Methods for identifying and stratifying cancer and cancer patients based on p2x4 receptor expression

Abstract

The present invention relates to methods for identifying cancer, responders, predicting response and stratifying patients with respect to a combination therapy comprising the administration of a P2X4 receptor inhibitor and a cell death inducing chemotherapy. The methods according to the present invention are based on the detection of P2X4 receptor protein expression in cells of a patient-derived cancer sample, e.g. cells or organoids, to identify cancer, responders, predict responders and stratify patients, wherein an increase in protein expression identifies cancer, responders and predicts responders when applying the combination therapy and also allows to stratify the patients accordingly.

Description

[0001] This invention relates to methods for identifying cancer, responders, predicting responses, and stratifying patients with combination therapies comprising administration of a P2X4 receptor inhibitor and chemotherapy that induces cell death. The method according to the invention identifies cancer, responders, predicts responses, and stratifies patients based on detecting the expression of the P2X4 receptor protein in cells of a patient-derived cancer sample (e.g., cells or organoids), wherein increased protein expression, when the combination therapy is applied, identifies cancer, responders, and predicts responses, and also allows for the corresponding stratification of the patients. Background Technology

[0002] Timely and accurate prediction and / or identification of cancer cells and responses to anticancer therapies, along with corresponding patient stratification, plays a crucial role in clinical practice and drug development. For patients with cancer, rapid diagnosis and appropriate stratification for effective treatment are essential for preventing cancer progression and metastasis. Key issues related to cancer patient response and stratification include the complexity and heterogeneity of the molecular background behind specific cancers.

[0003] Solid tumors exhibit a dynamic balance between cell death and proliferation, ensuring sustained tumor maintenance and growth. Many chemotherapy therapies rely on promoting apoptosis to reduce tumor size. However, enhanced apoptosis in cancer cells has been found to trigger paracrine activation of cells in the tumor microenvironment, initiating tissue repair programs that support tumor growth, thereby triggering insensitivity (if not resistance) to apoptosis-targeted chemotherapy.

[0004] At the molecular level, cell death leads to the release of damage-associated molecular patterns (DAMPs), including various growth factors, inflammatory mediators, mitochondrial DNA (mtDNA), reactive oxygen species (ROS), and metabolites, which trigger inflammation and activate innate immunity to stop damage and coordinate tissue repair and wound healing.

[0005] Schmitt et al. (in “Colon tumor cell death causes mTOR dependence by paracrine P2X4 stimulation.” Nature 612.7939 (2022): 347-353.) disclosed, through the study of tumor organoids containing Lgr5+ cells, how this paracrine activation of dying cancer cells can potentially contribute to the molecular mechanisms of treatment resistance involving mTOR-dependent survival-promoting programs. Lgr5+ cells have been described as the originating cells and cancer stem cells in colorectal cancer (CRC). This suggests that when killed during chemotherapy and / or toxin therapy, tumor cells release messenger compounds (particularly ATP) into the tumor environment, whose mTOR and P2X4-based functions improve the survival of neighboring tumor cells.

[0006] mTOR is a serine-threonine kinase involved in the PI3K / mTOR pathway, which regulates cell growth and proliferation in response to the availability of growth factors and nutrients. Schmitt et al. demonstrated that extracellular ATP release, particularly from dying cancer cells, leads to paracrine mTORC1 activation. ATP-associated mTOR activation occurs via purinergic receptors of the P2 family, which includes seven P2X receptor subtypes and eight (seven in mice) P2Y receptor subtypes. Gene expression of all known subtypes was detected in both untransformed human and tumor organoids, as well as mouse tumor organoids, with significant expression of P2X4 identified. Immunohistochemistry confirmed P2X4 expression in unattacked colon and colon tumors. Additionally, 5-(3-bromophenyl)-1,3-dihydro-2H-benzofurano[3,2-e]-1,4-diaza... Selective P2X4 inhibition of 2-keto(5-BDBD)24 reduced ATP-induced S6 phosphorylation in PDTO, while selective P2X1 (NF279) and P2X7 inhibitors (A-438079) had no effect. This evidence collectively suggests that the P2X4 receptor plays a crucial role in the signaling pathways involved in mTOR-associated tumor cell survival.

[0007] EP21163448 discloses the use of a combination of P2X4 receptor protein inhibitors or P2X4 pathway inhibitors with cell-death-inducing therapies to sensitize tumors that are otherwise resistant to cell-death-inducing chemotherapy. However, combination chemotherapy still lacks predictive biomarkers for identifying sensitive cancers, responders, and / or for stratifying patients.

[0008] Therefore, one object of the present invention is to provide predictive biomarkers for identifying susceptible cancers, responders, and / or for stratifying patients to combination therapy involving P2X4 receptor inhibitors and chemotherapy.

[0009] Surprisingly, the inventors of this invention discovered that the expression level of the gene p2rx4, which encodes the P2X4 receptor protein, did not differ significantly between cancer types, making any transcriptomic analysis unsuitable for stratification (see Example 1). In contrast, although the differences in gene expression levels were negligible, the protein levels of the P2X4 receptor differed significantly between cancer subtypes (see Example 2).

[0010] According to this discovery, the above-mentioned objective in the first aspect of the invention is achieved by providing a method for identifying cancer cells sensitive to anticancer chemotherapy, said anticancer chemotherapy comprising administering a combination of: i) an effective amount of at least one P2X4 receptor inhibitor and / or an inhibitor of the P2X4 receptor signaling pathway and ii) an effective amount of at least one chemotherapeutic agent that induces cell death and / or a treatment that induces cell death, said method comprising the step of detecting the expression of P2X4 receptor protein in cancer cells, wherein the detection of P2X4 receptor protein expression in said cancer cells identifies said cancer cells as sensitive to said combination chemotherapy when compared with cancer control cells that do not express P2X4 receptor protein.

[0011] The above-mentioned objective in the second aspect of the invention is further achieved by providing a method for predicting a patient's response to anticancer chemotherapy, said anticancer chemotherapy comprising administering a combination of: i) an effective amount of at least one P2X4 receptor inhibitor and / or an inhibitor of the P2X4 receptor signaling pathway and ii) an effective amount of at least one chemotherapeutic agent that induces cell death and / or a treatment that induces cell death, said method comprising the step of detecting the expression of the P2X4 receptor protein in cancer cells from a cancer sample from said patient, wherein the detection of the P2X4 receptor protein expression in said sample predicts a positive response of said patient to said combination chemotherapy when compared with cancer control cells that do not express the P2X4 receptor protein.

[0012] The above-mentioned objective in the third aspect of the invention is further achieved by providing a method for identifying responders to anticancer chemotherapy, the method comprising the step of detecting P2X4 receptor protein expression in cancer cells in a cancer sample obtained from a patient treated with anticancer chemotherapy, the anticancer chemotherapy comprising administering a combination of: i) an effective amount of at least one P2X4 receptor inhibitor and / or an inhibitor of the P2X4 receptor signaling pathway and ii) an effective amount of at least one chemotherapeutic agent that induces cell death and / or a treatment that induces cell death, wherein the detection of P2X4 receptor protein expression in the sample, when compared with a control cancer sample, identifies the patient as a responder to the combination chemotherapy.

[0013] Also preferred is the method according to the invention, which further includes the step of stratifying patients into treatment groups to apply appropriate anticancer treatment, particularly combination chemotherapy, said combination chemotherapy comprising administering a combination of: i) an effective amount of at least one P2X4 receptor inhibitor and / or a P2X4 receptor signaling pathway inhibitor and ii) an effective amount of at least one chemotherapeutic agent that induces cell death and / or a treatment that induces cell death.

[0014] Also preferred is the method according to the invention, wherein the detection of P2X4 receptor protein expression comprises: determining the protein expression of P2X4 receptor protein in the cancer cells, cancer tissue samples containing the cells, organoids containing the cancer cells, or other suitable cancer samples.

[0015] Also preferred is the method according to the invention, wherein the detection includes an immunohistochemical analysis of P2X4 expression in tissues or organoids.

[0016] The above-mentioned objectives in the fourth aspect of the invention are further achieved by providing the use of a kit comprising a P2X4 receptor protein binding antibody or a fragment of the P2X4 receptor protein or a derivative thereof, said kit being used according to the method of the invention for identifying cancer cells sensitive to anti-cancer chemotherapy, for predicting patient response to anti-cancer chemotherapy, for identifying responders to anti-cancer chemotherapy, and / or for stratifying patients into treatment groups, and said kit optionally further comprising materials for performing said method, such as buffers and reagents, and user instructions.

[0017] The above-mentioned objective in the fourth aspect of the invention is further achieved by providing a method for treating cancer, the method comprising the steps of: performing the method according to the invention; and, based on the results of the method according to the invention, administering to a patient i) an effective amount of at least one P2X4 receptor inhibitor and / or an inhibitor of the P2X4 receptor signaling pathway and ii) an effective amount of at least one chemotherapeutic agent that induces cell death and / or a combination of cell death-inducing treatments.

[0018] As mentioned above, it was unexpectedly discovered in the context of this invention that the expression of the P2X4 receptor protein differs significantly among cancer subtypes and can therefore be used as a biomarker. Based on this discovery, the present invention provides methods and apparatus for identifying cancer, responders, predicting responses, and stratifying patients with combination therapies comprising administration of a P2X4 receptor inhibitor and chemotherapy inducing cell death. The method according to the invention identifies cancer, responders, predicts responses, and stratifies patients based on the detection of P2X4 receptor protein expression in cells of patient-derived cancer samples (e.g., cells or organoids), wherein when the combination therapy is applied, increased protein expression identifies cancer cells, responders, and predicts responders, and also allows for the corresponding stratification of the patients.

[0019] Therefore, the above-mentioned objective in the first aspect of the invention is achieved by providing a method for identifying cancer cells sensitive to anticancer chemotherapy. Anticancer chemotherapy according to the invention comprises administering a combination of: i) an effective amount of at least one P2X4 receptor inhibitor and / or an inhibitor of the P2X4 receptor signaling pathway and ii) an effective amount of at least one chemotherapeutic drug that induces cell death and / or treatment with that drug that induces cell death.

[0020] Generally, any suitable at least one P2X4 receptor inhibitor and / or P2X4 receptor signaling pathway inhibitor can be used. Preferably, according to the method of the present invention, the P2X4 receptor inhibitor is a specific inhibitor of the P2X4 receptor, and is preferably selected from PPADS, Suramin, KN-62, TNP-ATP, Brilliant Blue G, 5-BDBD, BX-430, carbamazepine der., PSB-12054, PSB-12062, PSB-15417, NP-1815-PX, NC-2600, UoS14919, paroxetine, duloxetine, BAY-1797, BAY-2328065, IgG#151-LO, anti-P2X4 receptor protein antibody or inhibitor binding fragment or its derivative, siRNA, shRNA, miRNA, ribozyme, aptamer, antisense nucleic acid molecule, and small molecules less than about 500 Da, preferably 5DBDB, and even more preferably BAY-2328065.

[0021] Also preferred is the method according to the invention, wherein the chemotherapeutic drug inducing cell death and / or the treatment for inducing cell death are selected from necrotic cell supernatant; cationic amphiphilic drugs (CAD); classical anticancer agents, including anthracyclines, antimetabolites, and platinum drugs; topoisomerase inhibitors, such as camptothecin; PDE3A inhibitors, such as anagrelide, alone or together with cell-degrading cytokines; cell-degrading cytokines, such as IFN-α, IFN-γ, TNF-α, or TRAIL; taxanes, paclitaxel, and fluorinated taxanes, such as SB-T-12851, SB-T-12852, SB-T-12853, SB-T-12854, 5-fluorouracil, tegafur, capecitabine, and deoxyfluorouridine; and preferably 5-fluorouracil (5-FU).

[0022] In the context of this invention, an "effective amount" of at least one P2X4 receptor inhibitor and / or P2X4 receptor signaling pathway inhibitor, as well as at least one chemotherapeutic agent that induces cell death and / or a treatment that induces cell death, should mean that the compound is administered to the patient in an amount that achieves therapeutic or preventative effects on the cancer being treated or prevented.

[0023] The method according to this embodiment then includes the step of detecting P2X4 receptor protein expression in cancer cells. Detecting P2X4 receptor protein expression includes determining the protein expression of the P2X4 receptor protein in a sample derived from a patient, said sample containing cancer cells, cancer tissue samples containing cancer cells, or organoids containing cancer cells (e.g., organoids obtained from (early) patient samples). Alternatively, any other suitable cancer sample obtained from the patient can be used.

[0024] The patient can be any suitable cancer patient, such as a mammal, such as a mouse, pet, cow, or human.

[0025] As described in this article, the detection of P2X4 receptor protein expression in cancer cells identifies cancer cells as sensitive to combination chemotherapy as described in this article when compared with cancer control cells that do not express P2X4 receptor protein, and further details are provided below.

[0026] The method according to the invention can be performed in vivo or in vitro.

[0027] As mentioned above, it has also been unexpectedly discovered, within the context of this invention, that the expression of the P2X4 receptor protein differs significantly between cancer subtypes in patients who are responders or non-responders to anticancer chemotherapy as described herein. Based on this finding, this invention provides a method for predicting a patient's response to anticancer chemotherapy as described herein. The method according to the invention includes the step of detecting the expression of the P2X4 receptor protein in cancer cells from a cancer sample from said patient, as described herein.

[0028] The detection of P2X4 receptor protein expression in a sample allows for prediction of a positive response to the combination chemotherapy of the present invention when compared to cancer control cells that do not express the P2X4 receptor protein. In the context of this invention, a “response” or “positive response” to anticancer chemotherapy as described herein should refer to cancer in a patient who can be treated with anticancer chemotherapy as described herein. Therefore, the lack of P2X4 receptor protein expression allows for prediction of no response or poor response to the combination chemotherapy of the present invention in the patient. A positive response can also refer to the desired outcome of treatment when applied to a patient, ideally complete recovery from the disease, symptom relief, or slowing of disease progression. In the specific case of solid tumors, a positive response preferably refers to a reduction in tumor size or a slowing of tumor growth.

[0029] Information on predicting a patient’s response to anticancer chemotherapy as described in this article allows the attending physician to adjust the treatment strategy for the patient or patient group accordingly (see also below).

[0030] As mentioned above, it has been unexpectedly discovered in the context of this invention that the expression of the P2X4 receptor protein differs significantly between cancer subtypes in patients who are responders or non-responders to anticancer chemotherapy as described herein. Based on this finding, the present invention provides a method for identifying responders to anticancer chemotherapy, comprising the step of detecting the expression of the P2X4 receptor protein in cancer cells in cancer samples obtained from patients treated with anticancer chemotherapy according to the invention as described herein, i.e., comprising administering a combination of: i) an effective amount of at least one P2X4 receptor inhibitor and / or an inhibitor of the P2X4 receptor signaling pathway and ii) an effective amount of at least one chemotherapeutic agent that induces cell death and / or a treatment that induces cell death.

[0031] The detection of P2X4 receptor protein expression in a sample, compared to a control cancer sample, identifies the patient as a responder to the combination chemotherapy. In the context of this invention, a “responder” to anticancer chemotherapy as described herein should refer to a patient who can be treated with anticancer chemotherapy as described herein. Therefore, a “non-responder” should refer to a patient who does not respond to treatment with anticancer chemotherapy as described herein.

[0032] Against the backdrop of the foregoing, the present invention also allows for monitoring the treatment and / or response of patients treated with anticancer chemotherapy as described herein. For this purpose, the method of the present invention is performed on samples obtained from a patient or patient group prior to and / or during treatment with anticancer chemotherapy as described herein. Information regarding cancer cells or tissue, and therefore the patient's response to anticancer chemotherapy as described herein, then allows the attending physician to adjust the treatment strategy for the patient or patient group accordingly (see also below).

[0033] Both the methods for predicting patient response to anticancer chemotherapy as described herein and the methods for identifying responders to anticancer chemotherapy as described herein allow the attending physician to adjust treatment strategies for patients or patient groups. Therefore, in a preferred aspect, the method according to the invention further includes the step of stratifying patients into treatment groups for the application of appropriate anticancer therapy, particularly the step of combination chemotherapy according to the invention, said combination chemotherapy comprising administering a combination of: i) an effective amount of at least one P2X4 receptor inhibitor and / or a P2X4 receptor signaling pathway inhibitor and ii) an effective amount of at least one chemotherapeutic agent that induces cell death and / or a treatment that induces cell death.

[0034] In the context of this invention, if a treatment is identified as positive for a patient, the patient is "stratified" by assigning them to a subgroup treated with anticancer chemotherapy as described herein. In the context of this invention, stratification may also be based on other clinical parameters, such as the sex, age, or stage of the cancer patient. Furthermore, stratification may be performed additionally based on a combination of provided parameters.

[0035] However, in another preferred aspect of the method according to the invention, the anticancer combination chemotherapy is a personalized combination chemotherapy for an individual patient.

[0036] As mentioned above, the method according to the invention includes detecting the expression of the P2X4 receptor protein in cancer cells, cancer tissue samples containing cells, organoids containing cancer cells, or other suitable cancer samples. Generally, any suitable method for detecting P2X4 receptor protein expression can be used; however, current data suggest that transcriptomic analysis appears unsuitable for determining changes in P2X4 expression. To date, only immunohistochemical analysis has allowed for the proper evaluation of P2X4 receptor protein expression as a basis for stratifying potential cancers, responders, and patients suitable for combination therapy.

[0037] Therefore, the method according to the invention is preferred, wherein the determination comprises performing a protein detection method selected from: optical methods, such as UV absorption, bicinchoninic acid (BCA), Bradford assay, fluorescence-based methods, spectroscopic methods, mass spectrometry methods, and antibody-binding-based methods, such as Western blotting. The method according to the invention is also preferred, wherein the detection comprises immunohistochemical analysis of P2X4 expression in cancer cells, tissues, or organoids.

[0038] Particularly preferred methods are those based on P2X4 receptor protein antibody-antigen binding. These methods involve the complete antibody or its antigen-binding fragment, such as the scFv fragment. More preferably, the binding site is suitably labeled, for example with a fluorescent group, isotope, dye, or enzyme.

[0039] In the context of this invention, the term "P2X4 receptor protein" should refer to a human amino acid sequence or a functional fragment thereof having the amino acid sequence according to UniProt accession number Q99571, wherein the amino acid sequence is:

[0040] .

[0041] The term should also include mammalian homologs of human proteins, their splicing variants, and proteins with modifications such as post-translational modifications, such as phosphorylation, ubiquitination, and / or acetalization.

[0042] In a more preferred embodiment of the method according to the invention, the method further includes a step of quantifying the P2X4 receptor protein. Such protein quantification techniques may be selected from, but are not limited to, optical detection methods (e.g., UV absorption BCA or Bradford assay), fluorescence-based methods, spectroscopic methods (e.g., mass spectrometry), and immunohistochemical analysis is preferred.

[0043] In a particularly preferred embodiment of the method according to the invention, the protein detection method includes a high-throughput tissue protein analysis method, preferably selected from organoids and / or tissue microarrays (TMA).

[0044] The methods according to the invention generally relate to the treatment of cancer or neoplastic diseases in mammalian patients. Preferred are cancers or neoplastic diseases characterized by P2X4 receptor protein expression as described herein. Preferably, the cancer according to the method is selected from solid tumors or metastases thereof, said solid tumors or metastases being selected from Lrp5+ cancer stem cell-associated cancers, prostate cancer, pancreatic cancer, breast cancer, gastric cancer, liver cancer, brain cancer, lung cancer, kidney cancer and their metastases, and preferably cancers of epithelial origin, selected from neuroendocrine tumors such as colorectal cancer, pancreatic ductal carcinoma, gastric cancer, gastroesophageal junction adenocarcinoma, lung adenocarcinoma, squamous cell carcinoma of the lung, endometrial cancer, salivary gland cancer and pancreatic adenocarcinoma. In some implementations, the cancer can be, for example, primary or secondary (i.e., metastatic) esophageal cancer, gastroesophageal junction cancer, gastroesophageal adenocarcinoma, gastric cancer, chondrosarcoma, colorectal adenocarcinoma, breast cancer, ovarian cancer, head and neck cancer, melanoma, gastric adenocarcinoma, lung cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, cervical cancer, brain tumor, multiple myeloma, leukemia, lymphoma, prostate cancer, bile duct cancer, endometrial cancer, small intestinal adenocarcinoma, uterine sarcoma, or adrenocortical carcinoma. In some embodiments, the cancer may be, for example, colorectal cancer. In some embodiments, the cancer may be, for example, colorectal adenocarcinoma. In some embodiments, the cancer may be, for example, small intestinal adenocarcinoma. In some embodiments, the cancer may be, for example, hepatocellular carcinoma. In some embodiments, the cancer may be, for example, head and neck cancer. In some embodiments, the cancer may be, for example, renal cell carcinoma. In some embodiments, the cancer may be, for example, ovarian cancer. In some embodiments, the cancer may be, for example, prostate cancer. In some embodiments, the cancer may be, for example, lung cancer. In some embodiments, the cancer may be, for example, uterine sarcoma. In some embodiments, the cancer may be, for example, esophageal cancer. In some embodiments, the cancer may be, for example, endometrial cancer. In some embodiments, the cancer may be, for example, bile duct cancer. In some embodiments, each cancer may be, for example, unresectable, advanced, refractory, recurrent, or metastatic. In a preferred embodiment, the cancer is epithelial in origin. Preferred cancers are solid cancers, especially epithelial-derived solid cancers, particularly hepatocellular carcinoma or colorectal cancer.

[0045] Another aspect of the invention provides the use of a kit comprising a P2X4 receptor protein-binding antibody or a fragment of the P2X4 receptor protein or a derivative thereof, said kit being used according to the method of the invention for identifying cancer cells sensitive to anti-cancer chemotherapy, for predicting patient response to anti-cancer chemotherapy, for identifying responders to anti-cancer chemotherapy, and / or for stratifying patients into treatment groups, and said kit optionally further comprising materials for performing said methods, such as buffers and reagents, and user instructions. Particularly preferred are kits comprising materials for use based on P2X4 receptor protein antibody-antigen binding. These uses and kits relate to the complete antibody or its antigen-binding fragment and portion, such as scFv fragments, etc. More preferably, the binding portion is suitably labeled, for example with a fluorescent group, isotope, dye, or enzyme.

[0046] Then, another important aspect of the invention relates to a method for treating or preventing cancer, the method comprising the steps of: performing the method according to the invention; and, based on the results of the method according to the invention, administering to a patient, as described herein, i) an effective amount of at least one P2X4 receptor inhibitor and / or an inhibitor of the P2X4 receptor signaling pathway and ii) an effective amount of at least one chemotherapeutic agent that induces cell death and / or a combination of treatments that induce cell death.

[0047] As used in this article, the term "treatment" means stabilizing or reducing adverse symptoms associated with the condition; reducing the severity of disease symptoms; slowing the rate of disease progression; inhibiting or stabilizing the progression of the disease condition; or altering measures associated with the disease state in a desired manner. The term "prevention" as used in this article means preventing the occurrence of adverse symptoms associated with the condition or disease before they occur.

[0048] Treatment can be performed in any suitable manner. Preferably, the method according to the invention is used in a manner where the combination is administered concurrently or sequentially. Also preferably, the method according to the invention includes, in which the anticancer combination chemotherapy further comprises prior and / or concomitant anticancer chemotherapy, such as cytotoxic agents, Lgr5 inhibitors, Lgr5+ cancer cell ablation, mTOR inhibitors, and IMPDH inhibitors.

[0049] Regardless of the chosen route of administration, the compounds / pharmaceuticals of the present invention used in the anticancer combination chemotherapy, and / or the pharmaceutical compositions of the present invention, which can be used in a suitable hydrated form, are formulated into pharmaceutically acceptable dosage forms using conventional methods known to those skilled in the art. The actual dose level of the active ingredient in the pharmaceutical composition can be varied to obtain an amount of active ingredient that is non-toxic to the patient and effectively achieves the desired therapeutic response for a particular patient, composition, and route of administration. The chosen dose level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition used in the present invention, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors known in the medical field.

[0050] Dosing regimens will be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any patient depends on many factors, including the patient's body size, body surface area, age, the specific compound / drug to be administered, sex, time and route of administration, general health, and other medications administered concurrently. Typical dosages can be, for example, from 0.001 to 1000 μg; however, dosages below or above this exemplary range can be contemplated, especially taking into account the factors mentioned above. Generally, the routine administration regimen for the pharmaceutical composition should be from 1 μg to 10 mg units / day. If the regimen is continuous infusion, it should also be from 1 μg to 10 mg units / kg body weight / minute. Progress can be monitored through periodic assessments. The compositions of the present invention can be administered topically or systemically. Administration is typically parenteral, such as intravenously; the pharmaceutical composition can also be administered directly to the target site, for example, via biological ballistic delivery to internal or external target sites, or via catheter delivery to a site in an artery. Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Some examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions, including saline and buffer media. Parenteral carriers include sodium chloride solutions, Ringer's dextran, dextran and sodium chloride, lactated Ringer's solution, or non-volatile oils. Intravenous carriers include fluids and nutritional supplements, electrolyte supplements (e.g., those based on Ringer's dextran), etc. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases. Furthermore, depending on the intended use of the pharmaceutical composition, the pharmaceutical compositions used in this invention may contain additional agents, such as interleukins or interferons.

[0051] In the context of this invention, P2X4 inhibitors or P2X4 receptor inhibitors should refer to inhibitors of the P2X4 receptor protein itself or inhibitors of the P2X4 receptor signaling pathway. A P2X4 inhibitor should be understood as any compound that inhibits the biological function of the P2X4 receptor upon contact with cells, particularly its purinergic signaling in the context of tumors or tumor environments. A considerable number of compounds have been described as having activity as P2X4 receptor inhibitors and / or P2X4 receptor signaling pathway inhibitors, and such compounds known to those skilled in the art should be covered in this invention.

[0052] Generally, any suitable at least one P2X4 receptor inhibitor and / or P2X4 receptor signaling pathway inhibitor can be used. Preferably, the method according to the invention is used in which the P2X4 receptor inhibitor is a specific inhibitor of the P2X4 receptor, and is preferably selected from PPADS, suramin, KN-62, TNP-ATP, brilliant blue G, 5-BDBD, BX-430, carbamazepine derivatives, PSB-12054, PSB-12062, PSB-15417, NP-1815-PX, NC-2600, UoS14919, paroxetine, duloxetine, BAY-1797, BAY-2328065, IgG#151-LO, anti-P2X4 receptor protein antibody or inhibitor binding fragment or derivative thereof, siRNA, shRNA, miRNA, ribozymes, aptamers, antisense nucleic acid molecules, and small molecules less than about 500 Da, preferably 5DBDB, and even more preferably BAY-2328065.

[0053] Generally, the method according to the invention may involve any suitable chemotherapeutic agent that induces cell death and / or treatment that induces cell death, provided that it can be combined with the above inhibitors of P2X4 receptor inhibitors that are components of the P2X4 receptor signaling pathway (provided together, in combination or alone, as long as they work together in the subject). Also preferred is the method according to the invention, wherein the chemotherapeutic agent inducing cell death and / or the treatment inducing cell death are selected from necrotic cell supernatant; cationic amphiphilic drugs (CAD); classical anticancer agents, including anthracyclines, antimetabolites, and platinum drugs; topoisomerase inhibitors, such as camptothecin; PDE3A inhibitors, such as anagrelide, alone or together with cell-death cytokines; cell-death cytokines, such as IFN-α, IFN-γ, TNF-α, or TRAIL; taxanes, paclitaxel, and fluorinated taxanes, such as SB-T-12851, SB-T-12852, SB-T-12853, SB-T-12854, 5-fluorouracil, tegafur, capecitabine, and deoxyfluorouracil; and preferably 5-fluorouracil (5-FU).

[0054] Preferably, according to the method of the invention, the combination is administered to the patient simultaneously or separately as a combined and / or individual dosage form.

[0055] Preferably, the method according to the invention includes the combination chemotherapy comprising inhibiting ATP-dependent P2X4 receptor and / or P2X4 receptor signaling pathway-mediated tumor escape mechanisms.

[0056] Preferred is the method according to the invention for combining a P2X4 receptor inhibitor or a P2X4 receptor signaling pathway inhibitor with a chemotherapeutic agent and / or a treatment for inducing cell death, wherein the treatment and / or prevention further includes prior and / or concomitant anticancer chemotherapy, such as cytotoxins, Lgr5 inhibitors, Lgr5+ cancer cell ablation, mTOR inhibitors, and IMPDH inhibitors. Generally, any suitable anticancer chemotherapy can be used in the context of this invention, provided that it can be combined with the above combinations of the invention (provided together, in combination, or alone, as long as they work together in the subject), which effectively blocks the cancer escape mechanisms disclosed herein and thereby blocks further tumor growth and / or the formation or recurrence of metastasis. Preferred is the method according to the invention for treatment comprising a combination of a P2X4 receptor inhibitor or a P2X4 receptor signaling pathway inhibitor with a fluoropyrimidine agent, wherein the combination is provided simultaneously or separately as a combined and / or separate dosage form.

[0057] The methods according to the invention generally relate to the treatment of cancer or neoplastic diseases in mammalian patients. Preferred are cancers or neoplastic diseases characterized by P2X4 receptor protein expression as described herein. Preferably, the cancer according to the method is selected from solid tumors or metastases thereof, said solid tumors or metastases being selected from Lrp5+ cancer stem cell-associated cancers, prostate cancer, pancreatic cancer, breast cancer, gastric cancer, liver cancer, brain cancer, lung cancer, kidney cancer and their metastases, and preferably cancers of epithelial origin, selected from neuroendocrine tumors such as colorectal cancer, pancreatic ductal carcinoma, gastric cancer, gastroesophageal junction adenocarcinoma, lung adenocarcinoma, squamous cell carcinoma of the lung, endometrial cancer, salivary gland cancer and pancreatic adenocarcinoma. In some implementations, the cancer can be, for example, primary or secondary (i.e., metastatic) esophageal cancer, gastroesophageal junction cancer, gastroesophageal adenocarcinoma, gastric cancer, chondrosarcoma, colorectal adenocarcinoma, breast cancer, ovarian cancer, head and neck cancer, melanoma, gastric adenocarcinoma, lung cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, cervical cancer, brain tumor, multiple myeloma, leukemia, lymphoma, prostate cancer, bile duct cancer, endometrial cancer, small intestinal adenocarcinoma, uterine sarcoma, or adrenocortical carcinoma. In some embodiments, the cancer may be, for example, colorectal cancer. In some embodiments, the cancer may be, for example, colorectal adenocarcinoma. In some embodiments, the cancer may be, for example, small intestinal adenocarcinoma. In some embodiments, the cancer may be, for example, hepatocellular carcinoma. In some embodiments, the cancer may be, for example, head and neck cancer. In some embodiments, the cancer may be, for example, renal cell carcinoma. In some embodiments, the cancer may be, for example, ovarian cancer. In some embodiments, the cancer may be, for example, prostate cancer. In some embodiments, the cancer may be, for example, lung cancer. In some embodiments, the cancer may be, for example, uterine sarcoma. In some embodiments, the cancer may be, for example, esophageal cancer. In some embodiments, the cancer may be, for example, endometrial cancer. In some embodiments, the cancer may be, for example, bile duct cancer. In some embodiments, each cancer may be, for example, unresectable, advanced, refractory, recurrent, or metastatic. In a preferred embodiment, the cancer is epithelial in origin. Preferred cancers are solid cancers, especially epithelial-derived solid cancers, particularly hepatocellular carcinoma or colorectal cancer.

[0058] In one embodiment, the solid tumor or its metastasis is selected from Lrp5+ cancer stem cell-related cancers, prostate cancer, pancreatic cancer, breast cancer, gastric cancer, liver cancer, brain cancer, lung cancer, kidney cancer, and their metastases. Even more preferably, the solid tumor or its metastasis is a cancer of epithelial origin, such as colorectal cancer, pancreatic ductal carcinoma, gastric cancer, gastroesophageal junction adenocarcinoma, lung adenocarcinoma, squamous cell carcinoma of the lung, endometrial cancer, salivary gland cancer, and neuroendocrine tumors.

[0059] This invention relates to the following items.

[0060] Project 1. A method for identifying cancer cells sensitive to anticancer chemotherapy, said anticancer chemotherapy comprising administering a combination of: i) an effective amount of at least one P2X4 receptor inhibitor and / or an inhibitor of the P2X4 receptor signaling pathway and ii) an effective amount of at least one chemotherapeutic agent that induces cell death and / or a treatment that induces cell death, said method comprising the step of detecting P2X4 receptor protein expression in cancer cells, wherein the detection of P2X4 receptor protein expression in said cancer cells identifies said cancer cells as sensitive to said combination chemotherapy when compared with cancer control cells that do not express P2X4 receptor protein.

[0061] Project 2. A method for predicting a patient's response to anticancer chemotherapy, said anticancer chemotherapy comprising administering a combination of: i) an effective amount of at least one P2X4 receptor inhibitor and / or an inhibitor of the P2X4 receptor signaling pathway and ii) an effective amount of at least one chemotherapeutic agent that induces cell death and / or a treatment that induces cell death, said method comprising the step of detecting the expression of P2X4 receptor protein in cancer cells from a cancer sample from said patient, wherein the detection of P2X4 receptor protein expression in said sample predicts a positive response of said patient to said combination chemotherapy when compared with cancer control cells that do not express P2X4 receptor protein.

[0062] Project 3. A method for identifying responders to anticancer chemotherapy, comprising the step of detecting the expression of a P2X4 receptor protein in cancer cells in a cancer sample obtained from a patient treated with anticancer chemotherapy, said anticancer chemotherapy comprising administering a combination of: i) an effective amount of at least one P2X4 receptor inhibitor and / or an inhibitor of the P2X4 receptor signaling pathway and ii) an effective amount of at least one chemotherapeutic agent that induces cell death and / or a treatment that induces cell death, wherein the detection of P2X4 receptor protein expression in the sample, when compared with a control cancer sample, identifies the patient as a responder to the combination chemotherapy.

[0063] Project 4. The method according to Project 2 or 3 further includes the step of stratifying the patient into treatment groups for the application of appropriate anticancer therapy, particularly combination chemotherapy, said combination chemotherapy comprising the administration of a combination of: i) an effective amount of at least one P2X4 receptor inhibitor and / or a P2X4 receptor signaling pathway inhibitor and ii) an effective amount of at least one chemotherapeutic agent that induces cell death and / or a treatment that induces cell death.

[0064] Item 5. The method according to any one of items 1 to 4, wherein the application of said combination is accompanied or sequential.

[0065] Project 6. The method according to any one of Projects 1 to 5, wherein the detection of P2X4 receptor protein expression comprises: determining the protein expression of the P2X4 receptor protein in the cancer cells, cancer tissue samples containing the cells, organoids or other suitable cancer samples containing the cancer cells.

[0066] Item 7. The method according to Item 6, wherein the determination includes performing a protein detection method selected from: optical methods, such as UV absorption, quinoline carboxylic acid (BCA), Bradford assay, fluorescence-based methods, spectroscopic methods, mass spectrometry methods, and antibody-binding-based methods, such as Western blotting.

[0067] Item 8. The method according to Item 6 or 7, wherein the detection includes immunohistochemical analysis of P2X4 expression in tissues or organoids.

[0068] Project 9. The method according to any one of Projects 6 to 8, further comprising the step of quantifying the P2X4 receptor protein.

[0069] Item 10. The method according to any one of Items 6 to 9, wherein the method is a high-throughput method, preferably including a tissue microarray (TMA).

[0070] Item 11. The method according to any one of Items 1 to 10, wherein the cancer is selected from solid tumors or metastases thereof, the solid tumors or metastases thereof being selected from Lrp5+ cancer stem cell-related cancers, prostate cancer, pancreatic cancer, breast cancer, gastric cancer, liver cancer, brain cancer, lung cancer, kidney cancer and their metastases, and preferably cancers of epithelial origin, and neuroendocrine tumors selected from colorectal cancer, pancreatic ductal carcinoma, gastric cancer, gastroesophageal junction adenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, endometrial cancer, salivary gland cancer and pancreatic adenocarcinoma.

[0071] Item 12. The method according to any one of Items 1 to 11, wherein the inhibitor of the P2X4 receptor is a specific inhibitor of the P2X4 receptor, and is preferably selected from PPADS, suramin, KN-62, TNP-ATP, brilliant blue G, 5-BDBD, BX-430, carbamazepine derivatives, PSB-12054, PSB-12062, PSB-15417, NP-1815-PX, NC-2600, UoS14919, paroxetine, duloxetine, BAY-1797, BAY-2328065, IgG#151-LO, antibody or inhibitor binding fragments or derivatives thereof, siRNA, shRNA, miRNA, ribozymes, aptamers, antisense nucleic acid molecules, and small molecules less than about 500 Da, preferably 5DBDB, and even more preferably BAY-2328065.

[0072] Item 13. The method according to any one of Items 1 to 12, wherein the chemotherapeutic agent inducing cell death is selected from necrotic cell supernatant; cationic amphiphilic drugs (CAD); classical anticancer agents, including anthracyclines, antimetabolites, and platinum drugs; topoisomerase inhibitors, such as camptothecin; PDE3A inhibitors, such as anagrelide, alone or together with cell-death cytokines; cell-death cytokines, such as IFN-α, IFN-γ, TNF-α, or TRAIL; taxanes, paclitaxel, and fluorinated taxanes, such as SB-T-12851, SB-T-12852, SB-T-12853, SB-T-12854, 5-fluorouracil, tegafur, capecitabine, and deoxyfluorouracil; and preferably 5-fluorouracil (5-FU).

[0073] Project 14. The method according to any one of Projects 1 to 13, wherein the anticancer combination chemotherapy further comprises prior and / or concomitant anticancer chemotherapy, said anticancer chemotherapy comprising, for example, cytotoxins, Lgr5 inhibitors, Lgr5+ cancer cell ablation, mTOR inhibitors, and IMPDH inhibitors.

[0074] Item 15. The method according to any one of Items 1 to 14, wherein the anticancer combination chemotherapy is a personalized combination chemotherapy for the patient.

[0075] Item 16. Use of a kit comprising a P2X4 receptor protein binding antibody or a P2X4 receptor protein fragment or a derivative thereof, said kit being used according to any one of Items 1 to 15 for identifying cancer cells sensitive to anti-cancer chemotherapy, for predicting patient response to anti-cancer chemotherapy, for identifying responders to anti-cancer chemotherapy, and / or for stratifying patients into treatment groups, and said kit optionally further comprising materials for performing said methods, such as buffers and reagents, and user instructions.

[0076] Item 17. A method for treating cancer, comprising the steps of: performing the method according to any one of Items 1 to 15; and, based on the results of the method according to any one of Items 1 to 15, administering to the patient i) an effective amount of at least one P2X4 receptor inhibitor and / or an inhibitor of the P2X4 receptor signaling pathway and ii) an effective amount of at least one chemotherapeutic agent that induces cell death and / or a combination of cell death-inducing treatments.

[0077] Item 18. The method according to Item 17, wherein the combination is administered to the patient simultaneously or separately as a combined and / or individual dosage form.

[0078] Item 19. The method according to Item 17 or 18, wherein the combination chemotherapy includes inhibition of ATP-dependent P2X4 receptor and / or P2X4 receptor signaling pathway-mediated tumor escape mechanisms.

[0079] Item 20. The method according to any one of Items 17 to 19, wherein the cancer is selected from solid tumors or metastases thereof, the solid tumors or metastases thereof being selected from Lrp5+ cancer stem cell-related cancers, prostate cancer, pancreatic cancer, breast cancer, gastric cancer, liver cancer, brain cancer, lung cancer, kidney cancer and their metastases, and preferably cancers of epithelial origin, and neuroendocrine tumors selected from colorectal cancer, pancreatic ductal carcinoma, gastric cancer, gastroesophageal junction adenocarcinoma, lung adenocarcinoma, squamous cell carcinoma of the lung, endometrial cancer, salivary gland cancer and pancreatic adenocarcinoma.

[0080] Project 21. The method according to any one of Projects 17 to 20, wherein the anticancer combination chemotherapy further comprises prior and / or concomitant anticancer chemotherapy, said anticancer chemotherapy comprising, for example, cytotoxins, Lgr5 inhibitors, Lgr5+ cancer cell ablation, mTOR inhibitors, and IMPDH inhibitors.

[0081] The invention will now be further described with reference to the accompanying drawings and in the following embodiments, but is not limited thereto. For the purposes of this invention, all references cited herein are incorporated herein by reference in their entirety.

[0082] Figure 1The analysis of P2RX4 among the four common molecular subtypes of CRC according to GSE39582 (TCGA) is shown: CMS 1 (n=72), CMS2 (n=162), CMS3 (n=73), and CMS4 (n=127).

[0083] Figure 2 An overview of the immunohistochemical analysis of P2X4 expression in various tumor entities is shown. Expression scores are 0, +, ++, or +++.

[0084] Figure 3 Immunoblot analysis of P2X4 in SU-DHL5 lymphoma cells and three human colorectal cancer organoids is shown (A). SU-DHL5 cells survive in response to doxorubicin in the absence or presence of 5-BDBD (B). Human CRC tumor organoids survive in response to 5-FU in the absence or presence of 5-BDBD (C) and (D). Example

[0085] Transcriptomic analysis of P2RX4 gene

[0086] Based on previous data from the inventors, which showed that chemotherapy-induced cell death in colorectal and pancreatic cancer induces P2X4 / mTOR-dependent vulnerability in adjacent epithelial cells, making these tumors sensitive to combination therapy (chemotherapy plus P2X4 targeting; Schmitt et al., Nature, 2022), the inventors now evaluated whether changes in the P2RX4 gene might be associated with specific molecular subtypes of colorectal cancer. However, the differences in P2RX4 gene expression among different colorectal cancer subtypes were not significant. Figure 1 ).

[0087] Immunohistochemical analysis of P2X4 protein levels

[0088] The inventors then examined whether P2X4 expression in colorectal cancer could differ at the protein level, and extended the analysis to other tumor entities derived from epithelial cells known to express P2X4 under steady-state conditions.

[0089] To this end, the inventors used tissue microarrays (TMA) of colon cancer (n=278), pancreatic ductal carcinoma (n=100), gastric cancer (n=71 tumors), gastroesophageal junction adenocarcinoma (n=37), lung adenocarcinoma (n=93), lung squamous cell carcinoma (n=183), endometrial cancer (n=85), salivary gland carcinoma (n=28), and pancreatic neuroendocrine tumor (n=48) for immunohistochemical analysis of P2X4. Among all the tumor entities analyzed, the inventors found highly heterogeneous P2X4 expression patterns, ranging from complete absence of expression to strong expression (…). Figure 2 ).

[0090] Differences in P2X4 expression are likely associated with better or worse prognosis. In summary, current data suggest that transcriptomic analysis appears unsuitable for identifying changes in P2X4 expression. To date, only immunohistochemical analysis has allowed for the proper evaluation of potential cancer, responders, and patient stratification for combination therapies.

[0091] Immunoblot analysis shows that only P2X4-expressing tumor cells are sensitive to the combination treatment

[0092] To definitively demonstrate that only tumor cells expressing P2X4 are actually sensitive to combination therapy, the inventors compared the responses to 5-FU+ / -P2X4 inhibitors in colorectal cancer organoids expressing or not expressing P2X4. The inventors had previously shown that blocking P2X4 sensitizes organoids to 5-FU treatment (Schmitt et al., Nature, 2022). The inventors have now determined that these organoids do indeed express P2X4 (… Figure 3 A, CRC-organoid #1). In stark contrast, no sensitization to P2X4 inhibition was observed in colorectal cancer organoids and lymphoma cells that do not express the P2X4 protein. Figure 3 (B, C, D). Despite significant cell death induced by chemotherapy, this occurred, while additional P2X4 inhibition had no effect. Overall, the data clearly demonstrate that combination therapy (chemotherapy plus a P2X4 inhibitor) will only benefit patients with tumors exhibiting tumor cell-specific P2X4 expression, and such patients can be identified by immunohistochemical analysis of P2X4 in primary tumor tissue and / or metastases.

Claims

1. A method for identifying cancer cells sensitive to anticancer chemotherapy, said anticancer chemotherapy comprising administering a combination of: i) an effective amount of at least one P2X4 receptor inhibitor and / or an inhibitor of the P2X4 receptor signaling pathway and ii) an effective amount of at least one chemotherapeutic agent that induces cell death and / or a treatment that induces cell death, said method comprising the step of detecting P2X4 receptor protein expression in cancer cells, wherein the detection of P2X4 receptor protein expression in said cancer cells identifies said cancer cells as sensitive to said combination chemotherapy when compared with cancer control cells that do not express P2X4 receptor protein.

2. A method for predicting a patient's response to anticancer chemotherapy, said anticancer chemotherapy comprising administering a combination of: i) an effective amount of at least one P2X4 receptor inhibitor and / or an inhibitor of the P2X4 receptor signaling pathway and ii) an effective amount of at least one chemotherapeutic agent that induces cell death and / or a treatment that induces cell death, said method comprising the step of detecting P2X4 receptor protein expression in cancer cells from a cancer sample from said patient, wherein the detection of P2X4 receptor protein expression in said sample predicts a positive response of said patient to said combination chemotherapy when compared with cancer control cells that do not express P2X4 receptor protein, said method preferably further comprising the step of stratifying said patient into treatment groups for the application of appropriate anticancer treatment, particularly combination chemotherapy, said combination chemotherapy comprising administering a combination of: i) an effective amount of at least one P2X4 receptor inhibitor and / or an inhibitor of the P2X4 receptor signaling pathway and ii) an effective amount of at least one chemotherapeutic agent that induces cell death and / or a treatment that induces cell death.

3. A method for identifying responders to anticancer chemotherapy, comprising the step of detecting the expression of a P2X4 receptor protein in cancer cells in a cancer sample obtained from a patient treated with anticancer chemotherapy, said anticancer chemotherapy comprising administering a combination of: i) an effective amount of at least one P2X4 receptor inhibitor and / or an inhibitor of the P2X4 receptor signaling pathway and ii) an effective amount of at least one chemotherapeutic agent that induces cell death and / or a treatment that induces cell death, wherein the detection of P2X4 receptor protein expression in the sample, when compared with a control cancer sample, identifies the patient as a responder to the combination chemotherapy, the method preferably further comprising the step of stratifying the patient into treatment groups for the application of appropriate anticancer treatment, particularly combination chemotherapy, said combination chemotherapy comprising administering a combination of: i) an effective amount of at least one P2X4 receptor inhibitor and / or an inhibitor of the P2X4 receptor signaling pathway and ii) an effective amount of at least one chemotherapeutic agent that induces cell death and / or a treatment that induces cell death.

4. The method according to any one of claims 1 to 3, wherein the application of said combination is accompanied or sequential.

5. The method according to any one of claims 1 to 4, wherein the detection of P2X4 receptor protein expression comprises: Determine the protein expression of the P2X4 receptor protein in the cancer cells, cancer tissue samples containing the cells, organoids containing the cancer cells, or other suitable cancer samples, wherein preferably the determination includes performing a protein detection method selected from: optical methods, such as UV absorption, quinolinic acid (BCA), Bradford assay, fluorescence-based methods, spectroscopic methods, mass spectrometry methods, and antibody-based methods, such as Western blotting.

6. The method according to claim 4 or 5, wherein the detection comprises immunohistochemical analysis of P2X4 expression in tissues or organoids.

7. The method according to any one of claims 4 to 6, further comprising the step of quantifying the P2X4 receptor protein.

8. The method according to any one of claims 4 to 7, wherein the method is a high-throughput method, preferably comprising a tissue microarray (TMA).

9. The method according to any one of claims 1 to 8, wherein the cancer is selected from solid tumors or metastases thereof, the solid tumors or metastases thereof are selected from Lrp5+ cancer stem cell-related cancers, prostate cancer, pancreatic cancer, breast cancer, gastric cancer, liver cancer, brain cancer, lung cancer, kidney cancer and their metastases, and preferably cancers of epithelial origin, and neuroendocrine tumors selected from colorectal cancer, pancreatic ductal carcinoma, gastric cancer, gastroesophageal junction adenocarcinoma, lung adenocarcinoma, squamous cell carcinoma of the lung, endometrial cancer, salivary gland cancer and pancreatic adenocarcinoma.

10. The method according to any one of claims 1 to 9, wherein the P2X4 receptor inhibitor is a specific inhibitor of the P2X4 receptor, and is preferably selected from PPADS, suramin, KN-62, TNP-ATP, Brilliant Blue G, 5-BDBD, BX-430, carbamazepine derivatives, PSB-12054, PSB-12062, PSB-15417, NP-1815-PX, NC-2600, UoS14919, paroxetine, duloxetine, BAY-1797, BAY-2328065, IgG#151-LO, antibody or inhibitor binding fragments or derivatives thereof, siRNA, shRNA, miRNA, ribozymes, aptamers, antisense nucleic acid molecules, and less than about 500 Small molecules of Da, preferably 5-DBDB, and even more preferably BAY-2328065; and / or the chemotherapeutic agent that induces cell death is selected from necrotic cell supernatant; cationic amphiphilic drugs (CAD); classical anticancer agents, including anthracyclines, antimetabolites, and platinum drugs; topoisomerase inhibitors, such as camptothecin; PDE3A inhibitors, such as anagrelide, alone or with cell-death cytokines; cell-death cytokines, such as IFN-α, IFN-γ, TNF-α, or TRAIL; taxanes, paclitaxel, and fluorinated taxanes, such as SB-T-12851, SB-T-12852, SB-T-12853, SB-T-12854, 5-fluorouracil, tegafur, capecitabine, and deoxyfluorouracil; and preferably 5-fluorouracil (5-FU).

11. The method according to any one of claims 1 to 10, wherein the anticancer combination chemotherapy further comprises prior and / or concomitant anticancer chemotherapy, said anticancer chemotherapy comprising, for example, cytotoxins, Lgr5 inhibitors, Lgr5+ cancer cell ablation, mTOR inhibitors, and IMPDH inhibitors.

12. The method according to any one of claims 1 to 11, wherein the anticancer combination chemotherapy is a personalized combination chemotherapy for the patient.

13. Use of a kit comprising a P2X4 receptor protein binding antibody or a P2X4 receptor protein fragment or a derivative thereof, said kit being used according to any one of claims 1 to 12 for identifying cancer cells sensitive to anti-cancer chemotherapy, for predicting patient response to anti-cancer chemotherapy, for identifying responders to anti-cancer chemotherapy, and / or for stratifying patients into treatment groups, and said kit optionally further comprising materials for performing said methods, such as buffers and reagents, and user instructions.

14. A method for treating cancer, comprising the following steps: Perform the method according to any one of claims 1 to 12; And based on the results of the method according to any one of claims 1 to 12, administering to the patient i) an effective amount of at least one P2X4 receptor inhibitor and / or a P2X4 receptor signaling pathway inhibitor and ii) an effective amount of at least one cell-death chemotherapeutic agent and / or a combination of cell-death induced treatments, wherein preferably the combination is administered to the patient simultaneously or separately as a combined and / or individual dosage form, and / or wherein the combined chemotherapy includes inhibition of ATP-dependent P2X4 receptor and / or P2X4 receptor signaling pathway-mediated tumor escape mechanisms.

15. The method of claim 14, wherein the anticancer combination chemotherapy further comprises prior and / or concomitant anticancer chemotherapy, said anticancer chemotherapy comprising, for example, cytotoxins, Lgr5 inhibitors, Lgr5+ cancer cell ablation, mTOR inhibitors, and IMPDH inhibitors.

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