Combination Therapies Comprising Inhibitors of the P2X4 Receptor for the Treatment and Prevention of Proliferative Diseases - Patent application

Abstract

The present invention relates to the use of an inhibitor of the P2X4 receptor or an inhibitor of the P2X4 receptor signaling pathway in combination with an effective amount of a "cell death-inducing chemotherapeutic agent" and / or a "cell death-inducing therapy" in the prevention and / or treatment of a solid tumor (in particular colorectal cancer (CRC)) or metastasis thereof in a subject.

Description

[Technical field]

[0001] The present invention relates to the use of an inhibitor of the P2X4 receptor or an inhibitor of the P2X4 receptor signaling pathway in combination with an effective amount of a cell death-inducing chemotherapeutic agent and / or a cell death-inducing therapy in the prevention and / or treatment of a solid tumor (in particular colorectal cancer (CRC)) or metastasis thereof in a subject. [Background technology]

[0002] Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is an intestinal stem cell marker. LGR5 is also a candidate marker for colorectal cancer (CRC) stem cells, and may be closely related to the progression and prognosis of CRC, one of the most common cancers worldwide (Non-Patent Document 1).

[0003] Lgr5+ cells have been described as the cells of origin and cancer stem cells in colorectal cancer (CRC). Silencing of LGR5 reduced proliferation, migration, and colony formation in vitro, and reduced tumorigenicity in vivo (Non-Patent Document 2). Unfortunately, transient deletion of Lgr5+ cells in subcutaneous CRC tumors caused tumor stagnation only for the days when Lgr5+ cells were absent, after which tumor growth continued rapidly once the Lgr5+ cell pool was re-established (Non-Patent Document 3).

[0004] Non-Patent Document 4 describes that LGR5 is an interesting therapeutic target due to the well-known dysregulation of Wnt / β-catenin signaling that occurs during the adenoma-carcinoma sequence in colorectal cancer (CRC). Furthermore, recent studies have demonstrated that CRC tumors contain an LGR5+ subset and retain some degree of normal tissue architecture, raising translational interest. Such reports have raised hopes that specific subpopulations or molecules within tumors may be therapeutic targets to prevent recurrence and induce long-term remission.

[0005] Non-Patent Document 5 discloses that an increase in Lgr5 level is associated with the progression and treatment response of CRC and may serve as a therapeutic target for CRC patients.

[0006] The mammalian target of rapamycin (mTOR) is a serine-threonine kinase that controls cell growth and proliferation in response to the availability of growth factors and nutrients. Treatment with rapamycin significantly reduced the proliferation of certain CRC cell lines (rapamycin-sensitive), whereas other cell lines were resistant to its effects (rapamycin-resistant). Studies have shown that everolimus has limited clinical activity in the treatment of advanced colorectal cancer, and the situation is complicated by increased toxicity. Because the PI3K / mTOR pathway plays a central role in cancer biology, the combination of other agents with mTOR inhibition has been proposed (Non-Patent Document 6).

[0007] Regarding mTOR, several attempts at therapeutic combination approaches are known. Non-Patent Document 7 describes that in vivo, the combination of irinotecan and AZD2014 dramatically reduced the growth of ectopic patient-derived colon tumors, and this combination was more potent than Folfox and Folfiri. Finally, this combination completely inhibited liver and lung metastases that arose from orthotopic transplantation of SW480 cells. Therefore, the combination of mTOR catalytic inhibitors with other chemotherapeutic agents such as irinotecan at low doses may be promising for colon cancer treatment.

[0008] Non-Patent Document 8 reports a phase I study investigating safety, dose-limiting toxicity, and efficacy in three cohorts treated with the mTOR inhibitor everolimus delivered in (1) a combination of 5-fluorouracil and leucovorin (5-FU / LV), (2) mFOLFOX6 (5-FU / LV + oxaliplatin), and (3) mFOLFOX6 + panitumumab in patients with refractory solid tumors. Among 24 patients enrolled with refractory metastatic colorectal cancer, the median treatment duration was 2.7 months, and 45% of patients remained on treatment with stable disease for at least 3 months. They found that the regimen of everolimus in addition to 5-FU / LV and mFOLFOX6 appeared safe and tolerable, but the further addition of panitumumab produced unacceptable levels of toxicity and further study could not be recommended. Further studies are needed to better elucidate the role of mTOR inhibitors in patients with refractory solid tumors, with future studies focusing specifically on mCRC as a potential combination of this targeted therapy with cytotoxic therapy.

[0009] Non-Patent Document 9 describes that S-allylmercaptocysteine ​​(SAMC), a natural plant-derived product, has been studied in cancer treatment as a single or combination chemotherapeutic agent. The combination of SAMC with rapamycin, an mTOR inhibitor with limited efficacy anticancer potential due to drug resistance, was studied to explore the underlying mechanisms. They combined rapamycin with SAMC for colorectal cancer treatment in HCT-116 cancer cells and xenograft mouse models. In vivo studies were established by xenografting HCT-116 cells in BALB / c nude mice. The combination therapy was found to enhance tumor suppression capacity by upregulating Bax / Bcl-2 ratio as a result of activating apoptosis, inhibiting autophagy activity, and preventing Akt phosphorylation. The combination of SAMC and rapamycin enhanced anticancer potential and could be used to treat colorectal cancer. The discovered underlying mechanism of the autophagy / p62 / Nrf2 pathway may provide new directions for drug development, especially for traditional Chinese medicine development.

[0010] Non-patent literature 10 provides the antitumor effect of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) signaling, in combination with 5-fluorouracil treatment on CT-26 colorectal adenocarcinoma cells transplanted into BALB / c mice. The combination of rapamycin and 5-fluorouracil significantly reduced tumor size, suppressed the expression of B-cell lymphoma 2, increased tumor apoptosis, and inhibited mTOR signaling activity by dephosphorylation of S6K. The combination of rapamycin and 5-fluorouracil treatment had a synergistic tumor-inhibiting effect. It is said that future research on rapamycin is necessary to develop new therapeutic strategies.

[0011] Similarly, in Non-Patent Document 11, rapamycin liposomes were prepared using the ethanol injection method. The cellular uptake and biodistribution were detected by LC-MS and an in vivo imaging system. The antitumor effects of rapamycin liposomes in vitro were evaluated by MTT assay, transwell migration experiment, flow cytometry, and Western blot analysis. Furthermore, the therapeutic effects of rapamycin liposomes in vivo were evaluated using HCT-116 tumor-bearing mice. They demonstrated the excellent antitumor effects of rapamycin liposomes in HCT-116 xenograft mice in vivo. Furthermore, it was disclosed that rapamycin liposomes and 5-FU synergistically improved the efficacy of colorectal cancer via the Akt / mTOR and P53 pathways.

[0012] P2X receptors (P2XRs) are a family of ATP-gated ion channels expressed in many excitable and non-excitable cells. Although significant progress has been made in understanding the structure and function of these receptors over the past few decades, there is still a lack of understanding of specific and potent antagonists for P2XR subtypes, particularly P2X4R. 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) has been found to be a specific P2X4R antagonist that acts on ATP-induced currents (see, for example, Non-Patent Document 12).

[0013] Patent Document 1 discloses a method of inhibiting cancer cells by exposing the cancer cells to a purinergic receptor antagonist that targets one or more purinergic receptors of the cancer cells. The targeted purinergic receptors may include P2 purinergic receptors, such as P2X purinergic receptor subtypes (e.g., P2X3, P2X4, or P2X5). In some embodiments, the inhibited cancer cells are associated with hepatocellular carcinoma.

[0014] Patent Document 2 relates to an inhibitor of P2Y2 receptor or an inhibitor of P2Y2 receptor signaling pathway for use in preventing tumor metastasis or for use as a lead compound for developing a drug for preventing tumor metastasis. Figure 4 shows the effect of P2X4 receptor antagonist 5-BDBD (1 μM) on transendothelial migration of B16 cells stimulated by platelets.

[0015] Non-Patent Document 13 describes the effect of P2X4R inhibition on the viability, proliferation, migration, invasion, and apoptosis of prostate cancer (PCa) (PC3 and C4-2B4 cells) using selective P2X4R antagonists 5-BDBD and PSB-12062. The results demonstrated that inhibiting P2X4R impairs PCa cell proliferation and migration, but not apoptosis. They suggest that P2X4R has a role in promoting PCa tumorigenesis and is a clinically targetable candidate, whose inhibitors are already available and may suppress disease progression.

[0016] Non-Patent Document 14 outlines the purinergic system, a signal transduction pathway formed by nucleotides / nucleosides (mainly adenosine triphosphate (ATP), adenosine (ADO), and uridine triphosphate (UTP)), with their corresponding membrane receptors and defined transduction mechanisms. The dynamic equilibrium between ATP and ADO, achieved by the presence and control of a series of ectonucleotidases, defines the final outline of pro- or anti-tumor properties in tumors and cancer cell lines. So far, the purinergic system has been recognized as a potential therapeutic target for cancerous and neoplastic diseases.

[0017] Finally, Non-Patent Document 15 discloses that one of the most potent immunosuppressive factors is adenosine, which is generated in the tumor microenvironment by the degradation of extracellular ATP. Evidence accumulated over the past few years has shown that ATP is one of the main biochemical components of the tumor microenvironment and acts on P2 purinergic receptors expressed on both tumor and host cells. Stimulation of P2 receptors leads to various effects depending on the extracellular ATP concentration, the subtype of P2 receptor involved, and the type of target cell. Among the P2 receptors, the P2X purinergic receptor 7 (P2X7R) subtype is thought to play a major role in host-tumor cell interactions. Preclinical studies in several tumor models have shown that P2X7R targeting is potentially a highly effective anticancer therapy, and many pharmaceutical companies are currently developing potent and selective P2X7R small molecule inhibitors. In this review, we report multiple mechanisms by which extracellular ATP shapes the tumor microenvironment and how ATP stimulation of host and tumor cell P2 receptors contributes to tumor fate decisions. [Prior art documents] [Patent documents]

[0018] [Patent Document 1] WO 2017 / 070660A1 [Patent Document 2] WO 2013 / 139940 [Non-patent literature]

[0019] [Non-Patent Document 1] Wu XS, Xi HQ, Chen L. Lgr5 is a potential marker of colorectal carcinoma stem cells that correlates with patientsurvival. World J Surg Oncol. 2012;10:244 [Non-Patent Document 2] Hirsch D, Barker N, McNeil N, Hu Y, Camps J,McKinnon K, Clevers H, Ried T, Gaiser T. LGR5 positivity defines stem-like cells in colorectal cancer. Carcinogenesis. 2014 Apr;35(4):849-58. doi:10.1093 / carcin / bgt377. Epub 2013 Nov 26

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[0020] It is an object of the present invention to provide new and more effective combination therapeutic approaches for the prevention and / or treatment of cancerous diseases, particularly solid tumors (e.g., colorectal cancer) and their metastases. Other objects and advantages will be apparent to those skilled in the art upon consideration of the description of the present invention. [Means for solving the problem]

[0021] Generally, briefly described, the main aspects of the present invention can be described as follows:

[0022] In a first aspect of the present invention, the above object is solved by providing an effective amount of a P2X4 receptor inhibitor or an inhibitor of the P2X4 receptor signaling pathway for use in the prevention and / or treatment of a solid tumor or its metastasis in a subject, in combination with an effective amount of a cell death-inducing chemotherapeutic agent and / or a cell death-inducing therapy.Thus, preferably, said treatment and / or prevention comprises the inhibition of the tumor escape mechanism mediated by the ATP-dependent P2X4 receptor and / or the P2X4 receptor signaling pathway.

[0023] In a second aspect of the present invention, the above object is solved by a method for preventing and / or treating a solid tumor or metastasis thereof in a subject in need thereof, the method comprising simultaneously or sequentially administering to said subject (i) an effective amount of a P2X4 receptor inhibitor or an inhibitor of the P2X4 receptor signaling pathway, and (ii) an effective amount of a cell death-inducing chemotherapeutic agent and / or a cell death-inducing therapy.

[0024] Preferably, said treatment and / or prevention comprises the inhibition of tumor evasion mechanisms mediated by the ATP-dependent P2X4 receptor and / or the P2X4 receptor signaling pathway.

[0025] The elements of the present invention are described below. These elements are listed with specific embodiments. However, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be interpreted as limiting the present invention to only the explicitly described embodiments. The description should be understood to support and encompass embodiments that combine two or more of the explicitly described embodiments, or combine one or more of the explicitly described embodiments with any number of the disclosed elements and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application, unless the context indicates otherwise.

[0026] In a first aspect of the present invention, the above object is solved by providing an effective amount of a P2X4 receptor inhibitor or an inhibitor of the P2X4 receptor signaling pathway in combination with an effective amount of a fluoropyrimidine drug for use in the prevention and / or treatment of a solid tumor or metastasis thereof in a subject.

[0027] In the context of the present invention disclosed herein, the terms "P2X4 receptor inhibitor" and "inhibitor of P2X4 receptor signaling pathway" shall be understood to refer to any compound or means that inhibits the biological function of P2X4 receptor when contacted with cells, particularly its purinergic signaling associated with tumor or tumor environment.A significant number of compounds have been described that have activity as inhibitor of P2X4 receptor and / or inhibitor of P2X4 receptor signaling pathway, and such compounds known to those skilled in the art shall be included in the present invention. In a preferred embodiment, the P2X4 receptor inhibitor is a specific inhibitor of the P2X4 receptor, i.e. it does not bind or does not substantially bind to other P2X4 receptors or to P2Y receptors, and is preferably selected from the group consisting of 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, IgG#151-LO, antibodies or fragments or derivatives thereof, siRNA, shRNA, miRNA, ribozymes, aptamers, antisense nucleic acid molecules, small molecules, or modified versions of these inhibitors (see, for example, Braganca B, Correia-de-Sa P. Resolving the Ionotropic P2X4 Receptor Mystery Points Towards a New Therapeutic Target for Cardiovascular Diseases. Int J Mol Sci. 2020 Jul 15;21(14):5005. doi: 10.3390 / ijms21145005. PMID:32679900; PMCID: PMC7404342.) Preferably, the inhibitor is 5-BDBD.

[0028] In the context of the invention disclosed herein, the term "cell death-inducing chemotherapeutic agent" or "cell death-inducing therapy" refers to any suitable drug or therapy (e.g., chemotherapy, radiation, immunotherapy, apoptosis-inducing therapy, etc.) that induces cell death in cells (e.g., cancer cells, etc.). One preferred but illustrative example in the context of the present invention is the fluoropyrimidine drug 5-FU. Other examples are necrotic cell supernatants; cationic amphiphilic drugs (CAD); classical anticancer drugs such as anthracyclines, antimetabolites, and platinum drugs; topoisomerase inhibitors, e.g., camptothecin; PDE3A inhibitors such as anagrelide alone or in combination with cell death-inducing cytokines; death-inducing 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, and SB-T-12854. Other suitable molecules and compounds induce apoptosis (see, e.g., Pfeffer CM, Singh ATK. Apoptosis: A Target for Anticancer Therapy. Int J Mol Sci. 2018;19(2):448. Published 2018 Feb 2. doi:10.3390 / ijms19020448).

[0029] In the context of the present invention disclosed herein, the term "fluoropyrimidine drug" should be understood to refer to any compound having a chemical structure and pharmaceutical activity equivalent and / or substantially identical to the anticancer drug 5-fluorouracil (5-FU).Preferably, said fluoropyrimidine drug (FP) is selected from the group of 5-fluorouracil, tegafur, capecitabine, and doxifluridine, and is preferably 5-fluorouracil (5-FU).

[0030] In the context of the invention disclosed herein, the term "effective amount" is intended to mean the amount of drug in the composition administered to a subject that elicits a (desired) biological response. This is generally defined by a range between the minimal effective dose (MED) and the maximum tolerated dose (MTD). The MED is defined as the lowest dose level of a pharmaceutical agent that produces a clinically significant response in mean efficacy, which is statistically significantly better than the response produced by a placebo. Similarly, the MTD is the highest possible, yet tolerable dose level, with respect to a pre-specified clinical limit toxicity. Generally, these limits refer to the average patient population. Thus, a person skilled in the art will be able to easily identify such amounts (see also below).

[0031] As described herein, the present invention is based on the surprising discovery that when tumor cells die during chemotherapy and / or toxin therapy, they release messenger compounds, particularly ATP, into the tumor environment, which improve the survival of neighboring tumor cells based on the function of mTOR and P2X4.Therefore, the preventive and / or therapeutic treatment in the present invention is based on a combination approach that inhibits the tumor escape mechanism mediated by ATP-dependent P2X4 receptor and / or P2X4 receptor signaling pathway, together with the toxicity of the amount of cell death-inducing chemotherapeutic agent and / or cell death-inducing therapy.As exemplified by the preferred combination of 5-FU and 5-BDBD, this combination (possibly with other agents disclosed herein) effectively blocks this escape mechanism, thereby preventing further tumor growth and / or formation or recurrence of metastasis.In the context of the present invention disclosed herein, the cells surrounding the cells or tissue involved in proliferation disorder are the cells of the microenvironment of affected tissue, such as the tumor microenvironment.

[0032] Generally, any suitable inhibitor of P2X4 receptor or inhibitor of P2X4 receptor signaling pathway can be used in the context of the present invention, as long as it can be used in combination with cell death-inducing chemotherapeutic agent and / or cell death-inducing therapy (as long as they function together in a subject, are provided together, jointly, or individually).Preferably, it is an inhibitor of P2X4 receptor that is a specific inhibitor of P2X4 receptor or a specific inhibitor of a component of P2X4 receptor signaling pathway.In the context of the present invention, "specific inhibitor" means that the inhibitor compound does not bind or does not substantially bind to other receptors and / or receptor signaling pathways in target cells.In particular, specific inhibitor is preferred when it shows stronger binding to human P2X4 receptor or component of P2X4 receptor signaling pathway than other members of P2X and / or P2Y family and their signaling pathways, more than 2-fold, more than 3-fold, more than 10-fold, more than 35-fold, more than 50-fold, more than 100-fold, more than 1000-fold, or even more than 10000-fold.

[0033] Preferably, the inhibitor is selected from the group consisting of inhibitors of 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, IgG#151-LO, antibodies or fragments or derivatives thereof, siRNA, shRNA, miRNA, ribozymes, aptamers, antisense nucleic acid molecules, small molecules and chemically modified versions thereof, preferably selected from 5-BDBD. The therapeutic target defined in the disclosed invention, i.e. any inhibitor of the P2X4 receptor or its signaling pathway, such as a compound or composition, may be selected from a polypeptide, peptide, glycoprotein, peptidomimetic, antibody or antibody-like molecule (such as intrabody); nucleic acid such as DNA or RNA, e.g. antisense DNA or RNA, ribozyme, RNA or DNA aptamer, siRNA, shRNA, etc. (including variants or derivatives thereof, such as peptide nucleic acid (PNA)); genetic constructs for targeted gene editing, such as CRISPR / Cas9 constructs and / or guide RNA / DNA (gRNA / gDNA) and / or tracrRNA; heterobifunctional compounds (such as PROTAC or HyT molecules); carbohydrates, such as polysaccharides or oligosaccharides (including variants or derivatives thereof); lipids, such as fatty acids (including variants or derivatives thereof); or organic small molecules, including but not limited to small molecule ligands, or cell-permeable small molecules, may be used in the context of the present invention.

[0034] Generally, any suitable cell death-inducing chemotherapeutic agent and / or cell death-inducing therapy can be used in the context of the present invention, so long as it can be used in combination with the above-mentioned P2X4 receptor inhibitors, inhibitors of components of the P2X4 receptor signaling pathway (provided together, jointly, conjointly or individually, functioning together in a subject). Preferably, the supernatant of necrotic cells; cationic amphiphilic drugs (CAD); classical anticancer drugs, including anthracyclines, antimetabolites, platinum drugs; topoisomerase inhibitors, for example, camptothecin; PDE3A inhibitors, such as anagrelide, alone or in combination with cell death-inducing cytokines; death-inducing 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) may be used as drugs, and when fluoropyrimidine drugs (FPs) are used, they are preferably selected from the group of 5-fluorouracil, tegafur, capecitabine, and doxifluridine and other FP derivatives, more preferably 5-fluorouracil (5-FU) is selected.

[0035] The use of P2X4 receptor inhibitor or P2X4 receptor signaling pathway inhibitor in combination with cell death-inducing chemotherapeutic agent and / or cell death-inducing therapy according to the present invention is preferred, and said treatment and / or prevention further comprises prior and / or combined anti-cancer chemotherapy, such as cytotoxin, Lgr5 inhibitor, Lgr5+cancer cell depletion, mTOR inhibitor and IMPDH inhibitor.Generally, any suitable anti-cancer chemotherapy can be used in connection with the present invention, as long as it can be combined with the above-mentioned combination of the present invention (as long as it functions together in a subject, is provided together, jointly or individually) to effectively block the cancer escape mechanism disclosed herein, and thereby prevent tumor further growth and / or prevent metastasis formation or recurrence.

[0036] In the context of the present invention, the subject treatable by the therapeutic method of the present invention is preferably distinguished as having a tumor that has progressed, in particular recurred, relapsed or failed to respond to chemotherapy, toxin therapy, and / or radiation therapy. For example, the tumor disease is a reappearing or recurrent tumor disease (metastasis).

[0037] As mentioned above, the therapy and / or additional treatment may include at least one or more (cell death-inducing) cancer therapies, including chemotherapy, radiation therapy, or immunotherapy. Treatment regimes and schedules may vary depending on the type of preconditioning and cancer treatment. For example, chemotherapy or radiation therapy may be administered to the patient at least 1 day, 3 days, 5 days, 7 days after tumor preconditioning is completed. In another example, in some embodiments, cell-based immunotherapy may be administered to the patient at least 1 day, 3 days, 5 days, 7 days before starting the therapy of the present invention. In yet other embodiments, additional treatments may be administered to the patient during the treatment of the present invention (e.g., at least 12 hours, at least 1 day, at least 3 days, etc. after the start of preconditioning). Thus, the treatment of the present invention may further include administration of at least one additional anti-proliferative therapeutic agent, such as a chemotherapeutic agent.

[0038] Inhibitors of the P2X4 receptor or inhibitors of the P2X4 receptor signaling pathway in combination with a fluoropyrimidine drug for use according to the present invention are preferred, said combination being provided simultaneously or separately, in combination and / or in separate dosage forms.

[0039] As used herein, "cancer" may include the term "solid tumor". As used herein, the term "solid tumor" refers to a condition such as cancer that forms an abnormal tumor mass, such as sarcoma, carcinoma, and lymphoma. Examples of solid tumors or metastases thereof are selected from prostate cancer, pancreatic cancer, breast cancer, gastric cancer, liver cancer, brain cancer, lung cancer, kidney cancer, colorectal cancer and metastases thereof, in particular colorectal cancer and metastases thereof, and the solid tumor disease may be, for example, adenocarcinoma, squamous cell carcinoma, large cell carcinoma, etc.

[0040] In certain embodiments, the cancer may be, for example, primary or secondary (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, cholangiocarcinoma, endometrial cancer, small intestine adenocarcinoma, uterine sarcoma, or adrenocortical carcinoma. In some embodiments, the cancer may be, for example, esophageal cancer, gastroesophageal junction cancer, gastroesophageal adenocarcinoma, 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, cholangiocarcinoma, endometrial cancer, small intestine adenocarcinoma, uterine sarcoma, or adrenocortical carcinoma. In some embodiments, the cancer is, for example, colorectal cancer. In some embodiments, the cancer is, for example, colorectal adenocarcinoma. In some embodiments, the cancer is, for example, small intestine adenocarcinoma. In some embodiments, the cancer is, for example, hepatocellular carcinoma. In some embodiments, the cancer is, for example, head and neck cancer. In some embodiments, the cancer is, for example, renal cell carcinoma. In some embodiments, the cancer is, for example, ovarian cancer. In some embodiments, the cancer is, for example, prostate cancer. In some embodiments, the cancer is, for example, lung cancer. In some embodiments, the cancer is, for example, uterine sarcoma. In some embodiments, the cancer is, for example, esophageal cancer. In some embodiments, the cancer is, for example, endometrial cancer. In some embodiments, the cancer is, for example, bile duct cancer. In some embodiments, each cancer may be, for example, unresectable cancer, progressive cancer, refractory cancer, recurrent cancer, or metastatic cancer. Preferred cancers are solid cancers, particularly hepatocellular carcinoma or colorectal cancer.

[0041] The invention disclosed herein in various aspects and embodiments suggests a combination treatment regimen for cancer treatment. In such aspects and embodiments, the following drug combination is most preferred: 5-fluorouracil (as a cell death inhibitor), and 5BDBD (as an inhibitor of P2X4 receptor).

[0042] In one preferred embodiment of the present invention, the above object is solved by a method for screening the effective, preferably synergistic combination of inhibitor of P2X4 receptor or inhibitor of P2X4 receptor signaling pathway and cell death-inducing chemotherapeutic agent and / or cell death-inducing therapy, said method comprises: contacting solid tumor or its metastasis with at least one of candidate inhibitor of P2X4 receptor or candidate inhibitor of P2X4 receptor signaling pathway in combination with candidate cell death-inducing chemotherapeutic agent and / or cell death-inducing therapy by solid tumor, and detecting at least one of the following: shrinkage of said tumor, increase in apoptosis of said tumor cells, death of tumor cells, and decrease in metastasis caused by said tumor cells, thereby identifying the effective, preferably synergistic combination of candidate compound or therapy.Candidate compound can be selected as above.

[0043] Preferably, said screening is performed in cell culture, e.g. 3D culture using organoids of tumor cells.

[0044] More preferably, the screening method further comprises the prior and / or simultaneous addition of a compound for anti-cancer chemotherapy, including, for example, a cytotoxin, an Lgr5 inhibitor, an Lgr5+ cancer cell depletion, an mTOR inhibitor, and an IMPDH inhibitor. More preferably, the method according to the present invention, wherein the combination is provided simultaneously or separately as a combination and / or separate dosage form.

[0045] More preferably, the present invention, wherein said screening comprises detection of inhibition of tumor evasion mechanisms mediated by the ATP-dependent P2X4 receptor and / or the P2X4 receptor signaling pathway.

[0046] Identified combinations, preferably synergistic combinations, can be formulated into anti-cancer formulations as disclosed herein and used in the context of the present invention.

[0047] In another preferred embodiment of the present invention, the above object is solved by a method for preventing and / or treating a solid tumor or metastasis thereof in a subject in need of such prevention and / or treatment, the method comprising: (i) simultaneously or sequentially administering an effective amount of a P2X4 receptor inhibitor or an inhibitor of the P2X4 receptor signaling pathway, and (ii) simultaneously or sequentially administering to said subject an effective amount of a cell death-inducing chemotherapeutic agent and / or a cell death-inducing therapy.

[0048] Compounds suitable and / or suitable for treatment are as disclosed above.For use in treatment, compounds and compositions for use in the present invention can be formulated into pharmaceutical compositions suitable for easy administration to animals or humans.The term "pharmaceutical composition" refers to a mixture of substances that includes therapeutically active substances for medical use as disclosed herein (such as agonists or antagonists of the present invention).That is, in a further aspect, the present invention relates to the use of pharmaceutical compositions that include one or more compounds for use in the present invention, namely (i) an effective amount of an inhibitor of P2X4 receptor or inhibitor of P2X4 receptor signaling pathway, and (ii) an effective amount of a cell death-inducing chemotherapeutic agent and / or a cell death-inducing therapy, and pharmaceutical acceptable carriers, stabilizers and / or excipients.

[0049] A pharmaceutical composition for use in the present invention may comprise a combination therapeutic agent according to the treatment regimen disclosed herein, or alternatively, the present invention may comprise a combination of pharmaceutical compositions, each of which comprises a single component of the combination therapy of the present invention, e.g., preferably, one composition comprising an inhibitor and a pharma- ceutically acceptable carrier, stabilizer and / or excipient; another pharmaceutical composition comprising a fluoropyrimidine drug and a pharma- ceutically acceptable carrier, stabilizer and / or excipient. The compositions of the present invention in this embodiment may be combined into a treatment kit.

[0050] As an example, a pharmaceutical composition for use in the present invention may contain 0.1% to 100% (w / w), for example, about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 8%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%, preferably about 1% to about 20%, about 10% to 50%, or about 40% to 90%, of an active ingredient (e.g., an inhibitor of the P2X4 receptor or an inhibitor of the P2X4 receptor signaling pathway and / or a cell death-inducing chemotherapeutic agent and / or a cell death-inducing therapy).

[0051] The term "pharmaceutically acceptable" excipient, stabilizer or carrier used herein is intended to include any solvent, solubilizer, filler, stabilizer, binder, absorbent, base, buffer, lubricant, release control vehicle, diluent, emulsifier, humectant, dispersion medium, coating, antibacterial or antifungal agent, isotonic agent and absorption retardant, etc., that are compatible with drug administration.The use of such media and agents for pharmaceutically active substances is well known in the art.Unless conventional media or agents are incompatible with active compounds, their use in the composition is contemplated.Auxiliary agents can also be incorporated into the composition.

[0052] A pharmaceutical composition of the present invention (or for use with the present invention) is typically formulated to be compatible with its intended route of administration. Examples of routes of administration include oral, parenteral, e.g., intrathecal, intraarterial, intravenous, intradermal, subcutaneous, oral, transdermal (topical) and transmucosal administration. Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Kolliphor® EL (formerly Cremophor EL™; BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, injectable compositions should typically be sterile and fluid to the extent that easy syringability exists. They should usually be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Lipid particles may also be included (see above for rapamycin). Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0053] In some embodiments, the pharmaceutical composition comprising an inhibitor of the P2X4 receptor or an inhibitor of the P2X4 receptor signaling pathway is in a unit dose form of 10-1000 mg of the inhibitor. In some embodiments, the pharmaceutical composition comprising the inhibitor is in a unit dose form of 10-200 mg, or 200-400 mg, or even 400-600 mg, or 600-800 mg of the inhibitor of the P2X4 receptor or an inhibitor of the P2X4 receptor signaling pathway. Similar doses are used for the fluoropyrimidine drug according to the present invention.

[0054] The exemplary unit dosage form of pharmaceutical composition comprising inhibitor of P2X4 receptor or inhibitor of P2X4 receptor signaling pathway and / or cell death-inducing chemotherapeutic agent is tablet, capsule (e.g., as powder, granule, microtablet or micropellet), suspension, or disposable preloaded syringe.In certain embodiments, a kit is provided for producing a single-dose dosage unit.The kit can include both a first container that contains dry active ingredient and a second container that contains aqueous formulation.Alternatively, the kit can include single-chamber and multi-chamber preloaded syringes.

[0055] According to all aspects and embodiments of the pharmaceutical uses and methods of treatment provided herein, the effective amount of the compound or composition for use in the present invention administered at least once to a subject in need of treatment is typically about 0.01 mg / kg to about 100 mg / kg per administration, for example, about 1 mg / kg to about 10 mg / kg per administration. In some embodiments, the effective amount of the compound or composition of the present invention administered at least once to the subject is about 0.01 mg / kg to about 0.1 mg / kg per administration, about 0.1 mg / kg to about 1 mg / kg per administration, about 1 mg / kg to about 5 mg / kg per administration, about 5 mg / kg to about 10 mg / kg per administration, about 10 mg / kg to about 50 mg / kg per administration, or about 50 mg / kg to about 100 mg / kg per administration.

[0056] The appropriate dose of the compound or composition (or pharmaceutical composition comprising same) for use in the present invention for the prevention or treatment of solid tumors and / or recurrence and / or metastasis will vary depending on the type of disease being treated, the severity, course of the disease, whether the compound or composition and / or pharmaceutical composition of the present invention is administered for prophylactic or therapeutic purposes, previous therapy, the patient's medical history, age, size / weight and response to the compound or composition and / or pharmaceutical composition of the present invention, and the judgment of the attending physician. The compound or composition and / or pharmaceutical composition of the present invention is suitably administered to the patient at one time or over a series of treatments. When such a compound or composition and / or pharmaceutical composition of the present invention is administered over a series of treatments, the total number of administrations in a given course of treatment may consist of a total of about 2, 3, 4, 5, 6, 7, 8, 9, 10 or about 10 or more treatments. For example, treatment may be administered once every day (or 2, 3, or 4 times a day) for a week, a month, or even several months. In certain embodiments, the course of treatment may be continued indefinitely or until no longer necessary.

[0057] In the method of the present invention, the inhibitor of P2X4 receptor is preferably a specific inhibitor of P2X4 receptor, preferably selected from the group of 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, IgG#151-LO, antibodies or fragments or derivatives thereof, siRNA, shRNA, miRNA, ribozymes, aptamers, antisense nucleic acid molecules, small molecules, or modified versions of these inhibitors, preferably 5BDBD. For "specific" and other characteristics, see above.

[0058] In the method according to the present invention, preferably, the cell death-inducing chemotherapeutic agent is a fluoropyrimidine drug (FP), selected from the group of 5-fluorouracil, tegafur, capecitabine, and doxifluridine, and is preferably 5-fluorouracil (5-FU).Other preferred examples are supernatants of necrotic cells; cationic amphiphilic drugs (CAD); classical anticancer drugs such as anthracyclines, antimetabolites, and platinum drugs; topoisomerase inhibitors, for example camptothecin; PDE3A inhibitors, such as anagrelide, alone or in combination with cell death-inducing cytokines; death-inducing 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, and SB-T-12854.

[0059] Preferably, in the method of the present invention, said treatment and / or prevention comprises inhibition of tumor escape mechanisms mediated by ATP-dependent P2X4 receptor and / or P2X4 receptor signaling pathway, and / or said treatment and / or prevention further comprises prior and / or concomitant anti-cancer chemotherapy, including, for example, cytotoxins, Lgr5 inhibitors, Lgr5+ cancer cell depletion, mTOR inhibitors, and IMPDH inhibitors, also as detailed above.

[0060] As used herein, a "subject" includes all mammals, including but not limited to humans, but also non-human primates, such as cynomolgus monkeys. It also includes dogs, cats, horses, sheep, goats, cows, rabbits, pigs, and rodents, such as mice and rats. It will be understood that a particularly preferred subject according to the invention is a human subject, e.g., a human suffering from (or at risk of suffering from) a disorder, disease, or condition, e.g., a human patient.

[0061] As used herein, the term "comprises" should be interpreted as embracing both "comprises" and "consisting of," and both meanings are specifically intended, and thus include separately disclosed embodiments according to the present invention. As used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. For example, "A and / or B" shall be interpreted as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually described herein. In the context of the present invention, the terms "about" and "approximately" indicate a range of precision that a person skilled in the art would understand to ensure the technical effect of the feature in question. This term typically indicates a deviation of ±20%, ±15%, ±10%, e.g., ±5% from the indicated numerical value. As would be understood by a person skilled in the art, the specific such deviation from the numerical value of a given technical effect would depend on the nature of the technical effect. For example, in general, natural or biological technical effects are more likely to have such deviations than man-made or engineered technical effects. As would be understood by a person skilled in the art, the specific such deviation from the numerical value of a given technical effect would depend on the nature of the technical effect. For example, natural or biological technological effects are generally more likely to have such deviations than man-made or engineered technological effects. When an indefinite or definite article is used to refer to a singular noun such as "a," "an," or "the," this also includes the plural of that noun unless otherwise specified.

[0062] It should be understood that applying the teachings of the present invention to a particular problem or environment, and incorporating variations of the present invention or additional features thereto (such as further aspects and embodiments), is within the capabilities of one of ordinary skill in the art in view of the teachings contained herein.Unless otherwise indicated by context, the above feature descriptions and definitions are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described. [Brief description of the drawings]

[0063] [Figure 1-1]Figure 1 shows that loss of Lgr5+ cells is compensated by mTORC1 activation in neighboring cells. a) Schematic of the AOM / DSS protocol for induction of colon tumors and the time point of diphtheria toxin (DT) administration to deplete Lgr5+ cells. b) Mini-endoscopic images of colon tumors at days 0 and 9 after four injections of either DT (50 μg / kg body weight) or vehicle (HO) as control. Representative images from at least three independent experiments are shown. c) Average tumor size was measured on sequential hematoxylin and eosin (H&E) stained sections at day 9 after four injections of either DT or vehicle. Data are means ± SD, n ≥ 8 / treatment. p > 0.05 (ns: not significant) by two-tailed Student's t-test. d) Immunohistochemical analysis and quantification of Lgr5-EGFP expression in GFP-stained colon tumors from Lgr5 EGFP-DTR(+) mice treated with DT on day 9 as shown in a). Representative images are shown (n>8 / treatment, scale bar=300 μm). Data are mean ± SD, n>8 / treatment. ****p<0.0001 by two-tailed Student's t-test. e) Immunohistochemical analysis of Ki-67 expression in colon tumors 9 days after four injections of either DT or vehicle and quantification of Ki-67+ epithelial cells in the tumors. Representative images from four independent experiments are shown (n=4 / treatment, scale bar=200 μm). Data represent mean ± SD, n=4 / treatment. p>0.05 by two-tailed Student's t-test (ns: not significant). f) Flow cytometry analysis of Lgr5-EGFP+ tumor cells from Lgr5EGFP-DTR(+) tumor organoids 24 hours after DT treatment. Representative results are shown (n>3). g) AOM / DSS colon tumor organoids from Lgr5 EGFP-DTR(-) and Lgr5 EGFP-DTR(+) mice were treated with DT (20 ng / ml) for 24 hours. Representative images from two independent experiments are shown (scale bar=200 μm). h) Immunoblot analysis of Lgr5 EGFP-DTR(+) and Lgr5 EGFP-DTR(-) tumor organoids treated with DT for 24 hours. Representative results are shown (n=3).i) Immunofluorescence analysis of p-S6 (red) and GFP (Lgr5, green) expression in tumor tissues of vehicle- or DT (50 μg / kg)-treated AOM / DSS mice 24 h after injection. Representative images are shown (n=3, scale bar=50 μm). j) Lgr5 EGFP-DTR(+) tumor organoids were treated with DT (20 ng / ml), rapamycin (10 μM), or DT and rapamycin for 24 h. Representative images from three independent experiments are shown. k) Lgr5 EGFP-DTR(+) tumor organoids were treated with DT (20 ng / ml), rapamycin (10 μM), or DT and rapamycin for 24 h, reseeded and cultured for 72 h. The percentage of reseeding ability compared to vehicle-treated organoids is shown. Data are mean ± SEM, n =4. *** p<0.001 by one-way ANOVA with Bonferroni's multiple comparison test. l) Control and Raptor knockdown Lgr5 EGFP-DTR(+) tumor organoids were passaged and counted after 72 hours. The percentage of organoid formation capacity compared to control organoids is shown. Data from one representative experiment are shown and represent the mean ± SD, n=3. p>0.05 by two-tailed Student's t-test. m) Control and Raptor knockdown Lgr5 EGFP-DTR(+) tumor organoids were treated with DT (20ng / ml) for 24 hours, replated and cultured for 72 hours. The percentage of reseeding capacity compared to control organoids is shown. Data from one representative experiment are shown and represent the mean ± SD, n=3. *** p<0.001 by two-tailed Student's t-test. n) Efficiency of shRNA-mediated Raptor knockdown in Lgr5 EGFP-DTR(+) tumor organoids was determined by quantitative RT-PCR. Data are mean ± SD, n = 3. * p < 0.05 by two-tailed Student's t-test. [Figure 1-2] Same as above [Figure 1-3] Same as above [Figure 1-4] Same as above [Figure 2-1]Figure 2 shows that inhibition of mTOR induces tumor collapse upon depletion of Lgr5+ cells. a) Schematic of AOM / DSSATKN tumor organoid generation. b) Immunoblot analysis of AOM / DSSATKN tumor organoids treated with rapamycin (10 μM) alone or in combination with DT (20 ng / ml) for 24 h. Data from one representative experiment are shown (n=3). c) AOM / DSSATKN tumor organoids were injected subcutaneously and mice were treated with a single dose of DT (50 μg / kg), rapamycin (10 mg / kg), or both in combination for the indicated time points. Tumor tissues were subjected to immunofluorescence analysis of cleaved caspase 3 (red) and GFP (Lgr5, green) expression. Representative images from at least two mice / time point are shown (scale bar=100 μm). d) AOM / DSSATKN tumor organoids were injected subcutaneously, and when tumors reached 200 mm3, mice were treated with a single dose of DT (50 μg / kg), rapamycin (10 mg / kg), or a combination of both for 24 h. Representative images of H&E (n=3, scale bar=200 μm). e) Treatment regimen for subcutaneous AOM / DSSATKN tumors. Tumor organoids were injected up to 15 days before application of DT and rapamycin. DT was applied every 2 days, and rapamycin was injected once a day. f) Tumor growth kinetics of subcutaneous AOM / DSSATKN tumors in response to treatment as shown in (e). Mean tumor volume ± SEM is shown. n=5 for vehicle, n=5 for DT, n=6 for rapamycin, n=9 for rapamycin+DT. **p<0.01, ***p<0.001 by two-way ANOVA with Bonferroni's multiple comparison test. g) Representative images of subcutaneous AOM / DSSATKN tumors shown in (e) on day 8 treated with DT, rapamycin, or DT / rapamycin, (scale bar=5 mm). h) Representative images of p-S6 (red) stained small intestine sections from C57 / B6 WT mice treated with a single injection of AOM (10 mg / kg) or 12 Gray irradiation and sacrificed 8 hours after treatment (n=3).i) Immunoblot analysis of AOM / DSSATKN tumor organoids treated with chemotherapeutic agents 5-FU (20 μM), rapamycin (10 μM), or 5-FU and rapamycin for 24 h. Representative results are shown (n=3). j) Immunoblot analysis of human tumor organoids treated with 5-FU (20 μM), rapamycin (10 μM), or 5-FU and rapamycin for 24 h. Representative results are shown (n=3). k) AOM / DSSATKN tumor organoids were treated with chemotherapeutic agents 5-FU (20 μM), rapamycin (10 μM), or 5-FU and rapamycin for 48 h, replated and cultured for 72 h. The percentage of reseeding ability compared to vehicle-treated organoids is shown. Data are mean ± SD, n=3. One representative of three experiments is shown. *p<0.05, ***p<0.001 by one-way ANOVA with Bonferroni's multiple comparison test. l) Human tumor organoids were treated with 5-FU (20 μM), rapamycin (10 μM), or 5-FU and rapamycin for 48 h and then reseeded in ENR-containing medium for 72 h. The percentage of reseeding ability compared to vehicle-treated organoids is shown. Data are mean ± SD, n=3. One representative of three experiments is shown. *p<0.05, ***p<0.001 by one-way ANOVA with Bonferroni's multiple comparison test. m) Treatment regimen for subcutaneous AOM / DSSATKN tumors. Tumor organoids were injected up to 15 days before applying 5-FU and rapamycin. 5-FU (20 mg / kg) was applied every 2 days for 4 doses and rapamycin (10 mg / kg) was injected once a day. n) Tumor growth rate of subcutaneous AOM / DSSATKN tumors in response to treatment as shown in (n). Mean tumor volume ± SD is shown (n>5 for each condition). *p<0.05, ***p<0.001, ****p<0.0001 by 2-way ANOVA with Bonferroni's multiple comparison test. o) Representative images of subcutaneous AOM / DSSATKN tumors on day 8 treated with 5-FU, rapamycin or 5-FU / rapamycin as shown in (e) (scale bar=5 mm). [Figure 2-2] Same as above [Figure 2-3] Same as above [Figure 2-4] Same as above [Figure 2-5] Same as above [Figure 3-1]Figure 3 shows that S6 phosphorylation is triggered by ATP release from dying cells. a) Immunoblot analysis of Lgr5 EGFP-DTR(-) tumor organoids treated for 24 h with supernatants from Lgr5 EGFP-DTR(+) tumor organoids treated for 24 h with vehicle or DT. Representative results are shown (n=2). b) Analysis of ATP content in supernatants of vehicle or DT treated organoids 6 h after treatment. Data are means ± SD, n=7. *p<0.05 by two-tailed Student's t-test. c) Immunoblot analysis of Lgr5 EGFP-DTR(-) tumor organoids treated for 30 min with ATP (100 μM), 2MeS ADP (100 μM) and adenosine (100 μM). Representative results are shown (n=3). d) Analysis of ATP content in control or necrotic medium. Data are means ± SD, n=3. *p<0.05 by two-tailed Student's t-test. e) Immunoblot analysis of Lgr5 EGFP-DTR(+) tumor organoids treated with necrotic cell supernatant for 4 h. Representative results are shown (n=3). f) Immunoblot analysis of Lgr5 EGFP-DTR(+) tumor organoids treated with DT for 6 h in the presence or absence of apyrase. Representative results are shown (n=3). g) Immunoblot analysis of Lgr5 EGFP-DTR(+) tumor organoids treated with necrotic medium for 2 h in the presence or absence of apyrase. Representative results are shown (n=3). h) Lgr5 EGFP-DTR(+) tumor organoids were treated with purinergic receptor inhibitors (50 μM theophylline, 20 ng / ml pertussis toxin and 30 μM PPADS) and 5-FU (10 μM) as indicated for 48 h, re-seeded and cultured for 72 h. Percentage of reseeding ability compared to vehicle-treated organoids is shown. Data are mean ± SD, n=4 replicates. One representative experiment out of four performed is shown. ***p<0.001 by one-way ANOVA with Bonferroni's multiple comparison test. i) Relative mRNA expression levels of the indicated genes in AOM / DSS colon tumor organoids as determined by qRT-PCR (n=3).j) Immunoblot analysis of APTAK colon tumor organoids treated with ATP (100 μM) for 15 min in the presence or absence of 5-BDBD (10 μM). Representative results are shown (n=3). k) AOM / DSSATKN tumor organoids were treated with the indicated concentrations of 5-BDBD and 5-FU (20 μM) for 48 h, replated and cultured for 72 h. The percentage of reseeding ability compared to vehicle-treated organoids is shown. Data are mean ± SD, n=3 replicates. A representative one of three experiments performed is shown. ns p>0.05, * p<0.05 ***, p<0.001, **** p<0.0001 by one-way ANOVA with Bonferroni's multiple comparison test. l) Immunoblot analysis of doxycycline-induced P2X4 knockdown APTAK colon tumor organoids treated with 5-FU (20 μM). Organoids were cultured in medium - / + doxycycline before the experiment. Representative results are shown (n=3). m) Doxycycline-induced P2X4 knockdown APTAK organoids were treated with 5-FU (20 μM) for 48 h, re-plated and cultured for 72 h. Organoids were cultured in medium - / + doxycycline before the experiment. Percentage of replating ability compared to vehicle-treated organoids is shown. Data are mean ± SD, n=3. One representative experiment out of three performed is shown. ns p>0.05, * p<0.05 ***, p<0.001, **** p<0.0001 by one-way ANOVA with Bonferroni's multiple comparison test. n) Efficiency of shRNA-mediated P2X4 knockdown in Lgr5 EGFP-DTR(+) tumor organoids was determined by quantitative RT-PCR. Data are mean ± SD, n=3. ****p<0.0001 by two-tailed Student's t-test. o) APTAKshP2x4 tumor organoids were injected subcutaneously, mice were treated with a single dose of 5-FU for 24 h in the presence or absence of doxycycline, and tumor tissues were analyzed by immunofluorescence for p-S6 (red). One representative image is shown (n=3, scale bar=50 μm). p) Treatment regimen of subcutaneous APTAKshP2x4 tumors.Tumor organoids were injected up to 15 days before 5-FU treatment and doxycycline treatment (0.5 mg / ml in drinking water) was started 3 days before the first 5-FU application. 5-FU (20 mg / kg) was applied every 2 days for 4 times. q) Tumor growth kinetics of subcutaneous APTAKshP2x4 tumors in response to treatment as shown in (p). Mean tumor volume ± SD is shown (n>5 for each condition). ns >0.05, * p<0.05, *** p<0.001, **** p<0.0001 by two-way ANOVA with Bonferroni's multiple comparison test). r) Representative images of subcutaneous APTAKshP2x4 tumors at day 8 treated with 5-FU in the presence or absence of doxycycline as shown in (p) (scale bar = 5 mm). s) Representative image (r) H&E staining of subcutaneous APTAKshP2x4 tumors on day 8 treated as described in (r) (scale bar = 200 μm). [Figure 3-2] Same as above [Figure 3-3] Same as above [Diagram 3-4] Same as above [Figure 3-5] Same as above [Figure 4-1]Figure 4 shows that paracrine activation of cell death and apoptosis induced by mTOR signaling blockade is counteracted by inhibition of ROS. a) AOM / DSSATKN tumor organoids were treated with chemotherapeutic agents 5-FU (20 μM), rapamycin (10 μM), or 5-FU and rapamycin in the presence or absence of the apoptosis inhibitor zVAD (50 μM) for 48 h, reseeded and cultured for 72 h. The percentage of reseeding ability compared to vehicle-treated organoids is shown. Data are mean ± SD, n=3. One representative experiment out of four is shown. ns p>0.05, * p<0.05, *** p<0.001 by one-way ANOVA with Bonferroni's multiple comparison test. b) AOM / DSSATKN tumor organoids were treated with the chemotherapeutic agents 5-FU (20 μM), rapamycin (10 μM) or 5-FU and rapamycin in the presence or absence of NAC (2 mM), anti-HMGB1 antibody (5 μg / ml), Enbrel (10 mg / ml), C176 / C178 (0.5 μM), anakinra (100 ng / ml) for 48 h, reseeded and cultured for 72 h. The percentage of reseeding ability compared to vehicle-treated organoids is shown. Data are mean ± SD, n=3. One representative experiment out of four is shown. ns p>0.05, * p<0.05, **** p<0.0001 by one-way ANOVA with Bonferroni's multiple comparison test. c) AOM / DSSATKN tumor organoids were treated with the chemotherapeutic agents 5-FU (20 μM), rapamycin (10 μM), or 5-FU and rapamycin in the presence or absence of NAC (2 mM), Trolox (1 mM), or BHA (10 μM) for 48 h, then replated and cultured for 72 h. The percentage of replated capacity compared to vehicle-treated organoids is shown. Data are mean ± SD, n=3. One representative experiment out of four is shown. ns p>0.05, * p<0.05, **** p<0.0001 by one-way ANOVA with Bonferroni's multiple comparison test.d) Fluorescence microscopy and FACS analysis of ROS production using human colon organoids derived from tumor tissue treated with DCFDA (green) and 5-FU (20 μM, 20 h) in control. One representative experiment out of four is shown (scale bar = 1 mM). e) Immunoblot analysis of AOM / DSSATKN tumor organoids treated with rapamycin (10 μM) alone or in combination with DT (20 ng / ml) for 24 h in the presence or absence of NAC (2 mM). Data from one representative experiment out of three independent experiments are shown. f) AOM / DSSATKN tumor organoids were injected subcutaneously and, when tumors reached 200 mm3, mice were treated with a single dose of DT (50 μg / kg), rapamycin (10 mg / kg) or both in combination in the presence or absence of NAC (0.5% in drinking water) for 12 h. Tumor tissues were then subjected to immunofluorescence analysis using an antibody against cleaved caspase 3. One representative experiment out of three independent experiments is shown (n=3, scale bar=200 μm). g) Treatment regimen of subcutaneous APTAKshP2x4 tumors. Tumor organoids were injected up to 15 days before 5-FU treatment and NAC treatment (0.5% in drinking water) was started 3 days before the first 5-FU application. 5-FU (20 mg / kg) was applied every 2 days for 4 applications. h) Tumor growth kinetics of subcutaneous APTAKshP2x4 tumors in response to treatment as shown in (g). Mean tumor volume ± SD is shown (n>5 for each condition). ns >0.05, *** p<0.001, **** p<0.0001 by two-way ANOVA with Bonferroni's multiple comparison test. i) Representative images of subcutaneous APTAKshP2x4 tumors at day 8 treated with 5-FU and rapamycin in the presence or absence of NAC as shown in (p) (scale bar = 5 mm). j) Representative image H&E staining of subcutaneous APTAKshP2x4 tumors at day 8 treated as described in (g) (scale bar = 200 μm). k) Schematic showing how dying cells activate the mTOR survival pathway in neighboring cells to counter apoptosis induction by elevated ROS levels and maintain tumor integrity. [Figure 4-2] Same as above [Figure 4-3] Same as above [Figure 4-4] Same as above [Figure 4-5] Same as above [Diagram 5] Figure 5 shows that inhibition of P2X4 enhances the sensitivity of patient-derived tumor organoids from pancreatic ductal adenocarcinoma to chemotherapy. Patient-derived tumor organoids (PDTO) were established from pancreatic ductal adenocarcinoma and treated individually or with a combination of mTOR inhibitor (rapamycin), 5-FU, and P2X4 inhibitor 5-BDBD. Inhibition of mTOR enhances 5-FU-induced cell death and reduces proliferation of surviving tumor organoids. Inhibition of P2X4 also enhances the sensitivity of tumor organoids to 5-FU administration, leading to reduced tumor organoid viability. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] To functionally confirm that the involved MAP kinase or mTOR activation is indeed involved in the survival of Lgr5+ cell-depleted organoids, we applied SP600125, PH797804, or rapamycin to inhibit JNK, p38, and mTOR, respectively, in the presence or absence of DT. While the two MAPK inhibitors did not affect the survival of tumor organoids, the combination of DT and rapamycin caused substantial cell death within 24 h selectively in tumor organoids expressing DTR (Fig. 1k). Furthermore, reseeding of DT / rapamycin co-treated organoids resulted in a marked decrease in their organoid formation capacity, which was not observed when either compound was applied individually (Fig. 1l).

[0065] Lgr5 EGFPDTR(+)Treatment of tumor organoids and subcutaneously implanted AOM / DSSATKN tumors with the IMPDH inhibitor mizoribine in combination with DT-mediated depletion of Lgr5+ cells produced severe disintegration of organoids in vitro and markedly reduced viable growth of AOM / DSSATKN tumors without affecting phosphorylation of S6-ribosomal protein. These data suggested that de novo purine synthesis downstream of mTOR activation is required to maintain tumor integrity in the absence of Lgr5+ cells. When Lgr5+ cells were sufficiently removed from tumors, we detected massive apoptosis in tumor tissues of animals treated with DT / rapamycin, whereas apoptosis was barely detectable in animals treated with DT alone (Figure 2d). As a consequence, prolonged rapamycin treatment and Lgr5+ cell depletion caused significant tumor shrinkage of established tumors, in contrast to single rapamycin or DT administration applied individually, which caused only tumor stasis (Figure 2e-g).

[0066] We also found that the combination of mTOR inhibition and cell death induction can be used therapeutically to enhance the response of tumors to cytotoxic therapy, and the combination of rapamycin and 5-FU significantly inhibited the reseeding ability of tumor organoids (Figure 2k,m) and significantly reduced tumor growth in subcutaneously implanted tumors compared to control or each treatment alone (Figure 2n-o).

[0067] In confirmation of the critical function of ATP for paracrine activation of mTOR signaling by dying Lgr5+ cells, we were able to reduce the level of S6 phosphorylation induced by DT treatment or necrotic supernatant by hydrolyzing released ATP to AMP and inorganic phosphate by using apyrase (Fig. 3f, g). Blocking the P2 family of purinergic receptors using a combination of nonselective P2 receptor antagonists in combination with 5-FU phenocopied the effect of 5-FU / rapamycin dual treatment on organoid survival (Fig. 3h). P2X4 is significantly more expressed in mouse tumor cells compared to all other P2 receptors (Fig. 3i), and chemical inhibition of P2X4 in combination with 5-FU treatment led to reduced organoid viability in AOM / DSS and APTAK tumor organoids when compared to each treatment alone (Fig. 3k). Similar results were seen with genetic knockdown of P2X4 in APTAK tumor organoids.

[0068] In conclusion, our experiments reveal how increased ATP release from dying tumor cells (due to cell death-inducing chemotherapeutic agents and / or cell death-inducing therapies) activates mTOR survival signals in neighboring tumor cells in a P2X4 receptor-dependent manner. This activation is essential for remaining tumor cells to counteract increased proapoptotic ROS signals within the tumor in order to avoid cell death and maintain tumor integrity (Figure 4k). These findings open up new strategies for the effective treatment and / or prevention of solid tumors and metastases.

[0069] The present invention is further described in the following examples, without however being limited thereto, with reference to the accompanying drawings, in which: For purposes of the present invention, all references cited herein are incorporated by reference in their entirety. EXAMPLES

[0070] method

[0071] animal research

[0072] All experiments involving animals were reviewed and approved by the Regierungsprasidium Darmstadt, Darmstadt, Germany. EGFP-DTR(+) Mice were as described in references (3,4). scid Il2rg tm1Wjl NSG mice were purchased from the Jackson Laboratory and colonies were maintained in the animal facilities of the Georg-Speyer-Haus in Frankfurt or the MFD Diagnostics in Wendelsheim. Male or female NSG mice aged 8–10 weeks were used for the experiments. NOD / Shiscid, IL-2Rγnull (NOG) mice (7–12 weeks old, male) were obtained from the Central Institute for Experimental Animals (CIEA, Japan) and maintained in the animal facility of Keio University.

[0073] AOM / DSS-induced colon tumorigenesis and mini-endoscopy

[0074] Colon tumors were induced as described in ref. (33). Mice (8–10 weeks old) were injected intraperitoneally (ip) with AOM (10 mg / kg; Sigma, #A5486) and, 5 days later, were given 2% DSS (molecular weight, 36–50 kDA, MP Biochemicals, #SKU0216011090) in drinking water for 5 days followed by regular water for 14 days. DSS treatment was repeated for two more cycles. Miniendoscopy was performed on anesthetized mice at approximately day 80 and after treatment using a mouse miniendoscopy system (Karl Storz). Videos recorded from the endoscopy system were opened and viewed in VLC Media Player, and still images were captured from these videos.

[0075] Xenograft experiments

[0076] Organoids were collected using ice-cold Cell Recovery Solution (Corning, #354253) and mechanically dissociated into small clusters. Dissociated organoid cells were resuspended in PBS, mixed with 50% Matrigel (Corning, #356231) in a final volume of 100 μl, and injected subcutaneously into the right flank of NSG mice. Tumor size was monitored with a digital caliper, and tumor volume was calculated according to the formula (length × width × width) / 2. At the end of the experiment, mice were humanely euthanized and tumors were harvested.

[0077] Diphtheria toxin and other in vivo therapeutics

[0078] DT (Millipore, #322326) treatment was performed as described in references (3,4). Briefly, DT was injected intraperitoneally at a dose of 50 μg / kg for the times indicated in individual experiments. The mTOR inhibitor rapamycin (LC Labs, #-5000) was dissolved in 5% ethanol, 5% PEG400, and 5% Tween80 in PBS and injected intraperitoneally at 10 mg / kg once daily for the times indicated. 0.5% N-acetylcysteine ​​(Sigma-Aldrich, #A91165) was added to drinking water ad libitum. 0.5 mg / ml doxycycline (Sigma#D9891) was added to drinking water with 3% sucrose ad libitum. 5-FU (Sigma#F6627) was dissolved in sterile HO and injected intraperitoneally at a concentration of 20 mg / kg for the times indicated in individual experiments.

[0079] Tumor organoid culture

[0080] Colon tumor organoids were cultured as described in reference (27). AOM / DSS-induced colon tumors were harvested by longitudinally opening the colon and enzymatically dissociated for 30–40 min in digestion medium containing 1 mg / ml collagenase I (Sigma-Aldrich), 0.5 mg / ml dispase (Roche), and 50 μg / ml DNase (Sigma-Aldrich) in RPMI medium containing 2% FCS. The supernatant was washed and filtered through a 70 μM cell strainer. Cells were then embedded in Matrigel (Corning, #356231) and cultured in Advanced Dulbecco's Modified Eagle Medium / F12 (Invitrogen, #12634-028) supplemented with Glutamax (Invitrogen, #35050038), Hepes (Invitrogen, #15630056), Pen / Strep (Invitrogen, #15140122), N2 supplement (Invitrogen, #17502048), B27 supplement (Invitrogen, #17504044) and n-acetylcysteine ​​(Sigma-Aldrich, #A91165). EGF (Invitrogen, #PMG8045), RspoI, and noggin were added as niche factors. To culture ATKN organoids, puromycin (2 μg / ml), hygromycin (200 μg / ml) and blasticidin (5 μg / ml) were added to the medium.APTAK tumor organoids were generated from Apc Δ / Δ; Trp53 Δ / Δ; Tgfbr2 Δ / Δ; wild-type colon organoids by introduction of myristoylated human Akt and KrasG12D (Heichler et al. STAT3 activation through IL-6 / IL-11 incancer-associated fibroblasts promotes colorectal tumour development andcorrelates with poor prognosis. Gut. 2020 Jul;69(7):1269-1282. doi:10.1136 / gutjnl-2019-319200. Epub 2019 Nov 4. PMID: 31685519) and cultured in organoid medium containing puromycin (2 μg / ml) and hygromycin (200 μg / ml). LGR5-iCapspase9-T2A-tdTomato knock-in colon cancer organoids were established as described in reference (21). LGR5-iCT organoids were cultured without RspoI.

[0081] Organoid processing and organoid harvesting

[0082] For in vitro Lgr5+ cell depletion experiments, organoids were treated with 20 ng / ml DT (Millipore, #322326) in culture medium. For ablation of human LGR5+ cells, LGR5-iCT organoids were treated with 500 nM BB homodimerizer. Other treatments were performed as described in each figure legend. Rapamycin (10 μM, Selleckchem, #S1039), PH797804 (2 μM, Tocris, #5866), SP600125 (5 μM, Sigma, #S5567), 5-BDBD (1 μM and 10 μM, Tocris#3579), NF279 (10 μM, Tocris, #1199), PPADS (60 μM, Santacruz, #sc-202770), PTx (40 ng / ml, Tocris, #3097), NAC (2 mM, Sigma-Aldrich, #A91165), Trolox (1 mM, Tocris#6002), BHA (10 μM, Sigma#B1253), theophylline (50 μM, Sigma, #T1633), A438079 (10 μM, Sigma, #A9736), 2'3' cGAMP (10 and 30 μg / ml, Invivogen, #tlrl-nacga23), ATP (100 μM, Sigma, #A6419), 2MeS-ADP (100 μM, Tocris, #1624), adenosine (100 μM, Sigma, #A92251), 5-fluorouracil (20 μM, Sigma, #F6627-5G); C-176 and C-178 (500 nM, Ablasser Lab18), doxycycline (Sigma #D9891), anti-HMGB1 antibody (5 μg / ml), Enbrel (Etanercept, 10 Antibodies against sarcoma (100 mg / ml) and anakinra (Kineret, 100 ng / ml) were used. After treatment, organoids were harvested with ice-cold Cell Recovery Solution (Corning, #354253) and pelleted by centrifugation. To prepare protein lysates for Western blot analysis, organoid pellets were washed twice with cold PBS and pellets were either flash frozen or lysed in lysis buffer.

[0083] Measurement of ROS and ATP levels

[0084] ATP levels in organoid supernatants or necrosis media were measured using the CellTiter-Glo2.0 cell viability assay (Promega #G9242) according to the manufacturer's instructions. ROS levels were measured using H2DCFDA (ThermoFisher #D339). Briefly, organoids were treated with the indicated 5-FU, stained with H2DCFDA, and analyzed for H2DCFDA positivity using a fluorescent microscope or FACS.

[0085] Necrosis supernatant transfer experiment

[0086] Mouse colorectal cancer cells (CMT93 and CT26) and human colorectal cancer cells (HCT116 and RKO) were cultured overnight in DMEM medium containing 2% FCS, and culture supernatants with or without cells were collected, flash frozen for 5 min in liquid nitrogen, thawed at 37 °C, and filtered through a 0.45 μM filter. Filtered control supernatants (without cells) and necrotic supernatants (filtered supernatants of culture medium containing cells) were transferred to 60–70% confluent cells (cultured overnight in DMEM medium containing 2% FCS). Cells were collected in lysis buffer at 2 and 4 h time points for Western blot analysis. For the transfer experiment of necrotic supernatant in organoid control, necrotic supernatant was prepared from CT26 cells cultured overnight in serum-free Advanced DMEM / F12 and Lgr5 cells. EGFP-DTR(+) Tumor organoids were harvested after 4 hours and lysed in lysis buffer for Western blot analysis.

[0087] Western blotting

[0088] Harvested organoids were lysed in lysis buffer as described elsewhere (33). Protein content was quantified using Protein Assay Dye Reagent (Bio-Rad, #5000006), and 40 μg of total protein lysate in Laemmli buffer (Bio-Rad, #161-0747) was separated by SDS-PAGE, transferred to a 0.45 μm PVDF membrane (Millipore, #IPVH00010), and blocked for 1 h with 5% nonfat milk and 1% BSA in PBS. After blocking, the membrane was incubated with primary antibody overnight at 4 °C. The following antibodies were used: cleaved caspase 3 (#9661), cleaved PARP (#9548), phospho-histone H2a.X (#2577), phospho-p70 S6 kinase (#9205), p70S6 kinase (#9202), phospho-S6 (#2211), S6 (#2217), phospho-p38 (#9211), p38 (#9112), phospho-ERK (#4370), ERK (#9102), phospho-cJUN (#9261), cJUN (#9165), c-Myc (#5605), Cell Signaling-derived Cyclin D1 (#2922), P2X4 (Abcam#134559) and Sting (Abcam, #ab92605). Actin (Sigma, #A4700) was used as a loading control. After washing with PBS, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies for 1 h at RT, and the membranes were developed using SuperSignal West Pico Chemoluminescent Substrate (Thermo FisherScientific, #34580).

[0089] FACS

[0090] For FACS analysis, organoids were collected in ice-cold CellRecovery Solution, washed in cold PBS, dissociated into single cells using Accutase (Sigma, #A6964) for 5 min at room temperature, filtered through a 40 μM cell strainer, and resuspended in FACS buffer (cold PBS containing 1 mM EDTA and 1% FCS). Propidium iodide (Thermo Fisher, #BMS500PI) was added to the single cell suspension to exclude non-viable cells for flow cytometry. BD LSRFortessa 商標 Samples were acquired using a cell analyzer (BD Bioscience) and analyzed using FlowJo v9 (FlowJO LLC). For colon fibroblasts, DSS colon fibroblasts and colon tumor fibroblasts, tissues were harvested, washed in PBS containing Pen / Strep antibiotics, minced using a sterile scalpel and digested in digestion medium as above. The supernatant was washed and filtered through a 40 μM cell strainer. Cells were stained using the following antibodies: CD45 APC (#17-0451), CD31 eFlour450 (#48-0311), EpCAM PE-Cy7 (#25-5791-80), PDGFRα PE (#12-1401-81) (eBiosciensce). Viability dye eFluor 780 (eBiosciensce #65-0865-14) was included to exclude non-viable cells. BD FACSAria 商標 Samples were acquired and sorted using a Fusion sorter. For RNA extraction, cells were sorted directly in RLT lysis buffer and RNA was isolated using the RNeasy Micro Kit (Qiagen, # 74004).

[0091] siRNA-mediated knockdown

[0092] CT26 cells at 60–70% confluence were transfected with 25 nM siRNA smart pools against mouse Rheb (#L-057044-00), RagA (#L-057667-01) or RagB (#L-066440-01) siRNA (Dharmacon) using DharmaFECT (Dharmacon) and Lipofectamine 2000 (Invitrogen) transfection reagents. RNA was purified after 48 hours to confirm knockdown efficiency. For supernatant transfer experiments, medium was changed 16 hours after transfection and supernatants were collected after 72 hours, filtered and stored at 4°C.

[0093] RNA extraction and qRT-PCR

[0094] For RNA extraction, organoids were harvested directly in RLT buffer and RNA extraction was performed using the RNeasy Mini kit (Qiagen, #74106) according to the manufacturer's instructions. Total RNA (1–2 μg) was reverse transcribed into cDNA using SuperscriptII reverse transcriptase (Invitrogen, #18064-071) and OligodT (Invitrogen, #18418020). Real-time quantitative PCR was performed on a StepOne Plus Real-Time PCR system (Applied Biosystems) using FastStart Universal SYBR Master Mix (Roche, #4913914001) in a total volume of 20 μl. Relative gene expression levels were quantified using cyclophilin (2 [Ct cyclophilin-Ct target gene]) as a housekeeping gene.

[0095] Immunostaining and in situ hybridization

[0096] Immunostaining was performed as described in reference (35). Briefly, tissues were fixed with 4% paraformaldehyde. 6 μm OCT frozen tissue sections or 5 μm paraffin-embedded tissue sections were processed for immunostaining using standard histological protocols. The following primary antibodies were used: EGFP (Abcam, #ab6673), tdTomato (Rockland, #600-401-379), cleaved caspase 3 (Cell Signaling, #9579), Ki67 (Leica Bioststems, #NCL-L-Ki67-MM1), phospho-S6 (Cell Signaling, #2211), P2X4 (Abcam#134559). Sections were then incubated with HRP-conjugated antibodies (Dako) and signals were detected by DAB reaction. For immunofluorescence staining after primary antibody incubation, sections were incubated with fluorochrome-conjugated secondary antibodies (Invitrogen), and sections were mounted with ProLong Gold Antifade Mountant containing DAPI (Invitrogen, #P36931). Images were taken with a Zeiss microscope.

[0097] Plasmid, lentivirus, and retrovirus production

[0098] The top two P2X4 and Raptor shRNA sequences were obtained and cloned from the mir-E-based retroviral backbone as described in reference (36). K-ras G12D was cloned into pBABE-puro (Addgene plasmid #1764). IRIS-AKT (myristoylated) was cloned downstream of the hygromycin cassette in pMSCV-rtTA3-PGK-Hygro (kindly provided by Lars Zender, Tubingen). Retroviral particles were generated by cotransfecting the retroviral plasmid with the packaging plasmid pCL-ECO (Addgene plasmid #12371) into HEK293T cells using the calcium phosphate method. The Notch intracellular domain (NICD) was cloned into LentiCas9-BSD (Addgene plasmid #52962) by replacing Cas9. Lentiviral supernatant was prepared by co-transfection of Lenti-NICD-BSD plasmid, packaging vector psPAX2 (Addgene plasmid #12260), and envelope vector pMD2.G (Addgene, #12259) using Lipofectamine 2000. The medium was changed 12 hours after transfection, and viral supernatant was collected 48 hours later and filtered through a 0.45 μm PVDF filter (Millipore). The viral supernatant was concentrated by centrifugation at 12,000 rpm at 4°C overnight, and the viral pellet was resuspended in organoid medium. Single cells derived from organoids were infected with viral supernatant supplemented with polybrene (8 μg / ml polybrene, Sigma). Four hours after infection, cells were embedded in matrigel and cultured in organoid medium supplemented with 10 μM Y-27632 (Sigma). 48 h after infection, organoids were selected in 2 μg / ml puromycin, 200 μg / ml hygromycin or 5 μg / ml blasticidin for 5–7 days.Tgfr2gRNA was cloned into a gRNA cloning vector (Addgene plasmid #41824) as described in reference (37). Single cells derived from organoids were co-transfected with gRNA plasmid and Cas9 expression plasmid (Addgene plasmid #41815) using Lipofectamine 2000 as described in reference (38). Tgfbr2 mutant organoids were selected in the absence of Noggin and in the presence of 50 ng / ml TGF-β (R&D Systems, #7666-MB-005).

[0099] statistical analysis

[0100] No statistical methods were used to predetermine the sample size for the in vivo experiments. For the subcutaneous implantation experiments, tumor sizes were approximately 100–200 mm 3 Mice were distributed into treatment groups when the volume reached 100 μg / kg. Statistical significance between two groups was determined by two-tailed Student's t test, and for three or more groups, one-way or two-way ANOVA with Bonferroni's multiple comparison test was performed (GraphPad Prism 4.03, GraphPad Software, Inc.). All data in graphs are presented as mean ± SEM unless otherwise stated.

[0101] result

[0102] In experiments conducted in connection with the present invention, the present inventors used AOM / DSS to measure Lgr5 EGFP-DTR(+) We induced colon tumors in mice (4). In contrast to subcutaneous tumors, where depletion of Lgr5+ cells leads to tumor stagnation (3), we could not detect a significant difference in tumor size or a decrease in tumor cell proliferation when depleting Lgr5+ cells for 9 days in this orthotopic setting (Fig. 1a-e). We also could not identify any change in the number of apoptotic cells in the tumor 24 hours after depletion of Lgr5 cells (Fig. 1f).

[0103] Similarly, AOM / DSS processing Lgr5 EGFP-DTR(+) Deletion of Lgr5+ cells in mouse-derived CRC organoids did not affect the overall morphology or growth rate of tumor organoids (Fig. 1g). These results strongly indicate that inflammation-associated colon tumors are independent of Lgr5+ cells. Despite the controversial role of Lgr5+ cells as cancer stem cells, it is surprising that colon tumors are apparently unaffected by this rapid deletion of Lgr5+ cells, which represent an average of ∼30% of tumor cells in our CRC model (Fig. 1d). Instead, the rapid depletion of Lgr5+ cells resulted in a transient increase in Ki-67+ tumor cells at day 4 (i.e., 24 h after the second diphtheria toxin (DT) application).

[0104] We conclude that activation of survival programs in Lgr5-negative cells helps tumors tolerate Lgr5 cell loss and maintain tumor integrity. To test this finding and characterize the molecular mechanisms involved in Lgr5- cell survival, we examined Lgr5- cells for activation of known survival pathways 24 hours after Lgr5+ cell depletion. EGFP-DTR(-) We screened organoids derived from AOM / DSS-induced colon tumors in mice. Our immunoblot analysis showed that a single DT administration enhanced the phosphorylation of c-Jun, p38, p70 S6 kinase, and S6 ribosomal protein (Fig. 1h). Phosphorylation of c-Jun, p38, and S6 was observed in vivo 24 h after the first DT administration, as was an increase in c-Myc and Cyclin D1 (Fig. 1j).

[0105] Of note, depletion of Lgr5+ cells also led to S6 phosphorylation in human cancer organoids (21). To functionally confirm that the involved MAP kinase or mTOR activation is indeed involved in the survival of Lgr5+ cell-depleted organoids, we applied SP600125, PH797804, or rapamycin to inhibit JNK, p38, and mTOR, respectively, in the presence or absence of DT. While the two MAPK inhibitors did not affect the survival of tumor organoids, the combination of DT and rapamycin caused substantial cell death within 24 h selectively in tumor organoids expressing DTR (Fig. 1k). Moreover, reseeding of DT / rapamycin co-treated organoids significantly reduced their organoid formation capacity, which was not observed when either compound was applied individually (Fig. 1l).

[0106] Consistent with the critical role of mTORC1 in survival upon Lgr5+ cell deletion, shRNA-mediated knockdown of raptor significantly reduced the reseeding ability of DT-treated tumor organoids, although raptor expression was only reduced by approximately 40% (Figure 1m-o). To confirm that mTOR is activated in Lgr5- but not in the remaining Lgr5+ cells, we confirmed nearly 100% deletion of Lgr5+ cells in tumor organoids 24 h after DT treatment by FACS (Figure 1i). Furthermore, we performed double staining of Lgr5-EGFP and pS6 in AOM / DSS tumor tissues, and efficient Lgr5+ cell deletion and S6 phosphorylation were confirmed only in Lgr5- cells 24 h after DT injection (Figure 1j). Downstream of mTOR, we observed that application of DT / rapamycin significantly reduced the expression of c-myc and cyclin D1, which was also associated with a significant reduction in tumor cell proliferation in AOM / DSS-induced tumors.

[0107] Considering that the AOM / DSS model leads to the formation of only benign adenomas, we intended to confirm that mTOR activation is also relevant for tumor maintenance upon Lgr5+ cell depletion in more advanced stages of colon cancer. Therefore, we transduced constitutively active AKT (myristoylated AKT), mutant K-Ras (K-rasG12D), and active Notch1 (NICD) into Lgr5+ cells by either retroviral or lentiviral transduction. EGFP-DTR(+) The DT / rapamycin combination was introduced into AOM / DSS-induced tumor organoids derived from mice. Furthermore, Tgfbr2 was knocked out by CRISPR / Cas9-mediated gene editing, resulting in AOM / DSSATKN tumor organoids (Fig. 2a). Indeed, even in AOM / DSSATKN tumor organoids, depletion of Lgr5+ cells led to phosphorylation of S6 ribosomal protein, which could be prevented by rapamycin (Fig. 2b), and co-administration of DT / rapamycin led to the disappearance of proliferating tumor cells in subcutaneously implanted tumors within 24 h. Notably, histological evaluation of tumor tissues showed that no viable epithelial tumor cells were present in tumor tissues from DT / rapamycin-treated mice just 24 h after treatment (Fig. 2c).

[0108] Rapidly proliferating cells require an increased supply of nucleotides, which is achieved by de novo synthesis of nucleotides to be incorporated into RNA and into DNA in proliferating cells. Interestingly, both Myc and mTOR signaling can stimulate de novo synthesis of nucleotides (5, 6). Inhibitors of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in purine synthesis, have been used to target Myc- and mTORC1-dependent cancers (7, 8).

[0109] Given the activation of both mTOR and Myc signaling in Lgr5+ cell-depleted tumors, we investigated whether an IMPDH inhibitor could mimic the effects of mTOR inhibition. EGFPDTR(+)Treatment of tumor organoids and subcutaneously implanted AOM / DSSATKN tumors with the IMPDH inhibitor mizoribine in combination with DT-mediated depletion of Lgr5+ cells resulted in severe disintegration of organoids in vitro and markedly reduced viability and proliferation of AOM / DSSATKN tumors without affecting phosphorylation of S6-ribosomal protein. These data suggested that de novo purine synthesis downstream of mTOR activation is required to maintain tumor integrity in the absence of Lgr5+ cells.

[0110] Because activation of mTOR signaling is known to suppress apoptosis (Hui L, Abbas T, Pielak RM, Joseph T, Bargonetti J, Foster DA. Phospholipase D elevates the level of MDM2 and suppresses DNA damage-induced increases in p53. Mol Cell Biol. 2004 Jul;24(13):5677-86. doi:10.1128 / MCB.24.13.5677-5686.2004. PMID: 15199126; PMCID: PMC480910), we tested whether inhibition of the mTOR survival pathway following death of Lgr5+ would induce apoptosis in the remaining Lgr5- cells, leading to rapid and massive death of remaining tumor cells. To this end, we evaluated the kinetics of tumor cell apoptosis in the combination of Lgr5+ cell depletion and mTOR inhibition by immunostaining for cleaved caspase 3 on subcutaneously implanted tumor tissues of mice treated with DT combined with rapamycin for 6, 12 or 18 hours. As in DT-treated mice, in DT / rapamycin-treated mice, we could detect only a few Lgr5+ and Lgr5- apoptotic tumor cells 6 hours after treatment, a time point when Lgr5+ cells were still present in the tumor. However, at 12 hours after treatment, when Lgr5+ cells were fully depleted from the tumor, we detected extensive apoptosis in tumor tissues of animals treated with DT / rapamycin, but almost no apoptosis was detectable in animals treated with DT alone (Fig. 2d).

[0111] Eighteen hours after DT / rapamycin treatment, apoptosis was complete and most of the remaining Lgr5- tumor cells were dead, as indicated by the lack of cleaved caspase 3 and tumor tissue histology (Fig. 2d), suggesting that activation of mTOR signaling induced by Lgr5+ cell death is important for Lgr5- cell survival. Western blot analysis of colon tumor organoids using antibodies against p-γH2AX demonstrated increased DNA damage upon DT / rapamycin treatment, and antibodies against cleaved caspase 3 and cleaved PARP confirmed the increased apoptosis caused by the combined Lgr5+ ablation and mTOR inhibition. Consequently, in contrast to single doses of rapamycin or DT applied individually which only caused tumor stasis, prolonged rapamycin treatment and Lgr5+ cell depletion caused significant tumor regression of established tumors (Figures 2e–g), confirming that the requirement for mTOR activation for tumor maintenance is independent of the underlying mutational burden in the tumor.

[0112] The observed paracrine mTOR activation may not represent a specific response to tumor stem cell death alone. In line with this idea, we investigated whether tumor-free, untreated Lgr5 EGFP-DTR(+)We confirmed robust phosphorylation of S6 ribosomal protein in intestinal epithelial cells (IECs) when Lgr5+ cells were depleted in mice, and we also observed enhanced S6 phosphorylation in IECs when IEC apoptosis was induced in a stem cell-free manner in non-transformed wild-type mice using the carcinogen azoxymethane or single-dose total body irradiation (12 Gy) (Fig. 2h). Based on this, we conclude that the combination of mTOR inhibition and cell death induction can be therapeutically exploited to enhance cytotoxic therapeutic responses in tumors. As expected, 5-FU induced S6 phosphorylation in AOM / DSS tumor tissues and tumor organoids, which was suppressed by administration of rapamycin (Fig. 2i, j), and the combination of rapamycin and 5-FU significantly suppressed the reseeding ability of tumor organoids (Fig. 2k, m).

[0113] Importantly, co-treatment with 5-rapamycin also suppressed mTOR activation and organoid survival in human tumor organoids (Figure 2l,m).Similar to Lgr5+ cell depletion, 5-FU treatment in combination with rapamycin significantly reduced tumor growth in subcutaneously implanted tumors compared to controls or each treatment alone (Figure 2n-p).

[0114] Although it has been shown that Lgr5 expression confers resistance to 5-FU-based chemotherapy (Hsu HC, Liu YS, Tseng KC, Hsu CL, Liang Y, Yang TS, Chen JS, Tang RP, Chen SJ, Chen HC. Overexpression of Lgr5 correlates with resistance to5-FU-based chemotherapy in colorectal cancer. Int J Colorectal Dis. 2013Nov;28(11):1535-46. doi: 10.1007 / s00384-013-1721-x. Epub 2013 Jun 20), we aimed to confirm that mTOR activation by 5-FU treatment is independent of Lgr5+ cell death. The presence of Lgr5+ cells in our AOM / DSS tumor organoids was highly dependent on BMP signaling inhibition, and removing the BMP inhibitor Noggin from our standard organoid medium reduced the proportion of Lgr5+ to less than 1%. However, regardless of the absence of Lgr5+ cells, 5-FU treatment was able to induce phosphorylation of S6 in AOM / DSS tumors or organoids, indicating that activation of mTOR does not require the death of tumor stem cells. We further wished to prove that activation of mTOR signaling by cell death does not require a preceding inflammatory component.To this end, the inventors utilized genetically engineered APTAK tumor organoids, generated by introduction of myristoylated human Akt and KrasG12D in Apc Δ / Δ; Trp53 Δ / Δ; Tgfbr2 Δ / Δ; wild-type colon organoids (Heichler et al. STAT3 activation through IL-6 / IL-11 incancer-associated fibroblasts promotes colorectal tumour development and correlates with poor prognosis. Gut. 2020 Jul;69(7):1269-1282. doi:10.1136 / gutjnl-2019-319200. Epub 2019 Nov 4. PMID: 31685519). Similar to previous results in AOM / DSS tumor organoids, here 5-FU induced mTOR-dependent phosphorylation of S6, and inhibition of mTOR signaling upon 5-FU treatment significantly reduced the reseeding ability of organoids compared to treatment with 5-FU alone.

[0115] Next, we aimed to evaluate how depletion of Lgr5+ cells could induce mTOR activation in neighboring Lgr5- cells. Cell death leads to the release of danger-associated molecular patterns (DAMPs), including various growth factors, inflammatory mediators, dsDNA, and metabolites (9-11), which trigger inflammation, activate innate immunity to prevent damage, and coordinate tissue repair and wound healing (12). To test whether DT-mediated death of Lgr5+ cells leads to the release of DAMPs that may be responsible for paracrine mTOR activation, we investigated the mechanism by which DT-mediated depletion of Lgr5+ cells could induce mTOR activation in neighboring Lgr5- cells. EGFP-DTR(+) Using supernatant from tumor organoids, Lgr5 EGFP-DTR(-)Tumor organoids were treated for 22 hours to confirm that phosphorylation of S6 ribosomal protein was increased in organoids exposed to supernatant from DT-treated cells (Fig. 3a). Given that DT induces apoptosis but not other forms of cell death, we hypothesized that the release of either dsDNA or ATP could trigger phosphorylation of S6 ribosomal protein. Since we had observed an upregulated interferon response upon killing of Lgr5+ cells in AOM / DSS-induced tumors, as well as in subcutaneously implanted tumors (Extended Data Fig. 3a), which could also be observed in DT-treated tumor organoids, we first focused on dsDNA, which is known to initiate transcription of type I interferon (IFN) and interferon-stimulated genes (ISGs) (14-17) by activating the cGAS-STING pathway, which has also been recently shown to confer pro-tumorigenic and pro-metastatic properties when activated in tumor cells (13). Indeed, exogenous cGAMP induced phosphorylation of S6 ribosomal protein in a dose-dependent manner. EGFP-DTR(+) Neither the selective STING inhibitors C-176 and C-17818 nor CRISPR-mediated STING knockout in AOM / DSS-induced tumor organoids was able to prevent phosphorylation of S6 ribosomal protein after Lgr5+ cell depletion, ruling out an essential contribution of dsDNA to mTOR activation. Another DAMP released by dying cells is ATP, which we could detect as elevated in the supernatant of DT-treated organoids (Figure 3b). Extracellular ATP can be hydrolyzed by ATP hydrolases present in the tumor microenvironment to generate ADP and adenosine, which act as natural ligands for purinergic receptors (P2R) and adenosine receptors (P1R), and addition of either ATP or its hydrolysis products increased the phosphorylation of Lgr5+ cells. EGFP-DTR(-) Phosphorylation of S6 ribosomal protein in tumor organoids is induced (Figure 3c).

[0116] Importantly, exposure of both mouse and human colorectal tumor cells to the supernatant of necrotic cells generated by freeze-thaw cycles contained ATP and induced significant S6 phosphorylation (Figure 3d), emphasizing the idea that paracrine mTOR activation occurs independently of the type of cell death. EGFP-DTR(+) Incubation of tumor organoids with necrotic medium also resulted in S6 phosphorylation (Figure 3e).

[0117] In confirmation of the critical function of ATP for paracrine activation of mTOR signaling by dying Lgr5+ cells, we were able to reduce the levels of S6 phosphorylation induced by DT treatment or necrotic supernatants by hydrolyzing released ATP to AMP and inorganic phosphate using apyrase (Fig. 3f, g). Mechanistically, mTOR activation by ATP was dependent on Rheb rather than RagGTPases, as shown by the reduced ATP-induced S6 phosphorylation when Rheb was downregulated by siRNA in CRC cells. Downregulation of Rag A and Rag B had no obvious effect on p-S6 levels.

[0118] ATP signals through the P2 family of purinergic receptors, which include seven subtypes of P2X receptors and eight subtypes of P2Y receptors (20). Blocking these receptors using a combination of nonselective P2 receptor antagonists in conjunction with 5-FU recapitulated the effect of 5-FU / rapamycin dual treatment on organoid survival (Figure 3h). Furthermore, the nonselective P2 receptor antagonists PPADS and (PTx) blocked Lgr5+ cell death-induced S6 phosphorylation. We found that DT-treated Lgr5 cells were significantly more susceptible to S6 phosphorylation after 24 h of DT treatment. EGFP-DTR(-)We sequenced tumor cells from IECs and compared the expression of P2 receptors with cells from vehicle-treated mice, finding no obvious differences in the expression of P2X, P2Y receptors or connexin and pannexin hemichannels in DT-treated animals. Of note, P2X4 was significantly more highly expressed in mouse tumor cells compared to all other P2 receptors, as confirmed by our sequencing data and qPCR (Figure 3i). We also were able to detect a much higher expression of P2X4 in human tumor organoids compared to other P2 receptors, whereas organoids derived from healthy human tissues showed similarly high P2X4 expression levels, suggesting that in general, P2X4 is the most prominent P2 receptor in IECs. Immunohistochemistry for P2X4 in mouse intestinal tissue confirmed a strong and comparable expression of P2X4 in normal and tumorigenic colon tissues.

[0119] Given these expression data, we tested whether blockade of this single P2 receptor was sufficient to suppress ATP-induced activation of the mTOR survival pathway. Indeed, chemical inhibition with the selective P2X4 inhibitor 5BDBD reduced ATP-induced phosphorylation of S6 in AOM / DSS tumor organoids (Fig. 3j), whereas the use of inhibitors against P2x1 (NF279) or P2x7 (A-438079) did not reduce ATP-dependent S6 phosphorylation (Extended Data Fig. 3m). Thus, chemical inhibition of P2X4 in combination with 5-FU treatment led to reduced organoid viability in AOM / DSS and APTAK tumor organoids when compared to each treatment alone (Fig. 3k). Similarly, genetic knockdown of P2X4 in APTAK tumor organoids reduced mTOR activation induced by 5-FU and significantly reduced organoid survival compared to P2X4-competent organoids (Fig. 3l-n). In vivo knockdown of P2X4 in subcutaneously implanted tumors inhibited S6 phosphorylation by 5-FU treatment, resulting in tumor cell death and significantly reduced tumor growth compared to P2X4-competent tumors (Fig. 3o–r).

[0120] Taken together, our data show that ATP released by dying tumor cells triggers mTOR activation in neighboring tumor cells, which is critical for their survival. However, these cells must also receive a signal to trigger apoptotic cell death, which must be suppressed by mTOR. Consistent with this hypothesis, inhibition of apoptosis using zVAD (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) reduced the negative impact of 5-FU / rapamycin combination treatment on organoid survival (Figure 4a).

[0121] Since inhibition of apoptosis may rescue organoid death, we aimed to explore which upstream signals induce apoptosis and tumor organoid death upon 5-FU / rapamycin treatment. To this end, we screened inhibitors against potential pro-apoptotic DAMPs known to be released upon cell death, such as N-acetylcysteine ​​(NAC) against reactive oxygen species (ROS), anti-HMGB1 antibody, TNF signaling inhibitor Enbrel, STING inhibitor C176 / C178, and IL-1 signaling inhibitor Anakinra. Notably, only inhibition of ROS signaling by NAC was able to rescue the negative effect of 5-FU / rapamycin on organoid reseeding ability (Figure 4b), suggesting that ROS, when combined with inhibition of mTOR signaling, may be responsible for the increased DNA damage and apoptosis seen after chemotherapy. The identical effect of two other ROS inhibitors (vitamin E analog 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and butylated hydroxytoluene (BHA)) on organoid survival further indicated that ROS are key mediators of organoid death in our scenario (Figure 4c). By using the fluorescent dye 2',7'-dichlorofluorescin diacetate (DCFDA) to measure ROS levels in organoids, we were able to detect tumor organoid-specific ROS increase by 5-FU treatment, which can induce DNA damage and apoptosis in residual tumor cells (Figure 4d).

[0122] Thus, our results demonstrated reduced DNA damage and apoptosis in tumor organoids treated with DT and rapamycin plus NAC when compared to DT / rapamycin treatment alone, confirming that ROS are paracrine triggers of apoptosis (Figure 4e), and that in vivo NAC reduced apoptosis in tumor tissues of DT / rapamycin-treated animals (Figure 4f). Most importantly, and emphasizing the role of ROS as a mediator of tumor cell death, we were able to reverse tumor regression in 5-FU / rapamycin-treated mice by additional application of NAC (Figure 4g-j).

[0123] References

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[0125] Drawing Terminology ip intraperitoneal injection day Endoscopy sacrifice slaughter vehicle Average tumor size % of EGFP+ tumor cells % of Ki-67+ cells Propidium Iodide kinase β-actin Rapamycin organoids reseeding capacity Fold change Organoids Single cells noggin blasticidin Control cleaved Casp3 Tumor volume mouse human vs untreated in % ATP level Fold induction level fold induction Medium Necrotic medium Apyrase mRNA expression levels α-tubulin α-tubulin Doxycycline Treatment sc subcutaneous injection day -10 to -15 days anti-P2x4 anti-P2X4 Colon tumor organoids Dying tumor cell oxygen radicals mitochondrial ROS apoptosis

Claims

1. A pharmaceutical comprising a P2X4 receptor inhibitor or an inhibitor of the P2X4 receptor signaling pathway in combination with a cell death-inducing chemotherapeutic agent and / or a cell death-inducing therapy for the prevention and / or treatment of a solid tumor or metastasis thereof.

2. 2. The medicament comprising a P2X4 receptor inhibitor or an inhibitor of the P2X4 receptor signaling pathway in combination with a cell death-inducing chemotherapeutic agent and / or a cell death-inducing therapy according to claim 1, wherein said P2X4 receptor inhibitor is specific for P2X4 receptor inhibitors and is selected from the group of 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, IgG#151-LO, antibodies or fragments or derivatives thereof, siRNA, shRNA, miRNA, ribozymes, aptamers, antisense nucleic acid molecules, small molecules, or modified versions of these inhibitors.

3. 3. The medicament comprising a P2X4 receptor inhibitor or an inhibitor of the P2X4 receptor signaling pathway in combination with a cell death-inducing chemotherapeutic agent and / or cell death-inducing therapy according to claim 1 or 2, wherein the cell death-inducing chemotherapeutic agent is selected from the group consisting of supernatants of necrotic cells; cationic amphiphilic drugs (CAD); classical anticancer drugs including anthracyclines, antimetabolites, and platinum drugs; topoisomerase inhibitors, camptothecins; PDE3A inhibitors such as anagrelide alone or in combination with cell death-inducing cytokines; death-inducing cytokines including IFN-α, IFN-γ, TNF-α, or TRAIL; taxanes, paclitaxel, and fluorinated taxanes including SB-T-12851, SB-T-12852, SB-T-12853, and SB-T-12854, 5-fluorouracil, tegafur, capecitabine, and doxifluridine.

4. 4. The medicament comprising a P2X4 receptor inhibitor or an inhibitor of the P2X4 receptor signaling pathway in combination with a cell death inducing chemotherapeutic agent and / or a cell death inducing therapy according to any one of claims 1 to 3, wherein the solid tumor or metastasis thereof is selected from Lgr5+ cancer stem cell associated cancer, prostate cancer, pancreatic cancer, breast cancer, gastric cancer, liver cancer, brain cancer, lung cancer, renal cancer, colorectal cancer and metastases thereof.

5. 5. A medicament comprising a P2X4 receptor inhibitor or an inhibitor of the P2X4 receptor signaling pathway in combination with a cell death-inducing chemotherapeutic agent and / or a cell death-inducing therapy according to any one of claims 1 to 4, wherein the treatment and / or prevention further comprises prior and / or concomitant anti-cancer chemotherapy comprising cytotoxins, Lgr5 inhibitors, Lgr5+ cancer cell depletion, mTOR inhibitors, and IMPDH inhibitors.

6. 6. A medicament comprising a P2X4 receptor inhibitor or an inhibitor of the P2X4 receptor signalling pathway in combination with a cell death inducing chemotherapeutic agent and / or a cell death inducing therapy according to any one of claims 1 to 5, said combination being provided in combination and / or as separate dosage forms, simultaneously or separately.

7. 7. A medicament comprising a P2X4 receptor inhibitor or an inhibitor of the P2X4 receptor signalling pathway in combination with a cell death inducing chemotherapeutic agent and / or a cell death inducing therapy according to any one of claims 1 to 6, wherein said treatment and / or prevention comprises inhibition of a tumor evasion mechanism mediated by the ATP-dependent P2X4 receptor and / or mediated by the P2X4 receptor signalling pathway.

8. A medicament for the prevention and / or treatment of a solid tumor or metastasis thereof in a subject in need thereof, the medicament comprising: (i) simultaneously or sequentially administering an effective amount of a P2X4 receptor inhibitor or an inhibitor of the P2X4 receptor signaling pathway; (ii) A pharmaceutical comprising administering to said subject, either simultaneously or sequentially, an effective amount of a cell death-inducing chemotherapeutic agent and / or a cell death-inducing therapy.

9. 9. The pharmaceutical composition of claim 8, wherein the P2X4 receptor inhibitor is specific for P2X4 receptor inhibitors and is selected from the group of 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, IgG#151-LO, antibodies or fragments or derivatives thereof, siRNA, shRNA, miRNA, ribozymes, aptamers, antisense nucleic acid molecules, small molecules, or modified versions of these inhibitors.

10. 10. The pharmaceutical composition of claim 8 or 9, wherein the cell death-inducing chemotherapeutic agent is selected from the group consisting of supernatants of necrotic cells; cationic amphiphilic drugs (CAD); classical anticancer drugs including anthracyclines, antimetabolites, and platinum drugs; topoisomerase inhibitors, camptothecins; PDE3A inhibitors such as anagrelide alone or in combination with cell death-inducing cytokines; death-inducing cytokines including IFN-α, IFN-γ, TNF-α, or TRAIL; taxanes, paclitaxel, and fluorinated taxanes including SB-T-12851, SB-T-12852, SB-T-12853, and SB-T-12854, 5-fluorouracil, tegafur, capecitabine, and doxifluridine.

11. The method according to any one of claims 8 to 10, wherein the treatment and / or prevention comprises inhibition of a tumor evasion mechanism mediated by the ATP-dependent P2X4 receptor and / or the P2X4 receptor signaling pathway.

12. The pharmaceutical of any one of claims 8 to 11, wherein the treatment and / or prevention further comprises prior and / or concomitant anti-cancer chemotherapy comprising a cytotoxin, an Lgr5 inhibitor, Lgr5+ cancer cell depletion, an mTOR inhibitor, and an IMPDH inhibitor.

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