Production of organoid derived from ctc of small cell lung cancer patient, and IGF1r inhibitor as a molecular target drug for small cell lung cancer

Abstract

The present invention pertains to: a drug that is for small cell lung cancer and that contains, as an active ingredient, an insulin-like growth factor 1 receptor (IGF1R) inhibitor; and a method for determining whether or not administration of said drug is effective for treating a small cell lung cancer patient. Said method comprises a step for detecting expression of YAP1 or a factor downstream of same in cancer cells derived from the small cell lung cancer patient. As compared with control, a significantly high expression level of the YAP1 or a factor downstream of same indicates that administration of the drug is effective for treating the small cell lung cancer patient.

Description

Production of organoids derived from CTCs in small cell lung cancer patients and IGF1R inhibitors as molecular targeted therapeutic agents for small cell lung cancer

[0001] The present invention relates to the effectiveness of insulin-like growth factor 1 receptor (IGF1R) inhibitors for YAP1-positive small cell lung cancer, a subtype of small cell lung cancer, and a method for determining whether administration of an IGF1R inhibitor is effective in treating patients with small cell lung cancer. Furthermore, the present invention relates to a method for producing organoids derived from small cell lung cancer cells and a method for immunostaining YAP1 protein. This application claims priority based on Japanese Patent Application No. 2021-151032, filed in Japan on September 16, 2021, the contents of which are incorporated herein by reference.

[0002] In recent years, molecular targeted drugs targeting specific genes have been developed in the cancer field, and several drugs have improved the prognosis of cancer patients.

[0003] Small cell lung cancer (SCLC) accounts for approximately 15% of all lung cancers and has a poor prognosis. SCLC is a rapidly progressing cancer, and is often already inoperable at the time of diagnosis. This has made it difficult to collect sufficient patient samples, making it difficult to analyze its biological characteristics. To date, no molecular targeted therapeutic drug has been developed to treat SCLC, and patient prognosis has not improved.

[0004] Recently, it has been reported that small cell lung cancer can be classified into subtypes based on the expression of transcription factors ASCL1, NEUROD1, POU2F3, and YAP1 (see Non-Patent Document 1).

[0005] Baine MK, et al., SCLC Subtypes Defined by ASCL1, NEUROD1, POU2F3, and YAP1: A Comprehensive Immunohistochemical and Histopathologic Characterization, Journal of Thoracic Oncology, 15 (12), 1823-1835, 2020.

[0006] To develop a molecular targeted therapeutic drug and improve the prognosis of patients with small cell lung cancer, it is necessary to identify patient subgroups for which molecular targeted therapeutic drugs are effective, and to identify or develop effective therapeutic drugs for those subgroups. However, it has not been clear until now whether there are subgroups for which molecular targeted therapeutic drugs are effective, whether molecular targeted therapeutic drugs are effective for those subgroups, or whether there are methods for assessing the effectiveness of administering molecular targeted therapeutic drugs.

[0007] The present invention aims to provide a technique for identifying a subgroup of small cell lung cancer for which administration of a molecular targeted therapeutic agent is effective, identifying the molecular targeted therapeutic agent, and determining the effectiveness of administration of the molecular targeted therapeutic agent.

[0008] The present invention includes the following aspects: [1] A therapeutic agent for small cell lung cancer, comprising an insulin-like growth factor 1 receptor (IGF1R) inhibitor as an active ingredient. [2] The therapeutic agent according to [1], wherein the small cell lung cancer is small cell lung cancer in which the expression level of Yes1 Associated Transcriptional Regulator (YAP1) or a downstream factor thereof is significantly higher than that of a control. [3] The therapeutic agent according to [2], wherein the downstream factor of YAP1 is connective tissue growth factor (CTGF), cysteine-rich angiogenic inducer 61 (CYR61), or cyclin D1 (CCND1). [4] A method for determining whether administration of the therapeutic agent according to any one of [1] to [3] is effective in treating a patient with small cell lung cancer, the method comprising a step of detecting expression of YAP1 or a downstream factor thereof in cancer cells derived from the patient with small cell lung cancer, wherein a significantly higher expression level of YAP1 or a downstream factor thereof compared to a control indicates that administration of the therapeutic agent according to any one of [1] to [3] is effective in treating the patient with small cell lung cancer. [5] The method according to [4], wherein the downstream factor of YAP1 is CTGF, CYR61, or CCND1. [6] The method according to [4], wherein the detection of YAP1 expression is carried out by immunostaining of YAP1 protein. [7] The method according to any one of [4] to [6], wherein the cancer cells derived from the patient with small cell lung cancer are organoids derived from small cell lung cancer cells. [8] A method for producing organoids derived from small cell lung cancer cells, comprising a step of culturing cancer cells derived from a patient with small cell lung cancer in a medium.[9] The production method according to [8], wherein the medium contains at least two selected from the group consisting of insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2), EGF (epidermal growth factor) and epiregulin (EREG), and at least one selected from the group consisting of a Wnt agonist, a bone morphogenetic protein (BMP) inhibitor and a transforming growth factor-β (TGF-β) inhibitor.

[10] The production method described in [8] or [9], wherein the cancer cells are cancer tissue-derived cancer cells, pleural effusion-derived cancer cells, sputum-derived cancer cells, or blood-circulating cancer cells.

[0009] According to the present invention, it is possible to identify a subgroup of small cell lung cancer for which administration of a molecular targeted therapeutic agent is effective, to identify the molecular targeted therapeutic agent, and to provide a technique for determining the effectiveness of administration of the molecular targeted therapeutic agent.

[0010] FIG. 1 is a heat map created based on the results of RNA-seq analysis in Experimental Example 2. FIG. 2 is a graph showing the results of principal component analysis based on the results of RNA-seq analysis in Experimental Example 2. FIG. 3 is a micrograph of a representative small cell lung cancer organoid in Experimental Example 3. FIG. 4 is a diagram showing the relationship between IGF dependency, the type of transcription factor expressed, and the cluster of small cell lung cancer organoid lines in Experimental Example 3. FIG. 5 is a diagram showing the results of coexpression cluster analysis performed on IGF-dependent organoids and IGF-independent organoids in Experimental Example 3. FIG. 6 is a diagram showing the results of hierarchical cluster analysis performed on IGF-dependent organoids and IGF-independent organoids in Experimental Example 3. FIG. 7 is a photograph showing the results of immunostaining of small cell lung cancer organoids in Experimental Example 4. FIG. 8 is a graph showing the results of measuring the viable cell count in Experimental Example 5. FIG. 9 is a representative photograph of small cell lung cancer organoids 5 days after the addition of 1 μM linsitinib in Experimental Example 5. Fig. 10 is a graph showing the results of flow cytometry in Experimental Example 6. Fig. 11 is a graph showing the results of measuring tumor volume over time in Experimental Example 7.

[0011] As described in the Examples below, the inventors have invented a method for producing small cell lung cancer cell-derived organoids, which includes culturing cancer cells derived from small cell lung cancer patients in a medium. They were then able to produce small cell lung cancer cell organoids with high expression levels of YAP1 or its downstream factors, separate from POU2F3-expressing small cell lung cancer cell organoids. The inventors have also demonstrated that small cell lung cancer cells with high expression levels of YAP1 or its downstream factors require IGF1 for their proliferation, i.e., are IGF-dependent.

[0012] The inventors further demonstrated that small cell lung cancer cells expressing high levels of YAP1 or its downstream factors exhibit suppressed proliferation and induced apoptosis in the presence of an IGF1R inhibitor.

[0013] Therefore, administration of an IGF1R inhibitor is effective in treating small cell lung cancer patients with high expression levels of YAP1 or its downstream factors. The subgroup of small cell lung cancer cells for which administration of an IGF1R inhibitor is effective is small cell lung cancer cells with high expression of the YAP1 gene, as previously reported in Non-Patent Document 1, etc., and can also be called a YAP1-positive subtype. The inventors further clarified a method for determining whether administration of an IGF1R inhibitor is effective in treating small cell lung cancer patients, and completed the present invention.

[0014] [Method for producing organoids derived from small cell lung cancer cells] In one embodiment, the present invention provides a method for producing organoids derived from small cell lung cancer cells, comprising culturing cancer cells derived from a small cell lung cancer patient in a medium. Organoids are three-dimensional cellular tissues formed by the accumulation and self-organization of cells, and have a structure and function similar to that of organs in vivo.

[0015] Cancer cells derived from small cell lung cancer patients include cancer cells derived from cancer tissue, cancer cells derived from pleural effusion, cancer cells derived from sputum, and cancer cells circulating in the blood.

[0016] In the production method of this embodiment, the medium for culturing cancer cells derived from a patient with small cell lung cancer is preferably a medium obtained by adding to a basal medium at least two selected from the group consisting of insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2), EGF (epidermal growth factor) and epiregulin (EREG), and at least one selected from the group consisting of a Wnt agonist, a bone morphogenetic protein (BMP) inhibitor, and a transforming growth factor-β (TGF-β) inhibitor.

[0017] A medium for culturing cancer cells derived from a patient with small cell lung cancer preferably contains at least two selected from the group consisting of IGF1, FGF2, and EGF. The medium preferably contains IGF1, and more preferably contains FGF2 and EGF.

[0018] The medium for culturing cancer cells derived from a patient with small cell lung cancer contains at least one selected from the group consisting of a Wnt agonist, a BMP inhibitor, and a TGF-β inhibitor. While the Wnt agonist is not essential, it is preferable to include a BMP inhibitor and a TGF-β inhibitor.

[0019] Furthermore, the medium for culturing cancer cells derived from small cell lung cancer patients may contain serum, but is preferably serum-free.

[0020] Any serum-free basal cell culture medium can be used as the basal medium. Examples include defined synthetic media buffered to a pH of 7.2 or higher and 7.6 or lower with a carbonate buffer. More specifically, examples include Advanced Dulbecco's Modified Eagle's Medium / Ham's F-12 Mixed Medium (DMEM / F12) supplemented with glutamine, insulin, B27 supplement (Thermo Fisher Scientific), N-acetyl-L-cysteine ​​(Fujifilm Wako Pure Chemical Industries), penicillin, streptomycin, transferrin, etc. Alternatively, RPMI 1640 medium, Advanced RPMI medium, etc. may be used instead of DMEM / F12 medium.

[0021] (IGF1) IGF1, also known as somatomedin C, is a factor secreted mainly in the liver in response to stimulation by growth hormone (GH). It is known that most cells in the human body (particularly cells of muscle, bone, liver, kidney, nerve, skin, lung, etc.) are affected by IGF1. In addition to its insulin-like effects, IGF1 has the function of regulating cell growth (particularly nerve cells) and development, as well as cellular DNA synthesis.

[0022] The concentration of IGF1 contained in the culture medium is not particularly limited, but is preferably 5 ng / mL to 1 μg / mL, more preferably 10 ng / mL to 1 μg / mL, and even more preferably 50 ng / mL to 500 ng / mL.

[0023] (FGF2) FGF2 is a basic fibroblast growth factor that binds to the fibroblast growth factor receptor (FGFR) and promotes the proliferation and organization of vascular endothelial cells into tubular structures, i.e., angiogenesis. Human FGF2 is known to have two isoforms: low molecular weight (LWL) and high molecular weight (HWL). LWL is primarily present in the cytoplasm and acts via an autocrine mechanism, while HWL is present in the nucleus and is active via an intracellular intracrine mechanism.

[0024] The concentration of FGF2 contained in the culture medium is not particularly limited, but is preferably 5 ng / mL to 1 μg / mL, more preferably 10 ng / mL to 1 μg / mL, and even more preferably 50 ng / mL to 500 ng / mL.

[0025] Epidermal Growth Factor (EGF) is a potent mitogen for a variety of cultured ectodermal and mesodermal cells and has a profound effect on the differentiation of certain fibroblasts and specific cells. The EGF precursor exists as a membrane-bound molecule that is proteolytically cleaved to generate a 53-amino acid peptide hormone that stimulates cells.

[0026] The concentration of EGF contained in the culture medium is preferably 5 ng / mL to 500 ng / mL, more preferably 10 ng / mL to 400 ng / mL, and even more preferably 50 ng / mL to 200 ng / mL.

[0027] (Wnt Agonist) A Wnt agonist refers to a drug that activates T-cell factor (hereinafter also referred to as TCF) / lymphoid enhancer factor (hereinafter also referred to as LEF)-mediated transcription in cells. Therefore, Wnt agonists are not limited to Wnt family proteins, but also include Wnt agonists that bind to and activate Frizzled receptor family members, inhibitors of intracellular β-catenin degradation, and TCF / LEF activators. The Wnt agonist is preferably at least one selected from the group consisting of Wnt proteins, R-spondin, and GSK-3β inhibitors.

[0028] The medium for producing epithelial organoids preferably contains a Wnt agonist. The Wnt agonist more preferably contains a complex of a Wnt protein and its stabilizing substance, afamin, and even more preferably contains a complex of a Wnt protein and afamin and R-spondin. By including a complex of a Wnt protein and afamin and R-spondin in the medium, epithelial organoids can be formed with higher efficiency.

[0029] <<Wnt Protein>> The origin of the Wnt protein is not particularly limited, and Wnt proteins derived from various organisms can be used. Among them, Wnt proteins derived from mammals are preferred. Examples of mammals include humans, mice, rats, cows, pigs, rabbits, etc. Examples of mammalian Wnt proteins include Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, etc. In the culture medium for producing epithelial organoids, multiple types of Wnt proteins may be used in combination.

[0030] Methods for preparing Wnt proteins include, for example, methods using Wnt protein-expressing cells. The origin of the Wnt protein-expressing cells (e.g., biological species, culture form) is not particularly limited, as long as they stably express Wnt proteins, and they may also be cells that transiently express Wnt proteins. Examples of Wnt protein-expressing cells include L cells (ATCC CRL-2647) that stably express mouse Wnt3a and L cells (ATCC CRL-2814) that stably express mouse Wnt5a. Furthermore, Wnt protein-expressing cells can be produced using known genetic recombination techniques. Specifically, Wnt protein-expressing cells can be produced by inserting DNA encoding a desired Wnt protein into a known expression vector and then introducing the resulting expression vector into an appropriate host cell. The nucleotide sequence of the gene encoding the desired Wnt protein can be obtained from known databases, such as GenBank.

[0031] The Wnt protein expressed by the Wnt protein-expressing cells may be a fragment of the Wnt protein or may contain an amino acid sequence other than that of the Wnt protein, as long as it has Wnt activity. The amino acid sequence other than that of the Wnt protein is not particularly limited, and examples thereof include the amino acid sequence of an affinity tag. Furthermore, the amino acid sequence of the Wnt protein does not need to be completely identical to an amino acid sequence obtainable from a publicly known database such as GenBank; as long as it has Wnt activity, it may be substantially the same as an amino acid sequence obtainable from a publicly known database.

[0032] Examples of amino acid sequences that are substantially identical to the amino acid sequences of Wnt proteins that can be obtained from publicly known databases such as GenBank include amino acid sequences in which one to several amino acids have been deleted, substituted, or added to the amino acid sequences that can be obtained from publicly known databases.

[0033] An amino acid sequence in which one to several amino acids have been deleted, substituted or added means that the number of amino acids that can be deleted, substituted or added (preferably 10 or less, more preferably 7 or less, and even more preferably 6 or less) has been deleted, substituted or added by, for example, a known method for producing mutant peptides such as site-directed mutagenesis.

[0034] Furthermore, examples of substantially identical amino acid sequences include amino acid sequences that have an identity of at least 80% or more, preferably at least 85% or more, more preferably at least 90% or more, even more preferably at least 92% or more, particularly preferably at least 95% or more, and most preferably at least 99% or more with an amino acid sequence that can be obtained from a publicly known database.

[0035] The concentration of Wnt protein is preferably 50 ng / mL or more, more preferably 100 ng / mL to 10 μg / mL or less, even more preferably 200 ng / mL to 1 μg / mL or less, and particularly preferably 300 ng / mL to 1 μg / mL or less.

[0036] R-spondins include the R-spondin family consisting of R-spondin 1, R-spondin 2, R-spondin 3, and R-spondin 4. The R-spondin family is a secreted protein known to be involved in the activation and regulation of the Wnt signaling pathway. In the cell culture medium according to this embodiment, multiple types of R-spondins may be used in combination.

[0037] As long as it has R-spondin activity, it may be a fragment of R-spondin, or may contain an amino acid sequence other than that of R-spondin.

[0038] <<GSK-3β Inhibitors>> Examples of GSK-3β inhibitors include CHIR-99021 (CAS No.: 252917-06-9), CHIR-98014 (CAS No.: 252935-94-7), lithium, Kenpaullone (CAS No.: 142273-20-9), 6-bromoindirubin-30-acetoxime, SB216763 (CAS No.: 280744-09-4), SB415286 (CAS No.: 264218-23-7), FRAT family members that inhibit the interaction between GSK-3 and axin, and FRAT-derived peptides.

[0039] Afamin refers to a glycoprotein belonging to the albumin family, and is known to be present in blood and body fluids. Serum, which is typically added to culture media, contains afamin derived from the animal from which the serum was collected. Because serum contains impurities other than afamin, it is preferable to use afamin alone without serum.

[0040] The origin of afamin contained in the culture medium is not particularly limited, and afamin derived from various organisms can be used. Among these, afamin derived from mammals is preferred. Examples of mammals include those described above. The amino acid sequences of afamins from major mammals and the nucleotide sequences of the genes encoding them can be obtained from publicly known databases such as GenBank. For example, in GenBank, the amino acid sequence of human afamin is registered under the accession number AAA21612, and the nucleotide sequence of the gene encoding it is registered under the accession number L32140, while the amino acid sequence of bovine afamin is registered under the accession number DAA28569, and the nucleotide sequence of the gene encoding it is registered under the accession number GJ060968.

[0041] The afamin contained in the medium may be natural afamin contained in serum or the like purified by a known method, or it may be recombinant afamin. Recombinant afamin can be produced by appropriately using known genetic recombination techniques.

[0042] Recombinant afamin can be produced, for example, by inserting DNA encoding afamin into a known expression vector, introducing the resulting expression vector into an appropriate host cell to express the recombinant afamin, and purifying it using a known purification method. The recombinant afamin may also be afamin to which an affinity tag has been added. The affinity tag to be added is not particularly limited, and can be appropriately selected from known affinity tags. The affinity tag is preferably an affinity tag that can be recognized by a specific antibody, and examples include a FLAG tag, a MYC tag, an HA tag, and a V5 tag.

[0043] The Wnt proteins described above are highly hydrophobic because specific serine residues are modified with fatty acids (palmitoleic acid), and therefore Wnt proteins are prone to aggregation or denaturation in aqueous solutions, making them very difficult to purify and store.

[0044] Meanwhile, it has been reported that modification of this specific serine residue with a fatty acid is essential for the physiological activity of Wnt proteins and is involved in binding to members of the Frizzled receptor family.

[0045] It is also known that in aqueous solution, Wnt proteins bind one-to-one with afamin to form a complex, which is solubilized while maintaining high physiological activity. Wnt protein-afamin complexes can be produced by culturing cells that express both Wnt proteins and afamin, or by co-culturing Wnt protein-expressing cells and afamin-expressing cells.

[0046] The concentration of afamin contained in the medium is not particularly limited, but is preferably 50 ng / mL or more and 10 μg / mL or less, more preferably 100 ng / mL or more and 1 μg / mL or less, and even more preferably 300 μg / mL or more and 1 μg / mL or less.

[0047] (BMP Inhibitors) BMP binds as a dimeric ligand to a receptor complex consisting of two different receptor serine / threonine kinases, type I and type II receptors. The type II receptor phosphorylates the type I receptor, resulting in activation of the receptor kinase. The type I receptor then phosphorylates specific receptor substrates (SMADs), resulting in transcriptional activity via a signal transduction pathway. Generally, BMP inhibitors are, for example, drugs that bind to BMP molecules to form complexes that neutralize BMP activity, such as drugs that block or inhibit the binding of BMP molecules to BMP receptors. BMP inhibitors are also, for example, drugs that bind to BMP receptors and block or inhibit the binding of BMP molecules to the receptor, acting as antagonists or inverse agonists.

[0048] The BMP inhibitor preferably has an inhibitory activity of 50% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more, compared to the BMP activity level in the absence of the inhibitor.

[0049] The BMP inhibitor is preferably a natural BMP binding protein, and examples thereof include chordin-like proteins such as noggin, gremlin, chordin, and chordin domain; follistatin-related proteins such as follistatin and follistatin domain; DAN-like proteins such as DAN and DAN cysteine-knot domain; sclerostin / SOST, decorin, and α-2 macroglobulin.

[0050] Among the BMP inhibitors contained in the culture medium, chordin-like protein or DAN-like protein is preferred, with chordin-like protein being more preferred. As a chordin-like protein, noggin is preferred. Chordin-like protein and DAN-like protein are diffusible proteins that bind to BMP molecules with varying affinities and can inhibit the BMP molecules from approaching signaling receptors. When culturing epithelial stem cells, adding these BMP inhibitors to the cell culture medium can prevent stem cell loss.

[0051] The concentration of the BMP inhibitor contained in the culture medium is preferably 10 ng / mL or more and 100 ng / mL or less, more preferably 20 ng / mL or more and 100 ng / mL or less, and even more preferably 50 ng / mL or more and 100 ng / mL or less.

[0052] (TGF-β Inhibitors) TGF-β is a type of growth factor that is produced by almost all cells, including those in the kidney, bone marrow, and platelets. There are five subtypes of TGF-β (β1 to β5). TGF-β is known to promote the proliferation of osteoblasts and the synthesis and proliferation of connective tissues such as collagen, while suppressing the proliferation of epithelial cells and osteoclasts. In general, TGF-β inhibitors are, for example, drugs that block or inhibit the binding of TGF-β to TGF-β receptors, and bind to TGF-β to form a complex that neutralizes TGF-β activity. TGF-β inhibitors are, for example, drugs that bind to TGF-β receptors and block or inhibit the binding of TGF-β to the receptor, and act as antagonists or inverse agonists.

[0053] Examples of TGF-β inhibitors include A83-01 (CAS number: 909910-43-6), ALK5 inhibitor I (3-(pyridin-2-yl)-4-(4-quinonyl)-1H-pyrazole), LDN193189 (CAS No.: 1062368-24-4), SB-431542 (CAS No.: 301836-41-9), SB-505124 (CAS No.: 694433-59-5), SD-208 (CAS No.: 627536-09-8), SB-525334 (CAS No.: 356559-20-1), LY364947 (CAS No.: 396129-53-6), LY2157299 (CAS No.: 700874-72-2), TGF-β RI Kinase Inhibitor II 616452 (CAS No.: 446859-33-2), TGF-β RI Kinase Inhibitor III 616453 (CAS No.: 356559-13-2), TGF-β RI Kinase Inhibitor IX 616463 (4-((4-((2,6-dimethylpyridin-3-yl)oxy)pyridin-2-yl)amino)benzenesulfonamide), TGF-β RI Kinase Inhibitor VII 616458 (CAS No.: 666729-57-3), TGF-β RI Kinase Inhibitor VIII 616459 (CAS number: 356559-20-1), AP12009 (TGF-β2 antisense compound "Trabedersen"), Belagenpumatucel-L (TGF-β2 antisense gene-modified allogeneic tumor cell vaccine), CAT-152 (Glaucoma-lerdelimumab (anti-TGF-β-2 monoclonal antibody)), CAT-192 (Metelimumab (human IgG4 monoclonal antibody that neutralizes TGFβ1)), GC-1008 (anti-TGF-β monoclonal antibody), and the like. Of these, A83-01 is preferred as a TGF-β inhibitor.

[0054] The concentration of the TGF-β inhibitor contained in the medium is preferably 100 nM or more and 10 μM or less, more preferably 500 nM or more and 5 μM or less, and even more preferably 500 nM or more and 2 μM or less.

[0055] (Other Components) The medium may further contain a Rho-kinase (Rock) inhibitor. Examples of Rock inhibitors include Y-27632 (CAS No.: 129830-38-2), fasudil (HA1077) (CAS No.: 103745-39-7), and H-1152 (CAS No.: 871543-07-6). When Y-27632 is used as the Rock inhibitor, it is preferably added during the first two days of culturing the stem cells dispersed into single cells. The concentration of Y-27632 contained in the medium is preferably about 10 μM.

[0056] The medium may further contain at least one amino acid. Examples of amino acids include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and combinations thereof. The concentration of L-glutamine contained in the medium is 0.05 g / L or more and 1 g / L or less (usually 0.1 g / L or more and 0.75 g / L or less). The concentration of other amino acids contained in the medium is 0.001 g / L or more and 1 g / L (usually 0.01 g / L or more and 0.15 g / L or less). The amino acids may be synthetic.

[0057] The medium may further contain at least one vitamin, such as thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), calcium D-pantothenate (vitamin B5), pyridoxal / pyridoxamine / pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), calciferol (vitamin D2), DL-α-tocopherol (vitamin E), biotin (vitamin H), and menadione (vitamin K).

[0058] The medium may further contain at least one inorganic salt. The inorganic salt is used to help maintain the osmotic balance of the cells and to help regulate the membrane potential. Specific examples of inorganic salts include salts of calcium, copper, iron, magnesium, potassium, sodium, and zinc. Salts are usually used in the form of chlorides, phosphates, sulfates, nitrates, and bicarbonates. More specific salts include CaCl ２ , CuSO ４ -5H ２ O, Fe(NO ３ ) -9H ２ O, FeSO ４ -7H ２ O, MgCl, MgSO ４ , KCl, NaHCO ３ , NaCl, Na ２ HPO ４ , Na ２ HPO ４ -H ２ O, ZnSO ４ -7H ２ Examples include O.

[0059] The medium may further contain at least one sugar that can serve as a carbon energy source. Examples of sugars include glucose, galactose, maltose, and fructose. Among these, glucose is preferred, and D-glucose (dextrose) is particularly preferred. The concentration of sugar contained in the medium is preferably 1 g / L or more and 10 g / L or less.

[0060] The medium may further contain at least one trace element, such as barium, bromium, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, aluminum, or ions thereof.

[0061] The medium may further contain at least one additional agent, such as a nutrient or growth factor that has been reported to improve stem cell culture, such as cholesterol, transferrin, albumin, insulin, progesterone, putrescine, selenite, etc.

[0062] [Therapeutic Drug for Small Cell Lung Cancer] In one embodiment, the present invention provides a therapeutic drug for small cell lung cancer, the therapeutic drug containing an IGF1R inhibitor as an active ingredient. In the therapeutic drug of this embodiment, the small cell lung cancer is preferably small cell lung cancer in which the expression level of YAP1 or its downstream factors is significantly higher compared to a control. The downstream factors of YAP1 are the same as those described above, and include CTGF, CYR61, CCND1, etc.

[0063] The efficacy of IGF-1R inhibitors in the treatment of small cell lung cancer patients has been investigated at the basic research level since the early 2000s, but it has been difficult to identify appropriate patient subgroups and biomarkers (see, for example, G Sakuntala Warshamana-Greene, et al., The insulin-like growth factor-I receptor kinase inhibitor, NVP-ADW742, sensitizes small cell lung cancer cell lines to the effects of chemotherapy, Clin Cancer Res., 11 (4), 1563-1571, 2005; Rebekah L Zinn, et al., ERK phosphorylation is predictive of resistance to IGF-1R inhibition in small cell lung cancer, Mol Cancer Ther., 12 (6), 1131-1139, 2013).

[0064] In contrast, as described below in the Examples, the inventors have demonstrated that small cell lung cancer cells expressing high levels of YAP1 or its downstream factors experience growth suppression and apoptosis induction in the presence of an IGF1R inhibitor. Therefore, IGF1R inhibitors are effective in treating small cell lung cancer patients expressing high levels of YAP1 or its downstream factors.

[0065] The same applies to small cell lung cancer in which the expression level of YAP1 or its downstream factors is significantly higher than that of a control, and the same applies to an IGF1R inhibitor as described above.

[0066] The therapeutic agent of this embodiment may be formulated as a pharmaceutical composition for treating small cell lung cancer in which the expression level of YAP1 or its downstream factors is high. The pharmaceutical composition can be administered orally in the form of, for example, tablets, capsules, elixirs, microcapsules, etc., or parenterally in the form of injections, suppositories, topical skin preparations, etc. More specifically, topical skin preparations include dosage forms such as ointments and patches.

[0067] The pharmaceutically acceptable carrier may be any carrier typically used in the preparation of pharmaceutical compositions. More specifically, examples include binders such as hypromellose, dextrin, macrogol 400, gelatin, corn starch, tragacanth gum, and gum arabic; excipients such as lactose hydrate, D-mannitol, starch, crystalline cellulose, and alginic acid; solvents for injections such as water, ethanol, and glycerin; and adhesives such as rubber-based adhesives and silicone-based adhesives.

[0068] The therapeutic agent of this embodiment may contain additives, such as lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin, and maltitol; flavorings such as peppermint and rhododendron oil; stabilizers such as carmellose sodium, hydrogenated oil, light anhydrous silicic acid, povidone, glycerin fatty acid ester, benzyl alcohol, and phenol; buffers such as phosphates and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; and colorants such as yellow ferric oxide, ferric oxide, black ferric oxide, and titanium oxide.

[0069] A pharmaceutical composition for treating small cell lung cancer in which expression levels of YAP1 or its downstream factors are high can be formulated by appropriately combining the above-mentioned IGF1R inhibitor with the above-mentioned pharmaceutically acceptable carriers and additives, and mixing them in a unit dosage form required for generally accepted pharmaceutical practice. In the pharmaceutical composition, one IGF1R inhibitor may be used alone, or two or more may be used in combination.

[0070] An appropriate daily dose of a therapeutic agent or pharmaceutical composition for small cell lung cancer in which expression levels of YAP1 or its downstream factors are high is an amount containing the minimum effective dose of the active ingredient (IGF1R inhibitor) to produce a therapeutic effect. The effective minimum dose depends on various factors, including the activity of the active ingredient contained in the pharmaceutical composition, functional group modifications that determine lipid solubility / water solubility, administration route, administration time, excretion rate of the specific active ingredient used, treatment period, other concomitant drugs, age, sex, weight, disease, health condition, and medical history of the patient, and other factors well known in the medical arts.

[0071] Typically, the dosage of a therapeutic agent or pharmaceutical composition for a small cell lung cancer patient with high expression levels of YAP1 or its downstream factors is an amount containing about 0.0001 to about 100 mg / kg body weight of the active ingredient (IGF1R inhibitor) per day. The therapeutic agent or pharmaceutical composition may be administered once a day or in divided doses about 2 to 4 times a day.

[0072] [Method for determining whether administration of an IGF1R inhibitor is effective in treating a patient with small cell lung cancer] In one embodiment, the present invention provides a method for determining whether administration of an IGF1R inhibitor is effective in treating a patient with small cell lung cancer, the method comprising a step of detecting expression of YAP1 or a downstream factor thereof in cancer cells derived from the patient with small cell lung cancer, wherein a significantly higher expression level of YAP1 or a downstream factor thereof compared to a control indicates that administration of an IGF1R inhibitor is effective in treating the patient with small cell lung cancer.

[0073] Cancer cells derived from small cell lung cancer patients include cancer cells derived from cancer tissues, pleural effusions, sputum, and circulating blood cancer cells. Organoids prepared from these cancer cells can also be used. Organoids are three-dimensional cellular structures formed by the accumulation and self-organization of cells, and have structures and functions similar to those of organs in vivo.

[0074] The NCBI accession numbers for the cDNAs of the human YAP1 gene are NM_001130145.3, NM_001195044.2, NM_001195045.2, etc.

[0075] Downstream factors of YAP1 include CTGF, CYR61, CCND1, etc. The NCBI accession numbers for the cDNA of the human CTGF gene are NM_001901.4, etc. The NCBI accession numbers for the cDNA of the human CYR61 gene are NM_001554.5, etc. The NCBI accession numbers for the cDNA of the human CCND1 gene are NM_053056.3, etc.

[0076] As used herein, "the expression level of YAP1 or a downstream factor thereof in cancer cells is significantly higher than that in a control" means that the expression level of YAP1, CTGF, CYR61, CCND1, etc. at the gene level or protein level in cancer cells is significantly higher than that in a control. "Significantly higher" may mean "statistically significantly higher."

[0077] Examples of controls include IGF-independent small cell lung cancer cells, ASCL1-positive small cell lung cancer cells, and NEUROD1-positive small cell lung cancer cells.

[0078] The NCBI accession numbers for the cDNA of the human ASCL1 gene are NM_004316.4, etc. The NCBI accession numbers for the cDNA of the human NEUROD1 gene are NM_002500.5, etc. The NCBI accession numbers for the cDNA of the human POU2F3 gene are NM_001244682.2, NM_014352.4, etc.

[0079] In the method of this embodiment, detection of YAP1 expression may be performed by immunostaining of the YAP1 protein. Any antibody that recognizes YAP1 can be used as the antibody (primary antibody) for immunostaining. Furthermore, any secondary antibody that recognizes the primary antibody can be used as the secondary antibody.

[0080] It has been said that immunostaining of YAP1 usually does not work well. However, the inventors have found that immunostaining of YAP1 can be performed successfully by performing immunostaining under the conditions and methods described in the Examples.

[0081] An IGF1R inhibitor is a substance that inhibits downstream signal transduction of IGF1R. The NCBI accession numbers for the cDNA of the human IGF1R gene are NM_000875.5, NM_001291858.2, NM_152452.1, etc.

[0082] Specific examples of IGF1R inhibitors include linsitinib (CAS number: 867160-71-2), BMS-754807 (CAS number: 1001350-96-4), BVP-51004 (CAS number: 477-47-4), XL-228 (CAS number: 898280-07-4), and anti-IGF1R antibodies.

[0083] [Method for immunostaining YAP1 protein] In one embodiment, the present invention provides a method for immunostaining YAP1 protein in a biological sample, the method comprising the steps of using an anti-YAP1 rabbit monoclonal antibody as the primary antibody and an anti-rabbit IgG antibody diluted at a ratio of 1:50 to 1:500 as the secondary antibody. The antibody dilution can be changed as appropriate depending on the properties of the secondary antibody, and does not have to be at the above ratio.

[0084] The biological sample is not particularly limited, and examples include thin slices of cancer tissue and organoids. It has been said that immunostaining of YAP1 usually does not work well. However, the inventors have found that immunostaining of YAP1 can be performed successfully by performing immunostaining under the above conditions.

[0085] The dilution ratio of the anti-rabbit IgG antibody can be determined appropriately depending on the characteristics of the antibody used.

[0086] [Other Embodiments] In one embodiment, the present invention provides a method for treating small cell lung cancer, comprising: detecting the expression of YAP1 or a downstream factor thereof in cancer cells derived from a patient with small cell lung cancer; and, when the expression level of YAP1 or a downstream factor thereof is significantly higher as compared to a control, administering an effective amount of an IGF1R inhibitor to the patient with small cell lung cancer.

[0087] In one embodiment, the present invention provides a method for treating small cell lung cancer in which the expression level of YAP1 or a downstream factor thereof is significantly higher compared to a control, comprising administering an effective amount of an IGF1R inhibitor to a patient in need of treatment.

[0088] In one embodiment, the present invention provides an IGF1R inhibitor for use in treating small cell lung cancer in which the expression level of YAP1 or a downstream factor thereof is significantly higher compared to a control.

[0089] In one embodiment, the present invention provides use of an IGF1R inhibitor for the manufacture of a therapeutic agent for small cell lung cancer in which the expression level of YAP1 or a downstream factor thereof is significantly higher compared to a control.

[0090] In each of these embodiments, the YAP1 or a downstream factor thereof, the small cell lung cancer in which the expression level of YAP1 or a downstream factor thereof is significantly higher than that of a control, and the IGF1R inhibitor are the same as those described above.

[0091] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0092] Experimental Example 1 (Establishment of a Small Cell Lung Cancer Organoid Library) Small cell lung cancer samples were collected from surgical resection samples, endoscopic biopsy samples, circulating tumor cells (CTCs), sputum, and pleural effusion.

[0093] Small cell lung cancer organoids from surgical resection samples, endoscopic biopsy samples, sputum, and pleural effusion samples were established using a method similar to that described in Kawasaki K., et al., An Organoid Biobank of Neuroendocrine Neoplasms Enables Genotype-Phenotype Mapping, Cell, 183 (5), 1420-1435, 2020.

[0094] CTC isolation was performed as follows: First, 15 mL of blood sample was placed in a heparin tube. Next, 750 μL of RosseteSep Human CD36 Depletion Cocktail (Stem Cell Technology) was added to the blood and incubated for 20 minutes. Next, red blood cells and white blood cells were removed using Ficoll-Paque PLUS (Cytiva) by centrifugation at 1200 × g for 20 minutes, and CTCs were collected.

[0095] The recovered CTCs were then embedded in droplets of Matrigel (registered trademark), overlaid with complete medium, and cultured. The complete medium was prepared by adding niche factors whose composition is shown in Table 2 below to the basal culture medium whose composition is shown in Table 1 below. As a result, organoids were established from the CTCs.

[0096]

[0097]

[0098] Forty-one organoid lines were established from cancer cells derived from 28 small cell lung cancer patients, and a small cell lung cancer organoid library was obtained.

[0099] Experimental Example 2 (RNA-seq analysis of small cell lung cancer organoid library) Small cell lung cancer organoids were dissociated into single cells and cultured in the complete medium described above for 7 to 14 days. RNA was then extracted from each organoid using the RNeasy Plus Mini Kit (Qiagen).

[0100] Subsequently, the quality of the RNA was assessed using an Agilent 2100 bioanalyzer (Agilent). Subsequently, sequencing libraries were prepared using TruSeq RNA Library Prep Kit v2 (Illumina) and sequenced using a HiSeq 4000 (Illumina) or NovaSeq 6000 (Illumina).

[0101] Adapter sequences were removed from the raw fastq files using cutadapt (version 1.18), and the reads were then aligned to the reference human genome sequence, hg38, using STAR (version 2.6.1).

[0102] Gene sequence analysis was performed using the R Bioconductor package DESeq2. Read count matrices were normalized by a size factor, and variance-stabilizing transformations were performed using the vst function in DESeq2.

[0103] Cluster analysis was performed using the R Bioconductor package ConsensusClusterPlus based on the Euclidean distance and PAM algorithm. In the cluster analysis, genes with low variance (standard deviation of log2-normalized expression levels <1.5) were discarded.

[0104] Additionally, the nbinomLRT function of DESeq2 was used to identify differentially expressed genes.

[0105] Figure 1 is a heat map showing the expression of transcription factors ASCL1, NEUROD1, POU2F3, and YAP1 in each small cell lung cancer organoid based on the results of RNA-seq analysis. In Figure 1, the horizontal axis indicates each small cell lung cancer organoid line. As a result, it was revealed that each small cell lung cancer organoid could be classified into four subgroups based on the expression of these transcription factors.

[0106] Figure 2 shows the results of principal component analysis based on the RNA-seq analysis. The results revealed that each of the four subgroups of small cell lung cancer organoids had a distinct expression profile.

[0107] Experimental Example 3 (Investigation of IGF Dependence of Small Cell Lung Cancer Organoids) Small cell lung cancer organoids were dissociated into single cells using TrypLE Express (Thermo Fisher Scientific), and then filtered using a 20 μm cell strainer.

[0108] Next, 100 cells were seeded per well and cultured under different media conditions. The media used were the complete medium described above (Complete), complete medium minus EGF, IGF1, and FGF2 (-EIF), complete medium minus IGF1 (-IGF1), complete medium minus EGF (-EGF), and complete medium minus FGF2 (-FGF2). 10 μM Y-27632 was added to the medium for 2 days after seeding. Images of the cells were then taken 14 to 21 days after seeding using a BZX-710 digital microscope (Keyence).

[0109] Figure 3 shows a micrograph of a representative small cell lung cancer organoid. The results revealed that a subgroup of small cell lung cancer organoids is specifically dependent on IGF1. The arrows in Figure 3 indicate examples of organoids in which growth was particularly suppressed.

[0110] Figure 4 shows the relationship between IGF dependency, the type of transcription factor expressed, and the cluster for each small cell lung cancer organoid line. In Figure 4, the horizontal axis indicates each small cell lung cancer organoid line. As a result, it was revealed that YAP1-positive small cell lung cancer organoid lines are IGF-dependent.

[0111] Figure 5 shows the results of co-expression cluster analysis based on the results of RNA-seq analysis, revealing that the expression profiles of IGF-dependent and IGF-independent organoids differ.

[0112] Figure 6 shows the results of hierarchical cluster analysis based on the results of RNA-seq analysis. As a result, it was revealed that IGF-dependent organoids characteristically express YAP1 and its downstream factors, such as CTGF and CYR61.

[0113] [Experimental Example 4] (Immunostaining of small cell lung cancer organoids) Small cell lung cancer organoids were immunostained to detect YAP1 expression. The organoids were solidified with iPGell (GenoStuff) and then fixed, and thin sections were prepared. As the primary antibody, anti-YAP1 rabbit monoclonal antibody (product name "D8H1X", catalog number "#14074", Cell Signaling Technology) diluted 1:400 was used. In addition, Bond ＴＭ Anti-rabbit Poly-HRP-IgG from the Polymer Refine Detection kit (catalog number "DS9800", Leica Biosystems) was diluted 1:100 and used.

[0114] 7 is a photograph showing the results of immunostaining. Staining with the anti-YAP1 antibody was observed in the IGF1-dependent small cell lung cancer organoid lines KOR538, KOR317, and KOR273, but not in the IGF1-independent small cell lung cancer organoid lines KOR144, KOR203, and KOR457. These results confirmed that YAP1 expression can be detected by immunostaining.

[0115] Experimental Example 5 (Effect of IGF1R Inhibitor in Vitro) An IGF1R inhibitor was added to the culture medium of small cell lung cancer organoids to examine the effect on the growth of small cell lung cancer organoids. As small cell lung cancer organoids, multiple types of IGF-dependent small cell lung cancer organoid lines and IGF-independent small cell lung cancer organoid lines were used. Linsitinib (CAS number: 867160-71-2) was used as the IGF1R inhibitor. The viable cell count was measured using a commercially available kit (product name "CellTiter-Glo", Promega).

[0116] Small cell lung cancer organoids were dissociated into single cells using TrypLE Express (Thermo Fisher Scientific) and further filtered using a 20 μm cell strainer.

[0117] Subsequently, 3 x 10 per well ３ Cells were seeded and cultured in complete medium supplemented with 0.1, 1, 10, or 100 μM linsitinib. A control group was also prepared in which dimethyl sulfoxide (DMSO), the solvent used for linsitinib, was added. Five days after drug addition, viable cell counts were measured.

[0118] Figure 8 is a graph showing the results of measuring the number of viable cells. As a result, it was revealed that the addition of IGF1R inhibitors to the culture medium inhibited the growth of IGF-dependent organoids. Figure 9 is a representative photograph of small cell lung cancer organoids 5 days after the addition of 1 μM linsitinib.

[0119] [Experimental Example 6] (In vitro apoptosis assay) An IGF1R inhibitor was added to the culture medium of small cell lung cancer organoids, and the occurrence of apoptosis was measured.

[0120] First, small cell lung cancer organoids were dissociated into single cells using TrypLE Express (Thermo Fisher Scientific) and then filtered using a 20 μm cell strainer.

[0121] Subsequently, 5 × 10 cells were added to each well of a 3-well plate. ４ The cells were seeded and cultured for 5 days in the presence of 1 μM IGF1R inhibitor or 1 μM FGFR inhibitor. DMSO was added to the medium of control cells.

[0122] Subsequently, apoptosis induction was measured by flow cytometry using a BD FACSCalibur system (Becton Dickinson) using a commercially available kit (product name: "TACS Annexin V-FITC Apoptosis Detection Kit", R&D System).

[0123] Fig. 10 is a graph showing the results of flow cytometry, in which propidium iodide-positive and annexin V-positive cells are cells in which apoptosis was induced.

[0124] As a result, it was revealed that apoptosis was induced in IGF-dependent organoids by administering an IGF1R inhibitor to the culture medium.

[0125] Experimental Example 7 (Effect of IGF1R inhibitor in vivo) An IGF1R inhibitor was administered to tumor-bearing mice transplanted with organoids derived from IGF-dependent small cell lung cancer cells, the KOR317 strain (YAP1-positive subtype), and organoids derived from IGF-independent small cell lung cancer cells, the KOR457 strain (ASCL1-positive subtype), and the changes in tumor volume over time were observed.

[0126] Specifically, small cell lung cancer organoids (KOR317 and KOR457 strains) were first detached from Matrigel (registered trademark) using cell recovery solution (BD Biosciences). The detached small cell lung cancer organoids were kept on ice for 30 minutes while loosening them by pipetting every 10 minutes. Subsequently, approximately 1 × 10 ５ Each small cell lung cancer organoid was resuspended in approximately 50 μL of Matrigel, and the suspension was subcutaneously injected into 6- to 10-week-old NOD / Shi-scid, IL-2Rγ mice.

[0127] Approximately 3 months later, the tumor diameter (long diameter x short diameter x height / 2) was approximately 100-150 mm. ３ Once this was achieved, the subjects were randomly divided into a drug-treated group and a placebo-treated group, with n≧3 for each group.

[0128] Subsequently, linsitinib (50 mg / kg) was administered to the drug treatment group, and vehicle was administered to the placebo treatment group once daily, 5 days a week, for a total of 5 weeks. Linsitinib was dissolved in 30% PEG 400-0.5% Tween 80-5% propylene glycol (vehicle) and administered. Tumor diameter (long diameter x short diameter x height / 2) was measured once a week.

[0129] Figure 11 is a graph showing the results of measuring tumor volume over time after the start of drug or vehicle administration. As a result, as shown in Figure 11, tumor growth was suppressed in the group of mice transplanted with the YAP1-positive, IGF-dependent KOR317 strain and administered an IGF1R inhibitor. On the other hand, no effect of the IGF1R inhibitor was observed in mice transplanted with the ASCL1-positive KOR457 strain. This demonstrates that IGF1R inhibitors are therapeutic agents specific to YAP1-positive small cell lung cancer.

[0130] According to the present invention, it is possible to identify a subgroup of small cell lung cancer for which administration of a molecular targeted therapeutic agent is effective, to identify the molecular targeted therapeutic agent, and to provide a technique for determining the effectiveness of administration of the molecular targeted therapeutic agent.

Claims

1. A therapeutic agent for small cell lung cancer, comprising an inhibitor of insulin-like growth factor 1 receptor (IGF1R) as an active ingredient.

2. The therapeutic agent according to claim 1, wherein the small cell lung cancer is a small cell lung cancer in which the expression level of Yes1 Associated Transcriptional Regulator (YAP1) or its downstream factor is significantly higher compared to a control.

3. The therapeutic agent according to claim 2, wherein the downstream factor of YAP1 is connective tissue growth factor (CTGF), Cysteine-rich angiogenic inducer 61 (CYR61), or Cyclin D1 (CCND1).

4. A method for determining whether administration of the therapeutic agent according to any one of claims 1 to 3 is effective for the treatment of a patient with small cell lung cancer, the method comprising the step of detecting the expression of YAP1 or its downstream factor in cancer cells derived from the patient with small cell lung cancer, and a significantly higher expression level of the YAP1 or its downstream factor compared to a control indicates that administration of the therapeutic agent according to any one of claims 1 to 3 is effective for the treatment of the patient with small cell lung cancer.

5. The method according to claim 4, wherein the downstream factor of YAP1 is CTGF, CYR61, or CCND1.

6. The method according to claim 4, wherein the detection of the expression of YAP1 is performed by immunostaining of the YAP1 protein.

7. The method according to claim 4, wherein the cancer cells derived from the patient with small cell lung cancer are small cell lung cancer cell-derived organoids.

8. A method for producing small cell lung cancer cell-derived organoids, comprising the step of culturing cancer cells derived from a patient with small cell lung cancer in a medium.

9. The medium is at least two selected from the group consisting of insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2), EGF (Epidermal Growth Factor), and epiregulin (EREG), and The production method according to claim 8, comprising at least one selected from the group consisting of a Wnt agonist, a bone morphogenetic protein (BMP) inhibitor, and a transforming growth factor-β (TGF-β) inhibitor.

10. The production method according to claim 8 or 9, wherein the cancer cells are cancer tissue-derived cancer cells, pleural effusion-derived cancer cells, sputum-derived cancer cells, or blood-circulating cancer cells.