941.1x41686934-41682437 KRT10 [organ ism = Homo sapiens] [GeneID=3858] [chromosome=17]

[0743] KRT10, mRNA sequence SEQ ID NO. 90:

[0744] >NM_000421.5 KRT10 [organism=Homo sapiens] [GeneID=3858] [transcript=l]

[0745] SEQ ID NO. 91:

[0746] >NM_001379366.1 KRT10 [organism=Homo sapiens] [GeneID=3858] [transcript=2]

[0747] KRT10, Protein sequence

[0748] SEQ ID NO. 92:

[0749] >NP_000412.4 KRT10 [organ ism = Homo sapiens] [GeneID=3858] [isoform=l]

[0750] SEQ ID NO. 93:

[0751] >NP_001366295.1 KRT10 [organism=Homo sapiens] [GeneID=3858] [isoform=2]

[0752] KRT12 keratin 12 [ Homo sapiens (human) ]

[0753] Gene ID: 3859, updated on 5-Mar-2024

[0754] KRT12, Gene sequence HGNC:HGNC:6414

[0755] SEQ ID NO. 94:

[0756] >NC_000017.11x40867223-40861303 KRT12 [organism=Homo sapiens] [GeneID=3859] [chromosome=17]

[0757] SEQ ID NO. 95:

[0758] >NW_003871091.1x16491-10571 KRT12 [organism=Homo sapiens] [GeneID=3859] [chromosome=17]

[0759] SEQ ID NO. 96:

[0760] >NC_060941.1x41731542-41725618 KRT12 [organ ism = Homo sapiens] [GeneID=3859] [chromosome=17]

[0761] KRT12, mRNA sequence

[0762] SEQ ID NO. 97:

[0763] >NM_000223.4 KRT12 [organism=Homo sapiens] [GeneID=3859]

[0764] KRT12, Protein sequence

[0765] SEQ ID NO. 98:

[0766] >NP_000214.1 KRT12 [organism=Homo sapiens] [GeneID=3859]

[0767] KRT13 keratin 13 [ Homo sapiens (human) ]

[0768] Gene ID: 3860, updated on 3-Apr-2024

[0769] KRT13, Gene sequence HGNC:HGNC:6415

[0770] SEQ ID NO. 99:

[0771] >NC_000017.11x41505612-41500981 KRT13 [organism=Homo sapiens] [GeneID=3860] [chromosome=17]

[0772] SEQ ID NO. 100:

[0773] >NC_060941.1x42361165-42356534 KRT13 [organ ism = Homo sapiens] [GeneID=3860] [chromosome=17]

[0774] KRT13, mRNA sequence

[0775] SEQ ID NO. 101:

[0776] >NM_002274.4 KRT13 [organism=Homo sapiens] [GeneID=3860] [transcript=2]

[0777] SEQ ID NO. 102:

[0778] >NM_153490.3 KRT13 [organism=Homo sapiens] [GeneID=3860] [transcript=l]

[0779] KRT13, Protein sequence SEQ ID NO. 103:

[0780] >NP_002265.3 KRT13 [organism=Homo sapiens] [GeneID=3860] [isoform=b]

[0781] SEQ ID NO. 104:

[0782] >NP_705694.3 KRT13 [organism=Homo sapiens] [GeneID=3860] [isoform=a]

[0783] KRT14 keratin 14 [ Homo sapiens (human) ]

[0784] Gene ID: 3861, updated on 23-Mar-2024

[0785] KRT14, Gene sequence HGNC:HGNC:6416

[0786] SEQ ID NO. 105:

[0787] >NC_000017.11x41586895-41582279 KRT14 [organism=Homo sapiens] [GeneID=3861] [chromosome=17]

[0788] SEQ ID NO. 106:

[0789] >NC_060941.1x42442485-42437869 KRT14 [organ ism = Homo sapiens] [GeneID=3861] [chromosome=17]

[0790] KRT14, mRNA sequence

[0791] SEQ ID NO. 107:

[0792] >NM_000526.5 KRT14 [organism=Homo sapiens] [GeneID=3861]

[0793] KRT14, Protein sequence

[0794] SEQ ID NO. 108:

[0795] >NP_000517.3 KRT14 [organism=Homo sapiens] [GeneID=3861]

[0796] KRT15 keratin 15 [ Homo sapiens (human) ]

[0797] Gene ID: 3866, updated on 3-Apr-2024

[0798] KRT15, Gene sequence HGNC:HGNC:6421

[0799] SEQ ID NO. 109:

[0800] >NC_000017.11x41518890-41513745 KRT15 [organism=Homo sapiens] [GeneID=3866] [chromosome=17]

[0801] SEQ ID NO. 110:

[0802] >NC_060941.1x42379059-42369289 KRT15 [organ ism = Homo sapiens] [GeneID=3866] [chromosome=17]

[0803] KRT15, mRNA sequence

[0804] SEQ ID NO. Ill:

[0805] >NM_002275.4 KRT15 [organism=Homo sapiens] [GeneID=3866]

[0806] SEQ ID NO. 112:

[0807] >XM_011524784.4 KRT15 [organism=Homo sapiens] [GeneID=3866] [transcript=X2]

[0808] SEQ ID NO. 113:

[0809] >XM_017024614.3 KRT15 [organism=Homo sapiens] [GeneID=3866] [transcript=Xl]

[0810] SEQ ID NO. 114:

[0811] >XM_054316023.1 KRT15 [organism=Homo sapiens] [GeneID=3866] [transcript=X4]

[0812] SEQ ID NO. 115:

[0813] >XM_054316022.1 KRT15 [organism=Homo sapiens] [GeneID=3866] [transcript=X3]

[0814] SEQ ID NO. 116:

[0815] >XM_054316024.1 KRT15 [organism=Homo sapiens] [GeneID=3866] [transcript=Xl]

[0816] KRT15, Protein sequence SEQ ID NO. 117:

[0817] >NP_002266.3 KRT15 [organism=Homo sapiens] [GeneID=3866]

[0818] SEQ ID NO. 118:

[0819] >XP_011523086.1 KRT15 [organism=Homo sapiens] [GeneID=3866] [isoform=Xl]

[0820] SEQ ID NO. 119:

[0821] >XP_016880103.1 KRT15 [organism=Homo sapiens] [GeneID=3866] [isoform=X2]

[0822] SEQ ID NO. 120:

[0823] >XP_054171997.1 KRT15 [organism=Homo sapiens] [GeneID=3866] [isoform=Xl]

[0824] SEQ ID NO. 121:

[0825] >XP_054171998.1 KRT15 [organism=Homo sapiens] [GeneID=3866] [isoform=X3]

[0826] SEQ ID NO. 122:

[0827] >XP_054171999.1 KRT15 [organism=Homo sapiens] [GeneID=3866] [isoform=X2]

[0828] KRT16 keratin 16 [ Homo sapiens (human) ]

[0829] Gene ID: 3868, updated on 5-Mar-2024

[0830] KRT16, Gene sequence HGNC:HGNC:6423

[0831] SEQ ID NO. 123:

[0832] >NC_000017.11x41612767-41609778 KRT16 [organism=Homo sapiens] [GeneID=3868] [chromosome=17]

[0833] SEQ ID NO. 124:

[0834] >NC_060941.1x42468220-42465231 KRT16 [organ ism = Homo sapiens] [GeneID=3868] [chromosome=17]

[0835] KRT16, mRNA sequence

[0836] SEQ ID NO. 125:

[0837] >NM_005557.4 KRT16 [organism=Homo sapiens] [GeneID=3868]

[0838] KRT16, Protein sequence

[0839] SEQ ID NO. 126:

[0840] >NP_005548.2 KRT16 [organism=Homo sapiens] [GeneID=3868]

[0841] KRT17 keratin 17 [ Homo sapiens (human) ]

[0842] Gene ID: 3872, updated on 5-Mar-2024

[0843] KRT17, Gene sequence HGNC:HGNC:6427

[0844] SEQ ID NO. 127:

[0845] >NC_000017.11x41624575-41619442 KRT17 [organism=Homo sapiens] [GeneID=3872] [chromosome=17]

[0846] SEQ ID NO. 128:

[0847] >NC_060941.1x42480028-42474895 KRT17 [organ ism = Homo sapiens] [GeneID=3872] [chromosome=17]

[0848] KRT17, mRNA sequence

[0849] SEQ ID NO. 129:

[0850] >NM_000422.3 KRT17 [organism=Homo sapiens] [GeneID=3872]

[0851] KRT17, Protein sequence

[0852] SEQ ID NO. 130:

[0853] >NP_000413.1 KRT17 [organism=Homo sapiens] [GeneID=3872] KRT18 keratin 18 [ Homo sapiens (human) ]

[0854] Gene ID: 3875, updated on 5-Mar-2024

[0855] KRT18, Gene sequence HGNC:HGNC:6430

[0856] SEQ ID NO. 131:

[0857] >NC_000012.12:52948855-52952906 KRT18 [organ ism = Homo sapiens] [GeneID=3875] [chromosome=12]

[0858] SEQ ID NO. 132:

[0859] >NC_060936.1:52913414-52917465 KRT18 [organism=Homo sapiens] [GeneID=3875] [chromosome=12]

[0860] KRT18, mRNA sequence

[0861] SEQ ID NO. 133:

[0862] >NM_199187.2 KRT18 [organism=Homo sapiens] [GeneID=3875] [transcript=2]

[0863] SEQ ID NO. 134:

[0864] >NM_000224.3 KRT18 [organism=Homo sapiens] [GeneID=3875] [transcript=l]

[0865] KRT18, Protein sequence

[0866] SEQ ID NO. 135:

[0867] >NP_000215.1 KRT18 [organism=Homo sapiens] [GeneID=3875]

[0868] SEQ ID NO. 136:

[0869] >NP_954657.1 KRT18 [organism=Homo sapiens] [GeneID=3875]

[0870] KRT19 keratin 19 [ Homo sapiens (human) ]

[0871] Gene ID: 3880, updated on 5-Mar-2024

[0872] KRT19, Gene sequence HGNC:HGNC:6436

[0873] SEQ ID NO. 137:

[0874] >NC_000017.11x41528308-41523617 KRT19 [organism=Homo sapiens] [GeneID=3880] [chromosome=17]

[0875] SEQ ID NO. 138:

[0876] >NC_060941.1x42383869-42379161 KRT19 [organ ism = Homo sapiens] [GeneID=3880] [chromosome=17]

[0877] KRT19, mRNA sequence

[0878] SEQ ID NO. 139:

[0879] >NM_002276.5 KRT19 [organism=Homo sapiens] [GeneID=3880]

[0880] KRT19, Protein sequence

[0881] SEQ ID NO. 140:

[0882] >NP_002267.2 KRT19 [organism=Homo sapiens] [GeneID=3880]

[0883] KRT20 keratin 20 [ Homo sapiens (human) ]

[0884] Gene ID: 54474, updated on 5-Mar-2024

[0885] KRT20, Gene sequence HGNC:HGNC: 20412

[0886] SEQ ID NO. 141:

[0887] >NC_000017.11x40885242-40875889 KRT20 [organism=Homo sapiens] [GeneID=54474] [chromosome=17]

[0888] SEQ ID NO. 142: >NW_003871091.1x34510-25157 KRT20 [organism=Homo sapiens] [GeneID=54474] [chromosome=17]

[0889] SEQ ID NO. 143:

[0890] >NC_060941.1x41749557-41740198 KRT20 [organ ism = Homo sapiens] [GeneID=54474] [chromosome=17]

[0891] KRT20, mRNA sequence

[0892] SEQ ID NO. 144:

[0893] >NM_019010.3 KRT20 [organism=Homo sapiens] [GeneID=54474]

[0894] KRT20, Protein sequence

[0895] SEQ ID NO. 145:

[0896] >NP_061883.1 KRT20 [organism=Homo sapiens] [GeneID=54474]

[0897] MUC1 mucin 1, cell surface associated [ Homo sapiens (human) ]

[0898] Gene ID: 4582, updated on 17-Mar-2024

[0899] MUC1, Gene sequence HGNC:HGNC:7508

[0900] SEQ ID NO. 146:

[0901] >NC_000001.11x155192915-155185824 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [chromosome=l]

[0902] SEQ ID NO. 147:

[0903] >NC_060925.1x154331478-154325440 MUC1 [organism=Homo sapiens] [GeneID=4582] [chromosome=l]

[0904] MUC1, mRNA sequence

[0905] SEQ ID NO. 148:

[0906] >NM_001204291.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [transcript=15]

[0907] SEQ ID NO. 149:

[0908] >NM_001204292.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [transcript=16]

[0909] SEQ ID NO. 150:

[0910] >NM_001204295.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [transcript=19]

[0911] SEQ ID NO. 151:

[0912] >NM_001204286.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [transcript=10]

[0913] SEQ ID NO. 152:

[0914] >NM_001204290.2 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [transcript=14]

[0915] SEQ ID NO. 153:

[0916] >NM_001204294.2 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [transcript=18]

[0917] SEQ ID NO. 154:

[0918] >NM_001044393.3 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [transcript=8]

[0919] SEQ ID NO. 155:

[0920] >NM_001044390.3 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [transcript=5]

[0921] SEQ ID NO. 156:

[0922] >NM_001044391.3 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [transcript=6]

[0923] SEQ ID NO. 157:

[0924] >NM_001018017.3 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [transcript=3]

[0925] SEQ ID NO. 158:

[0926] >NM_001204293.2 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [transcript=17] SEQ ID NO. 159:

[0927] >NM_002456.6 MUC1 [organism=Homo sapiens] [GeneID=4582] [transcript=l]

[0928] SEQ ID NO. 160:

[0929] >NM_001204285.2 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [transcript=9]

[0930] SEQ ID NO. 161:

[0931] >NM_001204289.2 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [transcript=13]

[0932] SEQ ID NO. 162:

[0933] >NM_001204296.2 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [transcript=20]

[0934] SEQ ID NO. 163:

[0935] >NM_001044392.3 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [transcript=7]

[0936] SEQ ID NO. 164:

[0937] >NM_001204288.2 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [transcript=12]

[0938] SEQ ID NO. 165:

[0939] >NM_001018016.3 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [transcript=2]

[0940] SEQ ID NO. 166:

[0941] >NM_001204297.2 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [transcript=21]

[0942] SEQ ID NO. 167:

[0943] >NM_001204287.2 MUC1 [organism=Homo sapiens] [GeneID=4582] [transcript=ll]

[0944] SEQ ID NO. 168:

[0945] >NM_001371720.2 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [transcript=22]

[0946] MUC1, Protein sequence

[0947] SEQ ID NO. 169:

[0948] >NP_001018016.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [isoform=2 precursor]

[0949] SEQ ID NO. 170:

[0950] >NP_001018017.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [isoform=3 precursor]

[0951] SEQ ID NO. 171:

[0952] >NP_001037855.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [isoform=5 precursor]

[0953] SEQ ID NO. 172:

[0954] >NP_001037856.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [isoform=6 precursor]

[0955] SEQ ID NO. 173:

[0956] >NP_001037857.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [isoform=7 precursor]

[0957] SEQ ID NO. 174:

[0958] >NP_001037858.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [isoform=8 precursor]

[0959] SEQ ID NO. 175:

[0960] >NP_001191214.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [isoform=9 precursor]

[0961] SEQ ID NO. 176:

[0962] >NP_001191215.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [isoform=10 precursor]

[0963] SEQ ID NO. 177:

[0964] >NP_001191216.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [isoform=ll precursor]

[0965] SEQ ID NO. 178:

[0966] >NP_001191217.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [isoform=12 precursor] SEQ ID NO. 179:

[0967] >NP_001191218.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [isoform=13 precursor]

[0968] SEQ ID NO. 180:

[0969] >NP_001191219.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [isoform=14 precursor]

[0970] SEQ ID NO. 181:

[0971] >NP_001191220.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [isoform=15 precursor]

[0972] SEQ ID NO. 182:

[0973] >NP_001191221.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [isoform=16 precursor]

[0974] SEQ ID NO. 183:

[0975] >NP_001191222.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [isoform=17 precursor]

[0976] SEQ ID NO. 184:

[0977] >NP_001191223.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [isoform=18 precursor]

[0978] SEQ ID NO. 185:

[0979] >NP_001191224.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [isoform=19 precursor]

[0980] SEQ ID NO. 186:

[0981] >NP_001191225.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [isoform=20 precursor]

[0982] SEQ ID NO. 187:

[0983] >NP_001191226.1 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [isoform=21 precursor]

[0984] SEQ ID NO. 188:

[0985] >NP_001358649.2 MUC1 [organ ism = Homo sapiens] [GeneID=4582] [isoform=22 precursor]

[0986] SEQ ID NO. 189:

[0987] >NP_002447.4 MUC1 [organism=Homo sapiens] [GeneID=4582] [isoform=l precursor]

[0988] SEQ ID NO. 190

[0989] >H_cKIT_F_2

[0990] SEQ ID NO. 191

[0991] >H_cKIT_R_2

[0992] SEQ ID NO. 192

[0993] > Primer

[0994] SEQ ID NO. 193

[0995] > Primer

[0996] SEQ ID NO. 194

[0997] > Primer

[0998] SEQ ID NO. 195

[0999] >Primer CP2-BpuEI

[1000] SEQ ID NO. 196 >Primer Cp2-Bgll

[1001] The invention is also characterized by the following figures, figure legends and the following non-limiting examples. Brief Description of Drawings

[1002] Fig. 1 DCC detection methods.

[1003] (A) Workflow for the three different methods of detecting DCCs. (B) shows MCF7 cell line (left) positive for EpCAM and CK as well as a EpCAM and CK positive patient cell (brightness and contrast enhanced, respectively). Beside this is a dot-plot depicting the MGVs for CK and EpCAM of MCF7 cell line, PBMCs and patient samples, respectively. (C) shows a histogram of the DCC detection rates by different methods. (D) shows a dot-plot of found DCCD by the methods in the same patient samples (gray bar: median). (E) Correlation of found DCCDs between the two methods of detecting CK+DCCs. Degree of correlation (R) and significance level (7) is provided. (F) Correlation of both logio(DCCD+l) for the two methods of finding EpCAM+DCCs. Degree of correlation (R) and significance level (7) is provided.

[1004] Fig. 2 Clinical outcome by protocol.

[1005] Shown are Kaplan-Meier curves of the side-by-side comparison patient cohort for TSS for DCC detection by (A) Pl-CK- IC+, (B) P2-EpCAM-Depl-IF+, (C) P3-CK-IF+and (D) P3-EpCAM-IF+. P-values are provided.

[1006] Fig. 3 Clinical outcome by protocol.

[1007] Shown are Kaplan-Meier curves of the side-by-side comparison patient cohort for PFS (left) and OS (right) for DCC detection by (A) P1-CK-IC+, (B) P2-EpCAM-Depl-IF+, (C) P3-CK-IF+and (D) P3-EpCAM-IF+. P-values are provided.

[1008] Fig. 4 Identification of a marker for the detection of DCC with stem cell properties.

[1009] (A) Patients examined and number of patient samples included in the scRNA-seq study. (B) Verification of malignant origin of DCC by determination of copy number alterations (CNA) from gene expression. While plasma cells show no gains and losses, Ml (positive control) and MO cells of the EpCAM-high group show clear changes. (C) Projection of the isolated EpCAM+cells into the bone marrow atlas (S.B. Hay et al., 2018, Exp Hematol, 68:51-61) and identification of a separate "continent" from MO-DCC. Plasma cells that projected exactly onto the BM atlas plasma cells served as a positive control. (D) MO-DCC differ from Ml-DCC by the enrichment of GO terms with stem cell properties. (E) Determination of sternness scores using the cytotrace method (G.S. Gulati et al., 2020, Science, 367(6476):405-411). The DCC type of EpCAM-high DCC, which is predominant in NSCLC patients, achieves the highest values here.

[1010] Fig. 5 High sternness scores are characteristic for patients with rapid progression.

[1011] (A) UMAP of EpCAM+cells isolated from NSCLC patients. Cell clustering follows NSCLC-disease stage (M0 vs. Ml bone marrow derived cells), tissue origin (lymph node derived cells) and phenotype (cancer cells vs. plasma cells or LN cells).

[1012] (B) M0 or Ml stage DCC frequently display copy number variations (CNV) as inferred from gene expression. (C) Patients harboring EpCAM+DCC (lower curve in KM-analysis; upper curve displays EpCAM- patients) were divided into two groups: patients with relapse before 1500 days and patients without relapse (PFS, progression free survival). (D) Sternness scores of cells (denoted as "expression level") at the threshold of 1500 days (good, event after 1500 days ("without relapse"); bad, event before 1500 days ("relapse")) for tumor-specific survival (TSS) and overall survival (OS). Fig. 6 Gene expression according to patient split into good and bad prognosis.

[1013] (A) and (B) split according to TSS, (C) and (D) according to OS. (B) and (D) c-KTT expression in the respective subgroups. (E) Kaplan-Meier survival curve for patients with high and low c-KTT expression. (F) and (G) Kaplan-Meier survival analysis for patients harboring EpCAM+cells in bone that express the indicated transcripts of MUC1 or c-KTT. (F) effect of MUC1 expression alone in NSCLC; (G) effect of expression of either MUC1 or KIT in EPCAM+ cells. Note, that despite the very low number of events, all patients (breast cancer, i.e., "mamma") or four out of five (prostate cancer) progressing to metastasis are identified by EpCAM+DCC expressing c-KTT or MUC1 transcripts. "Distant relapse- free survival" as shown in (F) refers to a special case of progression -free survival (PFS), wherein only distant progression / relapse (in another organ) is considered. "Metastasis-free survival" as shown in (G) refers to a special case of PFS, wherein only a relapse / progress due to a metastasis is considered and not a local relapse.

[1014] Fig. 7 Gene expression according to patient split into good and bad prognosis.

[1015] Top 20 genes of non-surface proteins separating patients with good and bad TSS (A) and OS (B).

[1016] Fig. 8 Quantitative assessment of protein expression.

[1017] (A) Shown is a c-KTT+HEL (top, positive control) and patient cell (bottom). (B) shows the staining intensities for c- KTT for HEL cells, PBMCs and patient DCCs, respectively. (C) shows the validation of the image analysis for the detection of c-KTT+cells, whose MGV had to surpass the mean of the MGVs of 10 surrounding cells plus 3 SDs of these cells (MB+3SD). This was performed on patients which had c-KTT transcript+ cells (KIT+ cell). (D) number of C-KTT+ DCC found by increasing the number of screened mononucleated cells (MNCs) for a group of patients which were c- KTT+ after screening lxlO5MNCs (left, black) and a group of patients negative for c-KTT+DCC after screening lxlO5MNC (right, gray). (E) gives an example of a EpCAM-CK-c-KTT-positive cells. (F) shows the distribution of found cells to cell phenotypes and (G) the percentage of c-KTT+cells amongst found cells within the extended patient cohort. (H) shows the classification of patients based on found cell phenotypes within the extended patient cohort.

[1018] Fig. 9 Clinical outcome by protocol.

[1019] Shown are Kaplan-Meier curves of the side-by-side comparison patient cohort (i.e., the same 45 patients also assessed in Figure 2 and 3) for the TSS (top), PFS (middle), and OS (bottom). Shown is PFS, TSS, and OS for KTT+DCC detection vs. detection of KTT DCC (either CK+or EpCAM+) or DCC-. P-values are provided.

[1020] Fig. 10 Clinical outcome by protocol (extended patient cohort)

[1021] Shown are Kaplan-Meier curves of the extended patient cohort for PFS (left), TSS (mid) and OS (right). (A) shows PFS, TSS and OS for KTT+DCC (EpCAM+and / or CK+KTT+) detection, while (B) for KTT+DCC detection vs. detection of KIT- DCC. P-values are provided. (C) Forest plots for the HR based on clinicopathological variables and staining results for PFS, TSS, and OS. Hazard ratio as well as the lower and upper 95% confidence interval are shown. Variables are sorted by HR, highest to lowest. P-values are provided. Significant variables are shown. Fig. 11 Copy number alterations for DCC subpopulations

[1022] (A) Copy number alterations of a c-KTT+DCC after low coverage genome sequencing. Dots represent reads. Genomic regions without alteration scatter around a log ratio of 0 and are depicted in black. Grey values indicate aberration, here gains of chromosomal regions.

[1023] (B) Bar chart representing the percent of aberrant cells in total collective (n=49), single EpCAM -positive DCCs (n=27) and EpCAM-c-KTT-positive DCCs (n=18). (C) Cumulative CNA profiles for all aberrant cells, aberrant EpCAM -positive cells and EpCAM and / or CK cKTT-positive cells. Chromosomes are depicted on top. Gains in light grey and losses with darker grey. (D) Kaplan Meier plots depicting TSS based on presence of DCCs with aberrant versus non-aberrant genomes and TSS for patients with DCC+ / c-KTT+ cells with and without CNAs.

[1024] Fig. 12 Sanger sequencing of TP53 and KRAS mutations in DCC from NSCLC

[1025] Results for DCC isolated from bone marrow (BM) are shown in the first panel and results for DCC isolated from lymph node (LN) are shown in the second panel. TP53 was assessed for lung adenocarcinomas (LUAD) and non-LUAD. KRAS was assessed for LUAD only. For Ml patients, ID is highlighted in bold font. Number of cells (#cells), number of mutated cells (#mutated cells) and the sequencing results of the primary tumors (PT) are shown.

[1026] Fig. 13 Comparison of the impact of c-KIT gene expression vs mutation status in EpCAM+ DCC for tumor specific survival in NSCLC

[1027] Left panel displays the effect on survival when at least one of the DCC expressed high levels of c-KIT (same as Fig. 6E). The right panel displays the effect on survival when at least one of the DCC harbored a TP53 mutation. Time is shown in days.

[1028] Examples

[1029] The present invention is additionally described by way of the following illustrative non-limiting examples that provide a better understanding of the present invention and of its many advantages.

[1030] The examples are intended for the purpose of illustration and are not intended to limit the scope of the generality hereinbefore described.

[1031] Example 1 - General methods

[1032] A) Patients and bone marrow samples

[1033] NSCLC patients: From 2012 to 2017, patients undergoing potentially curative surgery for presumed or histologically verified non-small-cell lung cancer (NSCLC) at the Department for Thoracic Surgery of the University Hospital Regensburg or the Hospital Barmherzige Bruder Regensburg were prospectively enrolled in this study. Bone marrow aspirates were collected as described previously (M. Guzvic et aL, 2014, Cancer Res 74, 7383-7394). All aspects of this study were approved by the local ethics committee at the University of Regensburg (protocol number 07-079) and the patients provided written, informed consent.

[1034] Healthy donors, breast cancer and prostate cancer patients: Human disseminated cancer cells were obtained from BM aspirates of breast or prostate cancer patients without and with distant metastases. EpCAM+ cells were obtained from BM of patients without known malignant disease undergoing hip replacement surgery (HD, healthy donor). Human MSCs were obtained from BM aspirates of breast cancer patients or HDs. Written informed consent of cancer and control patients was obtained and the ethics committee of the University of Regensburg (ethics vote number 07 / 79) approved BM sampling and analysis of isolated cells.

[1035] B) Study exclusion criteria

[1036] NSCLC patients that presented with overt metastases (n=2) or with additional carcinoma within the last five years prior to the time of surgery (n=4), patients which underwent neoadjuvant chemotherapy or radiotherapy (n=2), patients who didn't undergo surgery (n=l), patients with an incomplete resection status (n=4) and patients who died within 60 days after surgery (n=l) were excluded from this study to exclude confounding variables, which resulted in a total of n = 56 patients. For the comparison of the disseminated cancer cell (DCC) detection methods only patients with results for Pl-CK-IC (see Example l.E), P2-EpCAM-Depl-IF (see Example l.D), and P3-CK / EpCAM-IF (see Example l.F) staining protocols were included (n=45).

[1037] C) Sample processing

[1038] Bone marrow (BM) aspirates were processed as described in (M. Guzvic et al., 2014, Cancer Res 74, 7383-7394). Briefly, the aspirates were washed twice in Hank's solution. For Pl-CK-IC (see Example l.E), and P3-CK / EpCAM-IF and P3-CK / EpCAM / c-KTT-IF staining (see Example l.F) density gradient centrifugation was performed using Percoll 60% (p = 1.077 g / ml) to enrich mononucleated cells (MNCs) and this cell suspension was applied to adhesion slides (Menzel, Braunschweig, Germany) at a concentration of 166,667 cells per field on an area of 154 mm2. Sedimentation of cells was allowed for 1-2 hours in a closed moist chamber. Thereafter, the supernatant was discarded, and the slides were air-dried for at least 8 hours or overnight. Slides were stored at -20°C until further processing.

[1039] For depletion enrichment of EpCAM-positive cells (P2-EpCAM-Depl-IF; see Example l.D), BM cells were placed on a Percoll 65% gradient (p = 1.083 g / ml) before depletion antibodies were added (see Example l.D).

[1040] D) P2-EpCAM-Depl-IF staining

[1041] To remove weakly EpCAM-positive erythroblastic cells (R. Lammers et aL, 2002, Exp Hematol 30, 537-545) and other BM populations, EpCAM-high DCCs were enriched using negative immunomagnetic depletion as described in (M. Guzvic et al., 2014, Cancer Res 74, 7383-7394). Briefly, autochthonous BM cells were captured on a LS MACS column after incubation with APC-conjugated CDllb, CD33, and CD45 antibodies (Miltenyi Biotec, Germany) and anti-CD235 (glycophorin A) microbeads. The flow-through cell fraction was counted using a hematocytometer.

[1042] E) Pl-CK-IC staining

[1043] Pl-CK-IC staining was performed according to the consensus protocol of DCC detection (T. Fehm et al., 2006, Cancer 107, 885-892). Briefly, adhesion slides were stained against cytokeratins (CK) using the monoclonal pan anti-CK antibody A45-B / B3 (Micromet, AS Diagnostik, Germany) at a final concentration of 2 pg / ml, detected by the APAAP technique or using avidin-biotin complex (ABC) method coupled with alkaline phosphatase Vectastain ABC kit (Vector Laboratories, USA). Isotype controls were performed on a slide from the same sample using the MOPC-21 antibody (Sigma Aldrich, USA). F) P3-CK / EpCAM -IF and P3-CK / EpCAM / c-KIT-IF staining

[1044] Adhesion-slides were blocked using 5% bovine serum albumin / PBS for 30 min. Primary antibodies (biotin-labelled anti- CK antibody A45-B / B3 (Micromet, AS Diagnostik, Germany; c=l,24 pg / ml), PE-labelled anti-EpCAM antibody HEA-125 (Miltenyi Biotec, Germany; c=0,25 pg / ml), BV421-labelled anti-c-KTT 104D2 (for P3-CK / EpCAM / c-KTT-IF staining; Biolegend, USA; c=0,25 pg / ml)) diluted in 5% bovine serum albumin / PBS were incubated for one hour at room temperature. Slides were washed three times using PBS. For detection of CK, incubation with AF488-labeled streptavidin was performed for half an hour at room temperature. Slides were washed three times using PBS, fixed in 0.5% parafbrmaldehyde / PBS for 5 minutes and washed 3 times with PBS.

[1045] G) Screening of bone marrow for disseminated cancer cells

[1046] Stained immunofluorescently labelled cell suspensions were manually screened for EpCAM -positive (EpCAM+) single cells using a microscope (Olympus) and DCC-positivity (DCC+) was assigned only for clearly bright and intensively stained cells, as it had been noted previously that weakly positive cells could confound the analysis (M. Guzvic et al., 2014, Cancer Res 74, 7383-7394, M. Werner-Klein et al., 2020, Nat Commun 11, 4977).

[1047] Immunofluorescently stained adhesion slides were manually screened for CK+or EpCAM+single cells using an inverted fluorescent microscope (Olympus). Pictures of these cells were subsequently analyzed by image analysis (see Example l.H) to determine positivity. Positivity was assigned only to cells displaying bright and homogenous surface-staining (for EpCAM or c-KTT) that remained clearly visible after photo acquisition with an exposure time of 1 sec. CK-positivity was assigned to bright and homogenous cytoplasmic staining. Cells displaying a dark or black appearance in brightf ield (BF) were excluded as possibly confounding dead cells. Furthermore, cells displaying autofluorescence in the FITC channel were excluded as possibly confounding cells.

[1048] Screening of immunocytological stained slides was performed using a microscope (Olympus or Zeiss) according to consensus criteria for DCC detection (T. Fehm et al., 2006, Cancer 107, 885-892) and followed validation and verification criteria according to ISO 17020 rules. CK-positivity was assigned to bright and homogenous intracellular staining.

[1049] The total number of positively stained cells was documented as the number of DCCs per million screened MNCs (disseminated cancer cell density, DCCD).

[1050] H) Image analysis

[1051] Images depicting positive cells, manually identified through P3-CK / EpCAM-IF or P3-CK / EpCAM / c-KTT-IF staining, were subjected to quantitative image analysis via Image! (version 1.54) software after screening to objectify the analysis. The stacked tif. images were initially separated into individual fluorescence channels and converted into grayscale. Potentially positive cells and 10 neighboring negative cells were manually labeled, and their respective mean gray values (MGVs) were quantified. The MGV of a cell of interest had to surpass the mean of the neighboring 10 cells plus three standard deviations of the MGVs of these cells for the respective fluorescent channel to be considered positive. Cells having a dark appearance in BF were excluded for this analysis as they often showed unspecific antibody / fluorophore binding. I) Whole genome amplification and analysis of copy number alterations

[1052] Positive cells identified by P3-CK / EpCAM / c-KTT-IF staining were manually picked from adhesion slides using a micromanipulator and subjected to whole genome amplification (WGA) using Amplil WGA kit (Menarini Silicon Biosystems) or as previously described in C.A. Klein et al, 2022, Mechanisms of Disease, 360:9334, P683-689. The quality of amplified DNA was evaluated with a set of 4 control primers using end-point PCR and assigned to genomic integrity index 0 to 4 (GII0-GII4) (B. Polzer et al., 2014, EMBO Mol Med 6, 1371-1386). Next generation sequencing libraries of amplified single cell genomes were prepared using the Amplil LowPass Kit (Menarini Silicon Biosystems) according to the manufacturer's instructions. Resulting libraries were quantified with the Qubit dsDNA HS Kit (Thermo Fisher Scientific) and fragment length distribution was analyzed with the High Sensitivity DNA Kit on the Bioanalyzer 2100 (Agilent Technologies, USA). Libraries were pooled in equal molar ratios and sequenced on Illumina NovaSeq 6000 platforms. The sequencing quality was then evaluated per sample with FastQC (version 0.11.8) (S. Andrews, 2010, FastQC: A Quality Control Tool for High Throughput Sequence Data, Available online at: http: / / www.bioinformatics.babraham.ac.uk / proiects / fastgc / ) and in a multisample comparison with MultiQC (version 1.8) (P. Ewelset al., 2016, Bioinformatics, 32:19, 3047-3048) before and after adapter trimming and contamination screening. Briefly, raw sequencing fastq data of single cells were trimmed, and remaining adapter sequences as well as low sequencing quality bases at the end of each read were removed using BBDuk (B. Bushnell, sourceforge.net / projects / bbmap / ). In order to increase the mapping quality (lowering false-positive alignments), read decontamination was performed using BioBloom Tools (J. Chuet aL, 2014, Bioinformatics, 30:23, 3402-3404) with filters for the genomes of Homo sapiens (hg38), Mus muscuius (mmlO), Escherichia coii (BL21), Mycoplasma pneumoniae (M129), Sphingobium sp. (SYK-6), Bradyrhizobium japonicum (USDA 110), Pichia pastoris (GS115), Maiessia giobosa (CBS 7966), Aspergillus fumigatus (Af293), and a set of viral genomes (RefSeq, 5k+ genomes). All reads that did not map exclusively to hg38 (GENCODE version J, GRCh38.plO) or did not map at all were defined as likely contaminations and discarded from downstream processing. Subsequently, the cleaned sample reads were aligned to the reference genome GRCh38 and GENCODE using bwa (version 0.7.17) (Li H. and Durbin R. (2009) Fast and accurate short read alignment with Burrows-Wheeler Transform. Bioinformatics, 25: 1754-60. [PMID: 19451168]) and CNNV profiles were created using QDNAseq (version 1.24.0) (Scheinin I et al., “DNA copy number analysis of fresh and formalin-fixed specimens by shallow whole-genome sequencing with identification and exclusion of problematic regions in the genome assembly.” Genome Research, 24, 2022-2032.) Finally, the LowPass-Seq was stratified into cell phenotype groups and submitted to the Progenetix user data tool (A.D. Donnenberg, et al, 2012, PLoS One 7, e52885) to generate individual frequency plots for different cell phenotypes (M. Baudis, M.L. Cleary, 2001, Bioinformatics 17, 1228-1229).

[1053] J) Whole transcriptome amplification

[1054] For whole transcriptome amplification (WTA) single cells in 1 pl of PBS were isolated using micromanipulation and deposited in tubes containing 6.4 pl of lysis buffer (Active Motif) containing 10 ng of tRNA (Roche), 1 pg of protease (Active Motif), and 1 pl of 37.5 pM solution of biotinylated oligo-dT peptide nucleic acids (PNAs; Active Motif).

[1055] Proteolytic digestion was performed by incubating the samples for 10 min at 45°C, followed by inactivation of protease at 75°C for 1 min, and annealing of PNA to poly-A tails of mRNAs, at 22°C for 15 min. PNA-mRNA complexes were precipitated in magnetic force field using streptavidin-conjugated metal beads. While precipitated in magnetic racks, bead pellets were washed with 10 pl of wash buffer 1 (50 mM Tris-HCI, 75 mM KCI, 10 mM DTT, 0.25% Igepal), 20 pl of wash buffer 2 (50 mM Tris-HCI, 75 mM KCI, 10 mM DTT, 0.5% Tween-20), and again with 20 l of wash buffer 1. These DNA-containing supernatant were transferred to a tube, containing 0.8 pl of polymerized 0.25% polyacrylamide as a carrier, for subsequent precipitation and WGA.

[1056] Reverse transcription on solid phase was performed for 45 min under rotation at 44°C, in 20 pl reaction mixture containing 0.5 mM of each dNTP (GE Healthcare), 200 U of SuperScript II reverse transcriptase (Invitrogen), 0.25% Igepal, 5 mM DTT, 30 pM of CisGTCTAGANs (SEQ ID NO. 192) primer, 15 pM of C15GTCTAGACTTGAGT24VN (SEQ ID NO. 193) primer (Metabion), and lx first strand buffer (Invitrogen). Primers were annealed at room temperature for 10 min, prior to addition of the enzyme.

[1057] Following reverse transcription, beads were precipitated in magnetic racks and washed in 20 pl of wash buffer 3 (50 mM KH2PO4, 1 mM DTT, 0.25% Igepal), and resuspended in 10 pl of buffer for tailing (4 mM MgCh, 0.1 mM DTT, 0.2 mM dGTP, 10 mM KH2PO4). Reaction mixture was overlaid with 40 pl of mineral oil, and the cDNA single strands released from beads by incubating the mixture at 95°C for 5 min, followed by incubation on ice for 3 min. Addition of dGTPs on 5' termini of single stranded cDNAs was performed by adding 10 U of terminal dNTP transferase (TdT; USB- Affymetrix) and incubating the mixture for 60 min at 37°C. After inactivation of TdT at 70°C for 5 min, 35 pl of WTA reaction mix 1 (4 pl of buffer I (Expand Long Template, Roche), 3% deionized formamide) was added.

[1058] Hotstart PCR was performed by incubating the sample to 78°C and adding 5.5 pl of WTA reaction mix 2 (3.2 mM each dNTP, 12 mM TCAGAATTCATGC15 (SEQ ID NO. 194) primer, and 7.5 U of PolMix (Expand Long Template, Roche)). WTA consisted of 40 cycles in MJ Research PCR cycler - 20 cycles of 15 s at 94°C, 30 s at 65°C, and 2 min at 68°C, and 20 cycles with an increase of elongation step for 10 s / cycle, followed by final cycle with 7 min of elongation.

[1059] K) NGS mRNA library preparation and sequencing

[1060] The majority of the (TTT)7and (CCC)s nucleotides forming the ends of cDNA products were removed by a limited-cycle PCR with primers introducing BpuEI and Bgll restriction sites (Protocol A) or a Bgll restriction site (Protocol B), followed by a restriction enzyme digestion.

[1061] For Protocol A, 1 pl each of two separate 1 / 5 dilutions of the original WTA sample was used in two sets of five separate PCR reactions (total of ten reactions) with a total volume of 20 pl per reaction with 1.44 pM final concentration of primer CP2-BpuEI (5'-TCA GAA TTC ATG (CCC)5GTC TTG AGT TTT TT-3'; SEQ ID NO. 195) and 1.44 pM final concentration of primer Cp2-BglI-13C (5'-TCA GAA TTC ATG (CCC)2 CGG (CCC)2-3'; SEQ ID NO. 196) for amplification. After an initial denaturation at 95 °C for 1 min, five cycles of 94 °C for 15 s, 55 °C for 1 min, and 65 °C for 180 s and three cycles of 95 °C for 15 s, 55 °C for 1 min, and 65 °C for 210 s (+10s / cycle) were carried out, followed by a final extension step of 7 min.

[1062] For Protocol B, 1 pl of a 1 / 5 dilution of the original WTA sample was used for five separate PCR reactions with a total volume of 20 pl per reaction with 2.4 pM final concentration of primer Cp2-BglI-13C (sequence above) for amplification. After an initial denaturation at 94 °C for 2 min, eight cycles of 94 °C for 15 s, 68 °C for 1 min, and 68 °C for 240 were carried out, followed by a final extension step of 7 min. Following the PCR, the single PCR reactions were pooled, and the remaining steps were identical for both protocols. Resulting cDNA products were purified with 1.8 volume of Ampure XP beads (Beckman Coulter, USA) according to the manufacturer's instructions and eluted in 40 pl of distilled water. Next, 5 pl of EcoRI buffer supplemented with 80 pM S-adenosyl methionine (New England Biolabs, Germany), 2.5 pl distilled water, and 2.5 pl BpuEI (5 U / pl) were added for a total volume of 50 pl and incubated at 37 °C for 1 h, followed by heat inactivation of the enzyme for 20 min at 65 °C. Subsequently, 1 pl of EcoRI buffer supplemented with 80 pM S-adenosyl methionine, 6.5 pl distilled water, and 2.5 pl Bgll (10 U / pl) were added for a final volume of 60 pl and incubated for 3 h at 37 °C, followed by heat inactivation of the enzyme for 20 min at 65 °C.

[1063] The complete restriction digest was purified with 1.8 volume of Ampure XP beads according to the manufacturer's instructions and eluted in 16 pl of 10 mM Tris-CI, pH 8.5 (Elution buffer, Qiagen, Germany). The length distribution of purified cDNA populations was determined on the Bioanalyzer 2100 (Agilent Technologies, USA). Optimal Covaris settings for fragmentation of each purified cDNA sample to 350 bp insert size were determined on the basis of the average length distribution. Subsequently, sequencing libraries were prepared according to the TruSeq DNA PCR-Free Library Prep kit (Illumina, USA). Resulting libraries were quantified with KAPA Library Quantification kit for Illumina Platforms (Kapa Biosystems, RSA), pooled in equal molar ratios, and sequenced on Illumina NovaSeq 6000 platforms.

[1064] L) Bioinformatic analysis of RNA-Seq data

[1065] The sequencing quality was evaluated per sample with FastQC (version 0.11.8) (S. Andrews, 2010, FastQC: A Quality Control Tool for High Throughput Sequence Data, Available online at: http: / / www. bioinfo rmatics.babraham.ac.uk / proiects / fastoc / ) and in a multisample comparison with MultiQC (version 1.8) (P. Ewelset al., 2016, Bioinformatics, 32:19, 3047-3048) before and after adapter trimming and contamination screening. Briefly, raw sequencing fastq data of single cells were trimmed, and remaining adapter sequences as well as low sequencing quality bases at the end of each read were removed using BBDuk (B. Bushnell, sourceforge.net / projects / bbmap / ). In order to increase the mapping quality (lowering false-positive alignments), read decontamination was performed using BioBloom Tools (J. Chuet aL, 2014, Bioinformatics, 30:23, 3402-3404) with filters for the genomes of Homo sapiens (hg38), Mus muscuius (mmlO), Escherichia coii (BL21), Mycoplasma pneumoniae (M129), Sphingobium sp. (SYK-6), Bradyrhizobium japonicum (USDA 110), Pichia pastoris (GS115), Maiessia giobosa (CBS 7966), Aspergillus fumigatus (Af293), and a set of viral genomes (RefSeq, 5k+ genomes). All reads that did not map exclusively to hg38 (GENCODE version J, GRCh38.plO) or did not map at all were defined as likely contaminations and discarded from downstream processing. Subsequently, the cleaned sample reads were aligned to the reference genome GRCh38 and GENCODE using STAR (version 2.7.9a) and gene level expected count estimated with RSEM (version 1.3.1) is used as expression of genes.

[1066] Seurate 4.4.0 pipeline was applied for further analysis. Briefly, cell quality was checked according to number of counts, number of expressed genes, and percentage of mitochondrial gene count for each cell. Cells with number of counts <10,000, number of expressed genes <1,000 or percentage of MT >50 were removed. For expression normalization and scaling, dimension reduction was performed with principal component analysis. According to elbowplot, the first 20 principal components (PCs) were used for cluster and further dimension reduction using umap. M) Identification of new DCC surface biomarkers

[1067] First the inventors checked if patients contained cells from each cluster and created the feature containClusterX for any patient that contain cells from cluster X. Each patient was assigned the value "yes" for the feature containClusterX if a cell had been found in the cluster under consideration, otherwise the value "no" was assigned. Kaplan-Meier survival analysis was performed with R package survminer(0.4.9) "surv_fit" and followed using Surv function from R package Survival(3.5.7).

[1068] Patients that contained BM derived cells in cluster with poor survival were further used to detect survival predictable surface markers. Patients with presence of EpCAM+DCCs in BM were grouped into progressive and stable group based on progression-free survival data. Patients with event in less than 1500 days were grouped as patient with poor survival, and without event in no less than 1500 days as good survival. Patient death or disease progression was counted as "event". Differential gene expression analysis was performed between poor and good survival using findMarker function, and candidate surface biomarkers were filtered using database surfaceome (D. Bausch-Fluck et al., 2018, PNAS, 115 (46) E10988-E 10997).

[1069] N) Statistical analysis and survival analysis

[1070] Statistical testing for associations of categorical variables was performed using the Chi-square (X2) test. For comparison of two groups, an (un-)paired t-test was performed. Correlation analysis was performed using the nonparametric Spearman correlation method. Survival curves were created with the Kaplan-Meier method and compared using the univariate log-rank test. Overall survival (OS) was calculated as a period from the date of surgery to the date of death from any cause or the date of last follow-up. Progression-free survival (PFS) was defined as the duration from the date of surgery until further disease progression or death. Tumor-specific survival (TSS) was defined as the duration from the date of surgery until death that was documented to be caused by lung cancer. When analyzing more than two groups, the results were corrected for multiple testing and considered statistically significant using the family-wise significance level of 0.05. To categorize expression of c-KIT and MUC1 into high and low expression levels "surv_cutpoint" and "surv_categorize" from R package survminer (0.4.9) were used. For the combined analysis KITorMUC, at least one of them needed to high to be classified as high. After that Kaplan-Meier survival analysis was performed on c-KIT, MUC1, and c-KITorMUC categories as above. For multivariable Cox regression analysis of the lung cancer cohort the inventors used the backwards feature selection "selectCox" function from R package pec (2023.4.12) to remove redundant features.

[1071] Except mentioned explicitly, all statistical analysis was performed by using the software GraphPad Prism 10 (GraphPad Software, San Diego, California USA), IBM SPSS Statistics (Version 29, IBM Corp., Chicago, IL), and Microsoft Excel (Version 16.82, Microsoft, Redmond, WA) or the mentioned R packages. A P-value of <0.05 was considered statistically significant.

[1072] Example 2 - DCC detection rates are protocol dependent

[1073] The impact of enrichment methods, choice of antibodies, and evaluation criteria for the detection of DCC has been a matter of debate since long (C.A. Klein, 2003, Adv Cancer Res 89, 35-67, S. Riethdorf, et al., 2008, Int J Cancer 123, 1991-2006, E. Borgen et aL, 1999, Cytotherapy 1, 377-388). While the varying outcomes were not entirely unexpected for studies using different DCC detection antigens, it was still unclear to what extent the clinical utility depends on presumably minor methodological variables. To address this, three approaches based on the classical DCC detection markers cytokeratin and EpCAM on an identical patient cohort were compared. For this bone marrow aspirates from 45 non-metastasized (n=21, 10 and 14 for UICC stage I, II, III, respectively) and histologically confirmed NSCLC patients was obtained (see Examples l.A and l.B; Table 3, side-by-side comparison). The impact of DCC detection by the three methods on survival was assessed after median follow-up times for progression-free survival (PFS), overall survival (OS) and tumor-specific survival (TSS) of 76.9, 90.8, and 90.8 months, respectively (range 5.1 - 123.0 months). During the follow-up period 23 of the 45 patients (51.1%) had further disease progression, 20 (44,4%) patients died, while the death of 12 (26.7%) patients was directly caused by NSCLC. Overall, 7 (15.6%) patients developed a local relapse or metastasis during the follow-up period.

[1074] Table 3: Patient cohort overview

[1075] Bone marrow aspirates of these 45 NSCLC patients were subjected to the following three approaches (Figure 1A). The Pl-CK-IC staining (protocol 1; Pl; see Example l.C and l.E) was based on the standard assay following the consensus protocol (T. Fehm et aL, 2006, Cancer 107, 885-892) and a widely used reference (S. Braun et aL, 2005, N Engl J Med 353, 793-802) and was run under routine diagnostic, standardized and accredited conditions (ISO 17020). It employs the pan-cytokeratin antibody A45-B / B3 (recognizing cytokeratin 8, 18, and 19) and is performed on slides after enrichment of mononuclear cells by density (1.077 g / ml) gradient centrifugation. Cells were detected by immunocytology (IC) using enzymatic alkaline-phosphatase polymer-enhanced staining with BCIP / NBT as substrate. P2-EpCAM-Depl-IF staining (protocol 2; P2; see Example 1.3 and l.D) included a density gradient (1.083 g / ml) centrifugation followed by MACS depletion of GlyA+, CD45+, CD11+, and CD33+cells and EpCAM immunofluorescent (IF) staining using HEA125-PE in suspension. EpCAM+DCC identified by this protocol had shown a higher prognostic impact in NSCLC than Pl-CK-IC staining (protocol 1; Pl) (T. Mederer et aL, 2022, Lung Cancer 167, 73-77), leaving it open whether this was due to the antigen (CK vs. EpCAM) or the method of enrichment. To address this point, the inventors established the P3-CK / EpCAM-IF staining protocol (protocol 3; P3; see Example l.C and l.F) using immunofluorescence-based double staining of cytokeratin and EpCAM on the same slides as prepared for Pl-CK-IC- staining (Pl) (i.e., cell isolation by density gradient 1.077 g / ml). The protocol differences are depicted in Table 4.

[1076] Table 4: The differences in sample processing and staining procedure of used methods.

[1077] To compare IC vs. IF on slides, CK / EpCAM double-staining was established using the breast cancer cell line MCF-7, spiked into peripheral blood mononuclear cells (PBMC). Thereafter, these samples served as positive control when the staining was applied to patient bone marrow samples that generally display lower expression levels of the applied markers than cultured MCF-7 cells, as can be seen in Figure IC. To enhance the objectivity of the CK / EpCAM doublestaining analysis, the inventors utilized Image! -derived mean gray values (MGV) of the cells of interest to determine positivity (see Example l.H). To mitigate inter-staining, inter-patient, and local variabilities, the MGVs of the cell of interest had to exceed the mean of the MGV of 10 neighboring cells plus three times the standard deviation of the MGVs of these 10 cells for each respective fluorescent channel to be called positive. For all protocols (Pl-CK-IC; P2-EpCAM-Depl-IF; P3-CK / EpCAM-IF) the disseminated cancer cell density (DCCD), i.e., the number of positive cells per million bone marrow cells was determined, by staining median numbers of 105- 2xl05bone marrow cells per protocol (see Examples l.G and l.H; Table 5). Detection rates as determined by the different methods as well as DCCDs of positive patients can be seen in Figures ID and IE. Using the P3-CK / EpCAM-IF staining, CK+DCCs were detected in significantly more bone marrow (X2, p=0.021) than with the Pl-CK-IC staining, while EpCAM+DCCs were detected in significantly more bone marrow than with the P2-EpCAM-Depl-IF staining (X2, p<0.001). For CK, IF did not detect a statistically higher DCCD compared to IC in CK+patients. Similarly, no significant difference in EpCAM detection was observed between MACS-depletion and density-gradient centrifugation methods. However, detection of CK+or EpCAM+cells was significantly correlated when Pl-CK-IC and P3-CK / EpCAM-IF were compared for CK+cells (P=0.0356; r=0.3141, Figure IF) or when P2-EpCAM-Depl-IF and P3-CK / EpCAM-IF were compared for EpCAM+cells (P<0.0001; r=0.7490; Figure 1G) indicating that first, the two DCC detection markers EpCAM and CK and second the antibodies used in different detection methods, such as immunocytology and immunofluorescence, identify overlapping cell populations.

[1078] Table 5: Number of screened cells per protocol

[1079] Example 3 - Outcome prediction varies between DCC detection protocols

[1080] Having established the differences as well as correlations in detection rates between the three protocols (see Example 2), the inventors performed Kaplan-Meier survival analysis to assess the prognostic value of each protocol for PFS, TSS, and OS for the same patient cohort (see Example l.N). Although trends could be observed for P2-EpCAM-Depl- IF and P3-CK / EpCAM-IF, both for CK and EpCAM as detection markers, none of the protocols were able to firmly predict outcome (shown for TSS in Figures 2A-2D and the other endpoints in Figure 3 and Table 6).

[1081] Table 6: Overview of the p-values for PFS, TSS, and OS for the detection of DCCs by Pl-CK-IC, P2-EpCAM- Depl.-IF and P3-CK / EpCAM-IF staining.

[1082] As evident from Table 6 (and from Figures 2 and 3) the prognostic value for PFS, TSS, and OS is not significant for each tested DCC detection protocol relying on the prior art DCC marker(s) EpCAM and / or CK. Thus, the detection of the DCC markers EpCAM and / or CK is not indicative of the cells measured being metastasis founder cells. The use of EpCAM and / or CK does not provide a negative prognostic metastasis marker (combination). Example 4 - In-depth molecular characterization of bone marrow DCC

[1083] The inventors systematically isolated EpCAM+DCC from the bone marrow of patients with breast, prostate, or lung cancer (Figure 4A; see also Example l.A. Due to the extreme rarity (approx. 1-2 DCC / million bone marrow cells), the cells were isolated using the micromanipulator. Based on a previously developed single cell transcriptome amplification method (see Example 1J) and with the availability of scRNA-seq methods (see Examples 1.1, 1 J, and l.K) the inventors were able to sequence the transcriptomes. DCC data were obtained for the three tumor types, breast, prostate, and lung cancer. From these patients, the inventors identified three types of DCC at stage MO (before manifestation of metastasis), i.e., EpCAM-high, EpCAM-low, and MOp cells. MOp cells were only found in prostate cancer patients, EpCAM-high cells in all three tumor types and EpCAM-low cells in breast and prostate, i.e., NSCLC patients only had EpCAM-high cells. A comparison with DCC from patients with manifest metastases (Ml-DCC) revealed a number of differences, the most surprising being the significantly higher stem cell potential of the MO cells (Figures 4C to 4E).

[1084] Example 5 - Identification of c-KIT and MUC1 as marker for metastasis founder cells

[1085] Of the three patient cohorts (breast, prostate, and lung cancer; see Example 4), survival data (see Example l. N) became first available for the most aggressive cancer, NSCLC. The inventors therefore analyzed NSCLC-derived DCC and found that -as in Example 4- cells derived from MO-stage patients cluster separately from Ml stage-derived DCC (Figure 5A) and frequently displayed copy number variations (CNVs) (Figure 5B). The inventors hypothesized that MO- stage DCC from bone marrow of patients that relapsed early may display characteristics different from DCC that were isolated from DCC-positive patients that did not relapse. Therefore, the inventors compared DCC from EpCAM -positive patients that relapsed vs. non-relapsing patients (Figure 5C). Unexpectedly, cells from patients with poor outcome displayed significantly higher sternness scores than DCC from patients with good outcome (Figure 5D).

[1086] Next, the inventors performed comparative analysis between EpCAM+DCC+patients with early relapse (progressors, i.e., bad prognosis) and late or no relapse (non-progressors, i.e., good prognosis) either for TSS (Figure 6A and 6B) and for OS (Figure 6C and 6D). Differential gene expression analysis was performed and genes encoding cell surface proteins were selected. Along with EpCAM, several novel candidate markers could be identified to distinguish DCCs from progressors and non-progressors (e.g., MUC1, KCNK5, SPN and c-KIT, Figure 6A and 6C). Surprisingly, patients with short TSS or OS (i.e., bad prognosis) had higher c-KIT transcript expression in EpCAM+DCC than patients with long TSS or OS (i.e., good prognosis; Figure 6B and 6D). High c-KIT expression identified patients at risk to progress or die, as shown by Kaplan-Meier survival analysis (Figure 6E). Accordingly, the inventors surprisingly found a negative prognostic marker for DCC, i.e., c-KIT, the expression or expression level of which in combination with at least one DCC marker such as EpCAM, is indicative of the cell being a metastasis founder cell.

[1087] The inventors performed a similar comparative analysis between EpCAM+DCC+patients with early relapse (progressors) and late or no relapse (non-progressors) either for TSS (Figure 7A) and for OS (Figure 7B) for genes not expressed on the cell surface. Also here, the inventors could identify characteristic and significantly differentially expressed genes. While EpCAM staining alone had a high sensitivity to detect patients at risk, it also was found in patients with good outcome. This means that EpCAM could not reliably distinguish patients with a positive outcome from patients with a negative outcome, and hence, had a low specificity. In addition to c-KTT, MUC1 was expressed on EpCAM -positive cells from patients with poor prognosis (Figure 6 A and 6C). Surprisingly, MUC1 gene expression correlated well with c-KTT gene expression (rho=0.49, p = 0.003). For all cancer types tested (i.e., breast, prostate, and lung cancer), MUC1 expression, either alone or in combination with c-KIT transcript (as an alternative marker, i.e., MUC1 or c-KTT expression) identified patients at risk to progress or die by Kaplan-Meier survival analysis (Figure 6F and 6G). Testing a MUC1 antibody (antibody clone 16A) on a bone marrow sample of a non-cancer patient, the inventors could not detect a single positive cell among 2 x 105bone marrow cells.

[1088] Therefore, the inventors unexpectantly found an additional negative prognostic marker for DCCs, i.e., MUC1, the expression or expression level of which alone or in combination with c-KIT or (an) additional DCC marker(s), such as EpCAM, is indicative of the cell being a metastasis founder cell.

[1089] The inventors first investigated the prognostic impact of c-KIT and tested its protein expression on candidate DCC (see Example 6).

[1090] Example 6 - c-KIT protein expression on DCC predicts outcome

[1091] As described in Example 5 the inventors identified c-KIT transcript to be expressed in NSCLC-DCC with high sternness scores and obtained hints for its prognostic relevance. The inventors next established quantitative assessment of protein expression (see Example l.F, l.G and l.H). The inventors used HEL cell line cells as internal positive control, whereas peripheral blood mononuclear cells (PBMC) served as negative controls and noticed that some cells within the patient samples showed similar expression of c-KIT as the HEL control cells (Figures 8A and 8B). Similar to the P3- CK / EpCAM-IF staining, the MGV of the cell of interest had to exceed the mean of the MGV of 10 neighboring cells plus three times the standard deviation of the MGVs of these 10 cells for the c-KIT channel to be called c-KIT positive (c- KTT+). This was then tested using samples from patients from whom c-KTT transcript positive DCCs had been isolated (Figure 8C). Confirming that the patient samples provided a subpopulation of cells that is c-KTT+.

[1092] The inventors subsequently added this c-KTT staining to P3-CK / EpCAM-IF (i.e., P3-CK / EpCAM / c-KTT-IF; see Example l.F) and compared this protocol to samples of the same 45 patients, subjected to staining protocol variants Pl-CK-IC, P2-EpCAM-Depl-IF, and P3-CK / EpCAM-IF (see Table 3, "side-by-side comparison"). Surprisingly, the inventors observed a strong prognostic impact of C-KTT+, detected as DCC either by CK or EpCAM labeling (Figure 9). An overview over the results of the survival analysis of the four methods used can be seen in Table 7.

[1093] Table 7: Overview of the p-values for PFS, TSS, and OS for the detection of DCCs by Pl-CK-IC, P2-EpCAM- Depl.-IF, P3-CK / EpCAM -IF, and P3-CK / EpCAM / c-KIT-IF staining.

[1094] The impact of total cell numbers screened on detection rates was then assessed. While increasing cell numbers from 0.5 - 2xl05resulted an almost linear increase of DCC numbers of c-KTT+patients, most patients negative at 0.5xl05bone marrow cells screened remained negative (Figure 8D).

[1095] The inventors then stained 11 additional patients BM cells (see Examples l.C to l.H) and re-assessed clinical impact, resulting in 56 patients (Table 3, extended cohort). Overall, 130 DCCs were detected from patients with typical DCCD (ranging 1-15 and excluding 2 patients with very high EpCAM-DCC numbers to avoid bias). Co-expression of c-KTT protein was found on EpCAM- or cytokeratin-single positive cells as well as on double positive cells, the latter being the most prevalent DCC population expressing c-KTT (Figure 8F). The total percentage of DCC (CK and / or EpCAM positive) displaying c-KTT was 36.9% (Figure 8G). Statistical associations of positive CK+, EpCAM+, and C-KTT+ samples with clinicopathological variables is depicted in Table 8. Subsequently, the inventors categorized patients into either c-KTT positive when at least one c-KTT positive DCC (expressing either cytokeratin or EpCAM or both) had been detected in bone marrow (Figure 8H).

[1096] Table 8: Statistical associations of positive CK+, EpCAM+, and c-KIT+samples with clinicopathological variables

[1097] Unexpectedly, patients harboring c-KTT+DCC in bone marrow displayed a significantly unfavorable outcome, for PFS, OS, and TSS (TSS, P=0.0009; PFS, P=0.0010; OS, P=0.0002; Figure 10A), when compared to patients without such cells (i.e., either DCC or DCC+c-KTT ). When comparing DCC+patients with and without C-KTT+ DCCs, the inventors noted that the prognostic information was strongly linked to c-KTT positivity (Figure 10B).

[1098] Finally, the inventors performed a multivariate Cox regression analysis and adjusted for confounding (clinicopathological) variables. Sex, age, histology, tumor size, presence of lymph node metastasis, grading, smoking habits, and the results of the P3-CK / EpCAM / c-KTT-IF-staining were included in this analysis (Figure IOC). In a backward procedure, all (for PFS) or most (in TSS and OS; exception. N-status) variables but c-KTT expression were removed from the model. The median hazard ratio caused by KIT expression was 3.4 (range: 1.6. -7.5 for PFS ), 7.7 (range: 2.3. -25) for TSS and 5 (range: 2.1-11.9) for OS. Surprisingly, a single c-KTT-positive DCC in bone marrow outcompeted all clinical variables, including tumor size, lymph node status or grading and was independent of adenocarcinoma- or squamous cell carcinoma histology.

[1099] The additional measurement of c-KTT protein expression in context of a staining protocol using EpCAM and / or CK to detect DCCs has, surprisingly, a negative predictive value, and specifically, allows to predict a poor survival of a patient. Since most cancer patients die from metastases (and the patients were free from metastases at the beginning of the study), the inventive marker combination (i.e., c-KTT in combination with EpCAM and / or CK), is further indicative of a cell being a metastasis founder cell.

[1100] Example 7 - c-KIT+and c-KIT DCC display similar genomic profiles

[1101] The inventors investigated whether c-KTT+DCCs represent a genomically different DCC subpopulation. For this, 49 DCCs from 25 patients were isolated, WGA and subsequent whole genome sequencing at low coverage was performed (Figure 11A), and the presence or absence of DNA gains and losses (copy number alterations, CNA) for each cell was determined. About 70% of the analyzed cells showed aberrations, with single EpCAM+cells (n=27) and c-KTT+DCCs cells (n=18) harboring CNA at similar frequency (22 / 27, 81.5% vs. 11 / 18, 61.1%, respectively, P =0.14). Figure 11B). Applying the Progenetix user data tool (A.D. Donnenberg, et al, 2012, PLoS One 7, e52885), characteristic aberration patterns for the c-KTT+(n=18) vs. c-KTT DCC (n=31) phenotypes (Figure 11C and 11D) among aberrant DCC could not be identified.

[1102] Example 8 - TP53 or KRAS mutations are not indicative of metastasis founder cells in contrast to the c- KIT + DCC phenotype.

[1103] As described in Examples 5 and 6, supra, the inventors found a so far unknown population of disseminated cancer cells defined by a high sternness score that is reflected, in particular, by a high expression level of c-KTT and / or MUC1. These cells are considered to be metastasis founder cells (MFC) as their detection is linked to a very poor prognosis; see Fig. 6, 9 and 10.

[1104] Since the 2017 landmark study from the TRACERX consortium (Jamal-Hanjani (2017), N Engl J Med ;376(22)) addressing the evolution of NSCLC in patients, it has become common knowledge that "driver mutations in EGFR, MET, BRAF, and TP53 were almost always clonal", and formed the group of early initiating events in the formation of NSCLC. Likewise, KRAS mutations were found to be among the initiating early events in lung adenocarcinomas (LUAD) and late events in lung squamous cell carcinomas LUSC). Based on the common general knowledge in the prior art, one would therefore expect to find KRAS and TP53 mutations in all DCC from primary tumors harboring these mutations (TP53 and KRAS mutations in LUAD, and TP53 mutations in non-LUAD). The inventors tested if this is indeed the case and whether DCC with TP53 or KRAS mutations expressed high c-KTT levels.

[1105] TP53 and KRAS mutation analysis in single DCC by Sanger sequencing of genomic DNA

[1106] Methods:

[1107] Single EpCAMiF+ disseminated cancer cells (DCCs) from bone marrow (BM) and lymph node (LN) samples of MO-stage and Ml-stage NSCLC patients were isolated by manual micromanipulation, as described in Examples 1 and 4. Combined whole transcriptome and genome amplification was performed as previously described (Klein (2002), Nat. BiotechnoL, 20(4)). TP53 hot spot region for single nucleotide variants (SNVs) exon 5-8 for LUAD and non-LUAD samples and the exonic region of KRAS for LUAD samples were amplified using endpoint PCR and sequenced as described in Elsner (2022), J PathoL, 258(3) on amplified cDNA or gDNA, whenever cDNA quality was not sufficient as assessed by the Amplil WTA QC multiplex PCR assay.

[1108] Results:

[1109] Analysis of DCC from BM and LN

[1110] Figure 12 displays the results of the TP53 and KRAS Sanger sequencing of EpCAMiF+ DCCs from bone marrow and lymph node samples. Surprisingly, TP53 mutations in bone marrow DCCs were only found in 2 / 32 (6.3%) patients and 2 / 64 (3.1%) single cells. In lymph node DCCs, TP53 mutations were found in 3 / 24 (12.5%) patients and 4 / 74 (5.4%) cells. Also unexpectedly, a KRAS mutation could only be detected in 1 / 12 (8.3%) patients (1 / 23 (4.3%) of bone marrow DCCs). None of the analyzed lymph node samples (18 DCCs isolated from 9 patients) harbored a KRAS mutation. Interestingly, the only KRAS mutation found was detected in a bone marrow DCC from an Ml patient (Figure 12). Comparison with the matched primary tumor (PT)

[1111] Of seven PT sequenced for TP53, three harbored mutations. Surprisingly, none of them were found in the DCC (Figure 12).

[1112] Conclusions:

[1113] Although TP53 and KRAS mutations belong to the most frequent mutations in primary NSCLC (35-50% frequency), their detection in DCC is, surprisingly, very rare, both in BM- and LN-derived DCC.

[1114] TP53 and KRAS mutation analysis in single DCC by RNA-seq reveals that the presence of these mutations is not indicative of metastasis founder cells

[1115] Methods:

[1116] Whole transcriptome amplification (WTA) products were subjected to next generation sequencing library preparation. In brief, WTA product was re-amplified by PCR and cleaned up using AMPure XP beads. Adapters were removed by restriction enzyme digestion, followed by concentration measurement using the Qubit dsDNA Broad Range Kit and fragment length analysis using an Agilent DNA High Sensitivity Kit on an Agilent 2100 Bioanalyzer machine. Samples of sufficient quality were fragmented to a mean fragment length of 350 bp using a Covaris M220X Systems. Library preparation was then done using the Illumina TruSeq DNA PCR-free library preparation kit and DNA unique dual indices. Samples were quantified by shallow sequencing on an Illumina MiSeq system or by qPCR quantification using the KAPA Library Quantification Kit. RNA-Seq was then performed on a NovaSeq 6000 systems with 2x150 bp sequencing mode. The sequencing quality was then evaluated with FastQC (version 0.11.8) and in a multisample comparison with MultiQC (version 1.8) before and after adapter trimming and contamination screening. Briefly, raw sequencing data of single cells were trimmed, and remaining adapter sequences as well as low sequencing quality bases at the end of each read were removed using BBDuk. In order to increase the mapping quality (lowering false-positive alignments), read decontamination was performed using BioBloom Tools with filters for the genomes of Homo sapiens (hg38), Mus musculus (mmlO), Escherichia coli (BL21), Mycoplasma pneumoniae (M129), Sphingobium sp. (SYK-6), Bradyrhizobium japonicum (USDA 110), Pichia pastoris (GS115), Malessia globosa (CBS 7966), Aspergillus fumigatus (Af293), and a set of viral genomes (RefSeq, 5k+ genomes). All reads that did not map exclusively to hg38 (GENCODE version J, GRCh38.plO) or did not map at all were defined as likely contaminations and discarded from downstream processing. Subsequently, the cleaned sample reads were aligned to the reference genome hg38 with STAR (version 2.5.1b). Uniquely mapped reads were counted per gene per sample using featurecounts from Subread. A total of 110 DCCs could then be processed for SNV analysis. Results were filtered for distribution of red depth (DP) > 10, allele depth (AD) > 5 and variant frequency (VF) > 0.1 was used.

[1117] Results:

[1118] Exonic TP53 SNVs were found in 13 / 110 (11.8%) of analyzed EpCAM+ DCCs and in 9 / 42 (21.4%) of analyzed patients (Table 9). Only two SNVs could be classified as potentially oncogenic mutations based on OncoKB, while one was likely oncogenic and the other likely neutral. Both mutations were found in the same cell. Interestingly, 5 / 13 (38.5%) of detected SNVs were found in DCCs derived from Ml-stage patients. No exonic KRAS mutations could be detected. 4 / 13 of DCC with TP53 mutations were c-KIT low. Table 9. Overview of exonic mutations in TP53 and KRAS genes detected among DCC based on SNV calling from single cell RNA-Seq data. NA, mutation not found in OncoKB; Unknown, mutation found in OncoKB, but pathogenicity is not known; Likely neutral, the mutation has no functional consequence; likely oncogenic, the mutation is likely pathogenic.

[1119] The inventors then tested whether patients with cells, in which mutations in the TP53 gene were identified (see Table 9 above) have a worse outcome than patients with TP53 wild type cells only. The inventors performed a direct comparison with the impact of c-KIT expression in EpCAM+ cells (Figure 6e / left panel of Figure 13), i.e. the same patients, for which they had generated expression data and mutational data, were compared for impact on survival.

[1120] As can be seen in Figure 13, c-KIT expression in DCC was clearly more meaningful for the outcome than mutation of TP53. The inventors conclude that phenotype overrides genotype at early stages of metastatic colony formation and that c-KIT expression in DCC marks metastasis founder cells (whereas TP53 or KRAS mutations do not).

[1121] Conclusion:

[1122] In the prior art it was concluded from sequencing of various primary tumor areas that cancer cells with KRAS and TP53 mutations are founders of metastasis. Unexpectedly, the present analysis of disseminated cancer cells (DCC) does not support this conclusion in the prior art. In context of the present invention is has been surprisingly found that KRAS and TP53 mutations are very rare in DCC: No KRAS and only one oncogenic TP53 mutation were detected among 110 DCC from NSCLC patients. Moreover, TP53 mutations in DCC were not predictive of patient survival. In contrast, the sternness phenotype of DCC as marked by elevated c-KIT and / or MUC1 expression, is highly predictive of the generation of metastases and poor patient survival. Therefore, in contrast to TP53 or KRAS mutations, high c-KIT and / or MUC1 expression in DCC marks metastasis founder cells.

[1123] Any cited documents are incorporated herein for reference in their entirety.

Claims

Claims1. A method for detecting metastasis founder cells in a sample, comprising measuring in single cells in a sample the expression or expression level of (i) a first marker which is c-KTT and (ii) at least one disseminated cancer cell (DCC) marker, wherein expression of c-KTT and at least one DCC marker in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of c-KTT and at least one DCC marker in a cell above respective thresholds are indicative of the cell being a metastasis founder cell.

2. The method of claim 1, wherein the cells in the sample are contacted with(i) an antigen-binding molecule binding c-KTT protein, or a probe or primer pair binding c-KTT RNA, preferably an antigen-binding molecule binding c-KTT protein; and / or(ii) at least one antigen-binding molecule binding at least one DCC marker protein, or at least one probe or primer pair binding at least one DCC marker RNA.

3. A method for analysing cells, comprising a step of contacting a sample comprising cells with(i) an antigen-binding molecule binding c-KTT protein, or a probe or primer pair binding c-KTT RNA, preferably an antigen-binding molecule binding c-KTT protein; and(ii) at least one antigen-binding molecule binding at least one DCC marker protein or at least one probe or primer pair binding at least one DCC marker RNA.

4. The method of claim 3, wherein expression or an expression level of c-KTT and at least one DCC marker is measured in single cells in the sample, and, preferably, wherein expression of c-KTT and at least one DCC marker in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of c-KTT and at least one DCC marker in a cell above respective thresholds are indicative of the cell being a metastasis founder cell.

5. The method of any one of claims 1 to 4, wherein the DCC marker(s) comprise(s) EPCAM, at least one cytokeratin, and / or MUC1; preferably wherein the DCC marker(s) comprise(s) at least EPCAM, and optionally further at least one cytokeratin or MUC1; and, preferably, wherein the cytokeratin(s) comprise(s) CK8, CK18 and / or CK19.

6. The method of any one of claims 1 to 5, wherein(I) the expression or expression level of c-KTT and at least one cytokeratin such as CK8, CK18 and / or CK19 is measured in single cells, and wherein (a) expression of c-KTT and at least one cytokeratin in a cell is indicative of the cell being a metastasis founder cell, or (b) expression levels of c-KTT and at least cytokeratin in a cell above respective thresholds are indicative of the cell being a metastasis founder cell;(II) the expression or expression level of c-KTT and EPCAM is measured in single cells, and wherein (a) expression of c-KTT and EPCAM in a cell is indicative of the cell being a metastasis founder cell, or(b) expression levels of c-KTT and EPCAM in a cell above respective thresholds are indicative of the cell being a metastasis founder cell;(III) the expression level of c-KTT and / or EPCAM is measured in single cells, and wherein in a cell the combination of (i) expression of c-KTT and EPCAM, and (ii) a significantly higher expression level of EPCAM than in hematopoietic cells and / or blood cells, is indicative of the cell being a metastasis founder cell; or(IV) the expression level of c-KTT, at least one cytokeratin and / or EPCAM such as CK8, CK18 and / or CK19 is measured in single cells, and wherein expression of c-KTT, at least one cytokeratin and EPCAM in a cell is indicative of the cell being a metastasis founder cell; preferably, wherein in a cell the combination of (i) expression of c-KTT, at least one cytokeratin and EPCAM, and (ii) a significantly higher expression level EPCAM than in hematopoietic cells and / or blood cells, is indicative of the cell being a metastasis founder cell.

7. The method of any one of claims 2 to 6, wherein said antigen-binding molecule(s) is / are antibody / ies or antigen-binding fragment(s) thereof, wherein said antigen-binding molecule(s), optionally, comprise(s) (a) detectable label(s) or is / are bound by (a) molecule(s) comprising (a) detectable label(s).

8. The method of any one of claims 2 to 7, wherein said antigen-binding molecules comprise) (i) an antigenbinding molecule, preferably an antibody or fragment, thereof binding c-KTT protein, and (ii) at least one antigen-binding molecule selected from the group consisting of: an antigen-binding molecule, preferably an antibody or fragment thereof, binding at least one cytokeratin protein, and an antigen-binding molecule, preferably an antibody or fragment thereof, binding EPCAM protein.

9. The method of any one of claims 1, 2 and 4 to 8, wherein the expression or expression level of c-KTT and at least one DCC marker is measured by imaging, flow cytometry, PCR, and / or sequencing; preferably wherein the expression or expression level of c-KTT, at least one cytokeratin and / or EPCAM is measured by imaging.

10. The method of any one of claims 1, 2 and 4 to 9, wherein the expression level of a marker corresponds to the level of a detectable signal, preferably a fluorescent signal, that is associated with the marker.

11. The method of any one of claims 1, 2 and 4 to 10, wherein the expression level of a marker is measured by imaging, and wherein the expression level of a marker in a cell is above the respective threshold when the level of a detectable signal associated with the marker in the cell is higher than the mean signal in 10 neighbouring cells plus three standard deviations of the signal in said 10 neighbouring cells.

12. The method of any one of claims 1 to 11, wherein the sample is a tissue sample, preferably, from a non- epithelial tissue or a body fluid, more preferably from bone marrow, blood, lymph nodes or cerebrospinal fluid, and most preferably, wherein the sample is a bone marrow sample or a blood sample.

13. The method of any one of claims 1 to 12, wherein(i) the sample comprises at least about lxlO4, at least about 5xl04, at least about lxlO5or at least about 5xl05, preferably at least about lxlO5, more preferably at least about 2xl05cells; and, preferably, wherein the sample is a bone marrow sample comprising at least about lxlO4, at leastabout 5xl04, at least about lxlO5or at least about 5xl05, preferably at least about lxlO5, more preferably at least about 2xl05mononucleated cells; and / or(II) the sample comprises from about lxlO4to about lxlO7or from about 5xl05to about 5xl05, preferably from about lxlO5to about 2xl05or from about 1.5xl05to about 3xl05, more preferably about 2xl05cells; and preferably, wherein the sample is a bone marrow sample comprising from about lxlO4to about lxlO7or from about 5xl05to about 5xl05, preferably from about lxlO5to about 2xl05or from about 1.5xl05to about 3xl05, more preferably about 2xl05mononucleated cells.

14. The method of any one of claims 1 to 13, wherein the method comprises prior to measuring the expression or expression level of c-KIT and the DCC marker(s) and / or prior to contacting the sample with an antigenbinding molecule binding c-KIT protein, at least one antigen-binding molecule binding at least one DCC marker protein, a probe or primer pair binding c-KIT RNA, and / or at least one probe or primer pair binding at least one DCC marker RNA, a step of(i) removing erythrocytes and / or leukocytes such as granulocytes from the sample, preferably by using a density gradient centrifugation, and / or(ii) removing cells expressing CD45, CD33, CD11, CD325a, GlyA, CD27 and / or CD319 from the sample, preferably by using a cell sorting method such as FACS, MACS or CELLSEARCH®.

15. The method of any one of claims 1, 2 and 4 to 14, wherein the metastasis founder cells are disseminated cancer cells (DCCs) or circulating tumor cells (CTCs).

16. The method of any one of claims 1, 2 and 4 to 15, wherein the metastasis founder cells are from an epithelial cancer such as a lung carcinoma, breast cancer or prostate carcinoma, preferably from a lung carcinoma, more preferably from a non-small-cell lung cancer (NSCLC).

17. A method for detecting metastasis founder cells in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) c-KIT and (ii) at least one DCC marker, wherein expression of c-KIT and at least one DCC marker in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of c-KIT and at least one DCC marker in a cell above respective thresholds are indicative of the cell being a metastasis founder cell, as defined in any one of claims 1, 2 and 4 to 16.

18. The method of claim 17, wherein the subject is a patient that has, previously had or is suspected of having a cancer, and / or wherein the patient previously had a metastasis and / or is suspected of having and / or developing a metastasis; and, preferably, wherein the cancer is an epithelial cancer such as a lung carcinoma, breast cancer or prostate carcinoma, preferably a lung carcinoma, more preferably a non-small-cell lung cancer (NSCLC).

19. The method of claim 17 or 18, wherein(i) the presence of metastasis founder cells in the subject indicates that the subject is likely to develop a metastasis, and / or the absence of metastasis founder cells in the subject indicates that the subject is not likely to develop a metastasis;(ii) the presence of metastasis founder cells in the subject indicates that the cancer is likely to relapse or progress, and / or the absence of metastasis founder cells in the subject indicates that the cancer is not likely to relapse or progress; and / or(iii) the presence of metastasis founder cells in the subject indicates a negative outcome of the cancer, and / or the absence of metastasis founder cells in the subject indicates a positive outcome of the cancer, preferably, a positive outcome comprises survival of the subject, and / or a negative outcome comprises death of the subject.

20. The method of any one of claims 17 to 19, wherein the subject is or has been treated with at least one anti - cancer drug, and preferably, wherein(i) the presence of metastasis founder cells in the subject indicates that the treatment with the anticancer drug(s) is or was not successful, and / or wherein the absence of metastasis founder cells in the subject indicates that the treatment with the anti-cancer drug(s) is or was successful; and / or(ii) the presence of metastasis founder cells in the subject indicates that at least one anti-cancer drug is to be administered to the subject, and / or wherein the absence of metastasis founder cells in the subject indicates that said anti-cancer drug(s) is / are not to be administered to the subject, wherein said anti-cancer drug(s) is / are as defined in claim J.

21. A method of prognosing the development of metastases in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) c-KIT and (ii) at least one DCC marker, as defined in any one of claims 1, 2 and 4 to 16,(a) wherein the presence of at least one cell expressing c-KIT and at least one DCC marker in the sample indicates that the subject is likely to develop a metastasis, and / or wherein the absence of a cell expressing c-KIT and at least one DCC marker in the sample indicates that the subject will likely not develop a metastasis, or(b) wherein the presence of at least one cell expressing c-KIT and at least one DCC marker at levels above respective thresholds in the sample indicates that the subject is likely to develop a metastasis, and / or wherein the absence of a cell expressing c-KIT and at least one DCC marker at levels above respective thresholds in the sample indicates that the subject will likely not develop a metastasis.

22. A method of prognosing the relapse of a cancer in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) c-KIT and (ii) at least one DCC marker, as defined in any one of claims 1, 2 and 4 to 16,(a) wherein the presence of at least one cell expressing c-KIT and at least one DCC marker in the sample indicates that the cancer is likely to relapse, and / or wherein the absence of a cell expressing c-KIT and at least one DCC marker in the sample indicates that the cancer is not likely to relapse, or(b) wherein the presence of at least one cell expressing c-KIT and at least one DCC marker at levels above respective thresholds in the sample indicates that the cancer is likely to relapse, and / or whereinthe absence of a cell expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the cancer is not likely to relapse.

23. A method of prognosing the progression of a cancer in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) c-KTT and (ii) at least one DCC marker, as defined in any one of claims 1, 2 and 4 to 16,(a) wherein the presence of at least one cell expressing c-KTT and at least one DCC marker in the sample indicates that the cancer is likely to progress, and / or wherein the absence of a cell expressing c-KTT and at least one DCC marker in the sample indicates that the cancer is not likely to progress, or(b) wherein the presence of at least one cell expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the cancer is likely to progress, and / or wherein the absence of a cell expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the cancer is not likely to progress.

24. A method for prognosing the outcome of a cancer in a subject, comprising measuring in single cells in a sample from the subject the expression or expression level of (i) c-KTT and (ii) at least one DCC marker, as defined in any one of claims 1, 2 and 4 to 16,(a) wherein the presence of at least one cell expressing c-KTT and at least one DCC marker in the sample is indicative of a negative outcome, and / or wherein the absence of a cell expressing c-KTT and at least one DCC marker in the sample is indicative of a positive outcome, or(b) wherein the presence of at least one cell expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample is indicative of a negative outcome, and / or wherein the absence of a cell expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample is indicative of a positive outcome; preferably, wherein a positive outcome comprises survival of the subject for more than about 18 months or for more than about 24 months, and / or wherein a negative outcome comprises death of the subject within about 18 months or within about 24 months, respectively.

25. A method of measuring the success of a treatment of a subject that is or had been treated with at least one anti-cancer drug, said method comprising measuring in single cells in a sample from the subject the expression or expression level of (i) c-KTT and (ii) at least one DCC marker, as defined in any one of claims 1, 2 and 4 to 16,(a) wherein the presence of at least one cell expressing c-KTT and at least one DCC marker in the sample indicates that the treatment with the anti-cancer drug(s) is or was not successful, and / or wherein the absence of a cell expressing c-KTT and at least one DCC marker in the sample indicates that the treatment with the anti-cancer drug(s) is or was successful, or(b) wherein the presence of at least one cell expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the treatment with the anti-cancer drug(s) is or was not successful, and / or wherein the absence of a cell expressing c-KTT and at least one DCCmarker at levels above respective thresholds in the sample indicates that the treatment with the anti - cancer drug(s) is or was successful.

26. A method of stratifying subjects for treatment with at least one anti-cancer drug, comprising measuring in single cells in a sample from a subject the expression or expression level of (i) c-KTT and (ii) at least one DCC marker, as defined in any one of claims 1, 2 and 4 to 16,(a) wherein the presence of at least one cell expressing c-KTT and at least one DCC marker in the sample indicates that the anti-cancer drug(s) are to be administered to the subject, and / or wherein the absence of a cell expressing c-KTT and at least one DCC marker in the sample indicates that the anticancer drug(s) are not to be administered to the subject, or(b) wherein the presence of at least one cell expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the anti-cancer drug(s) are to be administered to the subject, and / or wherein the absence of a cell expressing c-KTT and at least one DCC marker at levels above respective thresholds in the sample indicates that the anti-cancer drug(s) are not to be administered to the subject.Zl. One or more anti-cancer drugs for use in treating a patient having metastasis founder cells, wherein said anticancer drug(s) comprise(s)(a) a c-KTT inhibitor such as imatinib,(b) an antigen-binding molecule, preferably an antibody of fragment thereof, binding c-KTT, and, preferably, wherein said antigen-binding molecule blocks c-KTT signaling and / or is conjugated to a drug, preferably a cytostatic and / or cytotoxic drug such as Vedotin, Mafodotin, Deruxtecan, Govitecan, Tesirine, Ozagamyzin, Pasudotox, Soravtansine or Emtansine.(c) an antigen-binding molecule, preferably an antibody of fragment thereof, binding EPCAM, preferably, wherein said antigen binding molecule is conjugated to a c-KTT inhibitor as defined in (a),(d) an antigen-binding molecule, preferably an antibody of fragment thereof, binding c-KTT and EPCAM, preferably, wherein said antigen-binding molecule blocks c-KTT signalling and / or is conjugated to a drug as defined in (b) and / or a c-KTT inhibitor as defined in (a), and / or(e) CAR-T cells binding c-KTT and / or EPCAM, preferably, wherein said CAR T cells comprise(i) a CAR-T cell comprising a CAR binding c-KTT and additionally a CAR binding EPCAM,(ii) a CAR-T cell comprising a CAR binding c-KTT, and additionally a CAR-T cell comprising a CAR binding EPCAM, and / or(iii) a CAR-T cell comprising a CAR binding c-KTT and EPCAM.

28. The one or more anti-cancer drugs for use according to claim Zl, wherein the metastasis founder cells express c-KTT and at least one DCC marker, or have expression levels of c-KTT and at least one DCC marker above respective thresholds, as defined in any one of claims 1 to 20.

29. An antibody or fragment thereof binding c-KTT and EPCAM, which, preferably, blocks c-KIT signaling and / or which is, preferably, conjugated to a drug as defined in claim 27(b) and / or a c-KTT inhibitor as defined in claim 27(a).

30. A T cell comprising(i) a chimeric antigen receptor (CAR) binding c-KTT and additionally a CAR binding EPCAM, and / or(ii) a CAR binding c-KTT and EPCAM.

31. A population of CAR-T cells comprising a CAR-T cell binding c-KTT and a CAR-T cell binding EPCAM.

32. A kit for use in detecting metastasis founder cells, said kit comprising(a) an antigen-binding molecule binding c-KTT protein, an antigen-binding molecule binding EPCAM protein and / or an antigen-binding molecule binding at least one cytokeratin protein, as defined in any one of claims 2, 7 and 8; and / or(b) a probe or primer pair binding c-KTT RNA, a probe or primer pair binding EPCAM RNA and / or a probe or primer pair binding at least one cytokeratin RNA.

33. A kit-of-parts comprising (i) an antigen-binding molecule binding c-KTT protein, and (ii) at least one antigenbinding molecule binding at least one DCC marker protein, as defined in any one of claims 2, 7 and 8.

34. A method of isolating metastasis founder cells from a sample, comprising the steps of:(a) contacting cells in a sample with an antigen-binding molecule binding c-KTT protein, and (ii) at least one antigen-binding molecule binding at least one DCC marker protein, as defined in any one of claims 2, 7 and 8, and(b) isolating cells from the sample which express c-KTT and at least one DCC marker, wherein expression of c-KTT and at least one DCC marker in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of c-KTT and at least one DCC marker in a cell above respective thresholds are indicative of the cell being a metastasis founder cell, as defined in any one of claims 1, 2 and 4 to 20; and, preferably, wherein the DCC marker(s) comprise(s) or consist(s) of EPCAM.

35. An isolated cell expressing (i) a first marker which is c-KTT and (ii) at least one disseminated cancer cell (DCC) marker.

36. A population of isolated cells, wherein at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or all cells in the population express (i) a first marker which is c-KTT and (ii) at least one disseminated cancer cell (DCC) marker.

37. The cell of claim 35 or the population of cells of claim 36, wherein the DCC marker(s) comprise(s) or consist(s) of EPCAM and / or MUC1, preferably EPCAM.

38. A cell culture comprising the cell of claim 35 or 37 or the population of cells of claim 36 or 37, a medium for maintaining and / or proliferating the cell(s), and optionally, stromal cells; and preferably, wherein the mediumcomprises at least one ligand of c-KTT, and / or the stromal cells express or secrete at least one ligand of c- KTT; and, preferably, wherein the ligand(s) of c-KTT comprise(s) SCF and / or meteorin-like (METRNL).

39. A method of culturing cells, comprising culturing the cell of claim 35 or 37 or the population of cells of claim 36 or 37 in a medium for maintaining and / or proliferating the cell(s); optionally, wherein said cells are cultured on stromal cells; and preferably, wherein the medium comprises at least one ligand of c-KTT, and / or the stromal cells express or secrete at least one ligand of c-KTT of c-KTT; and, preferably, wherein the ligand(s) of c-KTT comprise(s) SCF and / or meteorin-like (METRNL).

40. Use of the cell of claim 35 or 37 or the population of cells of claim 36 or 37 or the cell culture of claim 38 for drug screening and / or for identifying drug targets.

41. A method of treating a subject in need thereof comprising the steps of(a) performing a method of detecting metastasis founder cells in a subject according to any one of claims 17 to 20, and(b) administering an effective amount of one or more anti-cancer drugs as defined in claim 1 to the subject, when the subject has metastasis founder cells.

42. A method for detecting metastasis founder cells in a sample, comprising measuring in single cells in a sample the expression or expression level of a first marker which is MUC1, wherein expression of MUC1 is indicative of the cell being a metastasis founder cell, or wherein the expression level of MUC1 in a cell above a threshold is indicative of the cell being a metastasis founder cell.

43. The method of claim 42, further comprising measuring in single cells in the sample the expression or expression level of a second marker which is c-KTT, wherein expression of MUC1 and / or c-KTT is indicative of the cell being a metastasis founder cell, or wherein expression levels of MUC1 and / or c-KTT in a cell above respective thresholds are indicative of the cell being a metastasis founder cell.

44. The method of claim 42 or 43, further comprising measuring in single cells in the sample the expression or expression level of at least one additional disseminated cancer cell (DCC) marker such as EPCAM, wherein expression of (i) MUC1 and / or c-KTT in combination with (ii) expression of the additional DCC marker(s) in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of MUC1 and / or c- KTT above respective thresholds in combination with expression level(s) of the additional DCC marker(s) above respective thresholds in a cell are indicative of the cell being a metastasis founder cell.

45. A method for analysing cells, comprising a step of contacting a sample comprising cells with(i) an antigen-binding molecule binding MUC1 protein or a probe or primer pair binding MUC1 RNA, preferably a probe or primer pair binding MUC1 RNA; and(ii) an antigen-binding molecule binding c-KTT protein or a probe or primer pair binding c-KTT RNA, preferably an antigen-binding molecule binding c-KTT protein; and optionally(iii) at least one additional antigen-binding molecule binding at least one DCC marker protein or at least one additional probe or primer pair binding at least one DCC marker RNA.

46. The method of claim 45, wherein expression or an expression level of MUC1 and c-KIT, and optionally at least one additional DCC marker, is measured in single cells in the sample; and, preferably, wherein expression of MUC1 and / or c-KIT, and optionally at least additional one DCC marker such as EPCAM, in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of MUC1 and / or c-KIT, and optionally at least one additional DCC marker such as EPCAM, above respective thresholds are indicative of the cell being a metastasis founder cell.

47. A method for analysing cells, comprising a step of contacting a sample comprising cells with(i) an antigen-binding molecule binding MUC1 protein or a probe or primer pair binding MUC1 RNA, preferably a probe or primer pair binding MUC1 RNA; and(ii) at least one antigen-binding molecule binding at least one additional DCC marker protein or at least one probe or primer pair binding at least one additional DCC marker RNA, preferably at least an antigen-binding molecule binding EPCAM; and optionally(iii) an antigen-binding molecule binding c-KIT protein a probe or primer pair binding c-KIT RNA, preferably an antigen-binding molecule binding c-KIT protein.

48. The method of claim 47, wherein expression or an expression level of MUC1 and at least one additional DCC marker, and optionally c-KIT, is measured in single cells in the sample, and, preferably, wherein expression of MUC1 and at least one additional DCC marker, and optionally c-KIT, in a cell is indicative of the cell being a metastasis founder cell, or wherein expression levels of MUC1 and at least one additional DCC marker, and optionally c-KIT, above respective thresholds are indicative of the cell being a metastasis founder cell.

49. The method of any one of claims 44 to 48, wherein the DCC marker(s) comprise(s) at least one cytokeratin and / or EPCAM, preferably at least EPCAM, and, preferably, wherein the cytokeratin(s) comprise(s) CK8, CK18 and / or CK19.

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