Cancer cell strain

Abstract

To provide a cancer cell strain that readily forms subcutaneous tumors and facilitates in vitro and in vivo therapeutic testing.SOLUTION: The present invention discloses the following: a cancer cell strain that can form subcutaneous tumors and exhibits RET fusion gene positivity; a cancer cell strain derived from pleural effusion of a human patient with RET fusion gene-positive lung cancer; a cancer cell strain deposited under an accession number NITE P-03936; and a cancer cell strain incorporating an additional luciferase gene.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a cancer cell line that is useful for testing such as investigating drug efficacy. [Background technology]

[0002] Cancer cell lines are widely used in research into the pathology and efficacy of cancer. For example, Patent Document 1 discloses a cell line derived from human hepatoid cell carcinoma in the papilla of vater. This technology is intended to obtain a cell line that can be used to evaluate the therapeutic effect of cancer therapeutic drugs or candidate substances for hepatoid cell carcinoma in the papilla of vater, or to search for and develop factors related to prognosis prediction.

[0003] Meanwhile, there is a gene known as an oncogene, the RET fusion gene. When the normal RET gene present in normal cells fuses with another gene to form the RET fusion gene, it is known that this causes the cell to become tumorigenic, resulting in the development of thyroid cancer, lung cancer, etc. By advancing research into the RET fusion gene, it is believed that it will be possible to study the pathology of thyroid cancer and lung cancer and to examine the efficacy of drugs.

[0004] As a cell line that can be used for the study of RET fusion genes, the RET fusion gene lung cancer cell line, LC-2 / ad, is known. LC-2 / ad is currently known as a commercially available RET fusion gene lung cancer cell line. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2013-244014 A Summary of the Invention [Problem to be solved by the invention]

[0006] Here, experimental animals such as mice are used to study pathology and drug efficacy. However, it has been pointed out that the LC-2 / ad cell line has difficulty engrafting subcutaneously in immunodeficient mice and is unable to form subcutaneous tumors. In other words, when using the LC-2 / ad cell line, it is not possible to form tumors in mice and use them to examine the pathology and efficacy of cancer. Therefore, the LC-2 / ad cell line was difficult to handle in experiments.

[0007] For this reason, there is a strong demand for RET fusion gene-positive cancer cell lines that can easily form subcutaneous tumors, for example, cell lines that can easily engraft and form tumors subcutaneously in mice.

[0008] The present invention has been made in consideration of the above-mentioned circumstances, and an object of the present invention is to provide a cancer cell line that can easily form subcutaneous tumors and that can easily perform in vitro and in vivo therapeutic experiments. [Means for solving the problem]

[0009] In order to solve the above problems, the present invention has the following aspects. Aspect 1 of the present invention is This is a cancer cell line that can form subcutaneous tumors and is RET fusion gene positive.

[0010] Aspect 2 of this embodiment is This is a cancer cell line derived from pleural effusion of a patient with human RET fusion gene-positive lung cancer, and is RET fusion gene-positive.

[0011] Aspect 3 of this embodiment is The cancer cell line according to embodiment 1 or 2, which is the cell line deposited under accession number NITE P-03936.

[0012] Aspect 4 of this embodiment is The cancer cell line according to embodiment 1 or 2, further comprising a luciferase gene introduced therein. Effect of the Invention

[0013] According to the present invention, a cancer cell line is obtained which is capable of easily forming subcutaneous tumors and which can easily be used for in vitro and in vivo therapeutic experiments. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic diagram showing the results of mRNA-seq analysis of the PB302p cell line of this example. [Diagram 2] FIG. 1 is a photograph showing the state of one of the mice in the meningeal carcinomatosis model transplanted with PB302 / luc cells of this example from days 1 to 28. [Diagram 3] FIG. 1 is a photograph of a mouse subcutaneously transplanted with PB302 / luc ivs LM-SC cells of this example. [Figure 4] FIG. 1 is a photograph of PB302 / luc ivs LM-SC cells of this example observed under a microscope. [Diagram 5] FIG. 1 shows the results of an MTT assay verifying the effect of a selective RET inhibitor on PB302 / luc ivs LM-SC cells in this example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, the cancer cell line according to the present invention will be described with reference to embodiments, however, the present invention is not limited to the following embodiments.

[0016] (cancer cell lines) The cancer cell line of this embodiment is capable of forming subcutaneous tumors and is RET fusion gene positive. The term "capable of forming a subcutaneous tumor" broadly refers to the ability to form subcutaneous tumor-like tissue. Furthermore, it is preferable that a subcutaneous tumor, i.e., a subcutaneous tumor-like tissue, can be formed within several tens of days, preferably 1 to 28 days, after subcutaneous injection into various animals. Various animals can be used as appropriate, but mammals, particularly laboratory animals such as mice, rats, rabbits, etc. are preferably used. Among these animals, immunodeficient animals, particularly immunodeficient mice, are preferred, but are not limited to these. Here, the immunodeficient animal is an animal in an immunodeficient state due to impaired function of T cells, B cells, etc. As such an animal, for example, nude mice, SCID mice, etc. can be used.

[0017] The cancer cell line of this embodiment may be derived from pleural effusion of a patient with human RET fusion gene-positive lung cancer. "Derived from the pleural effusion of a human RET fusion gene-positive lung cancer patient" refers to cells obtained from the pleural effusion of a human RET fusion gene-positive lung cancer patient, or cells obtained by induction therefrom (RET fusion gene-positive lung cancer cells). The cells obtained by induction therefrom may be, for example, cells obtained by gene transfer into the cells obtained from the pleural effusion, or cells having certain properties (e.g., properties other than human RET fusion gene positivity, growth ability, etc., such as high subcutaneous tumor formation ability) selected by screening and established as a cell line. For the establishment of cell lines, a conventionally known method can be used. For example, an isolated cell line may be cultured in a known tissue culture medium, the medium periodically replaced until the cell line stably grows, and then a cell line that stably grows for one year or more or for several dozens of passages may be selected as a candidate cell line.

[0018] For example, the above-mentioned cell line capable of forming a subcutaneous tumor can be obtained from a cancer cell line obtained from the pleural effusion of a patient suffering from human RET fusion gene-positive lung cancer. Specifically, a cell line capable of forming a subcutaneous tumor can be derived from the cells obtained from the pleural effusion, and obtained by screening, continuous culture, or the like. For screening, for example, in vivo screening can be used. For example, a cell line obtained from the pleural effusion can be subcutaneously transplanted into an experimental animal such as a mouse, and cells with a high ability to form a subcutaneous tumor can be extracted, cultured, and established.

[0019] In the present Example described later, the RET fusion gene-positive cancer cell line (PB302p in the Example described later) obtained from the pleural effusion of the human RET fusion gene-positive lung cancer patient can be used to induce and obtain a RET fusion gene-positive cancer cell line capable of forming a subcutaneous tumor (PB302 / luc ivs LM) and a RET fusion gene-positive cancer cell line capable of forming a subcutaneous mass (PB302 / luc ivs LM-SC(PB302)). All of these induced cell lines are also cell lines derived from the pleural effusion of a human RET fusion gene-positive lung cancer patient.

[0020] The cancer cell line of this embodiment may be the cell line deposited under accession number NITE P-03936. As described later in the Examples, the cell line deposited under Accession No. NITE P-03936 (PB302 / luc ivs LM-SC cell line, or simply PB302 cell line, in the Examples described later) was derived from pleural effusion of a patient with human RET fusion gene-positive lung cancer and is RET fusion gene-positive. This cell line can also form subcutaneous tumors. That is, it was obtained by screening RET fusion gene positive cells obtained from pleural effusion of human RET fusion gene positive lung cancer patients and establishing a cell line capable of forming subcutaneous tumors. This cell line also has a luciferase gene introduced into it. This cell line can also form subcutaneous tumors when implanted subcutaneously in animals.

[0021] The cancer cell line of this embodiment may be a cell line having biological properties equivalent to those of the cell line deposited under accession number NITE P-03936.

[0022] The cell line of this embodiment, particularly the PB302 / luc ivs LM-SC cell line, is characterized by a confluent state (approximately 1.0 to 5.0 × 10 5 pieces / cm 2 ) shows small round cells.

[0023] The cancer cell line of this embodiment may further include another gene introduced therein. The other gene can be introduced, for example, by infecting a cancer cell line with a viral vector into which the gene has been introduced. As a method for infecting a cancer cell line with a viral vector, a conventional method for infecting a viral vector can be appropriately used. Examples of genes that can be introduced include luciferase.

[0024] It is more preferable that the cancer cell line of this embodiment is one into which a luciferase gene has been further introduced. Luciferase is widely used as a gene for luminescence assays, and the location of the cancer cell line of this embodiment can be easily confirmed by examining the expression of luciferase, i.e., luminescence, making it useful for biological studies.

[0025] The cancer cell line of this embodiment may be capable of forming a tumor. The term "capable of easily forming a tumor" means, for example, that a tumor is formed within a few days, preferably 1 to 10 days, after transplantation of the cell line into the animal. In particular, the cell line may be capable of forming a subcutaneous tumor within 4 days after subcutaneous transplantation. A cell line capable of easily forming a tumor mass can be obtained from cells obtained from the pleural effusion of a patient with lung cancer positive for the human RET fusion gene by screening, continuous culture, etc. For example, the cell line obtained from the pleural effusion is subcutaneously transplanted into an experimental animal such as a mouse, and cells are extracted from tissues that have a high ability to form subcutaneous tumors and then cultured.

[0026] The cancer cell line of this embodiment preferably has the KIF5B and RET genes. In particular, it is preferable to have a fusion sequence of the KIF5B and RET genes on the genome. KIF5B is known to be a fusion partner with the RET gene in cancer tissues, such as non-small cell lung cancer, and is known to be a factor that frequently becomes a partner. The cancer cells of this embodiment have a gene that frequently becomes a partner with the RET gene, and therefore can be particularly preferably used in tests related to the analysis of the mechanism of cancer. The genes contained in the cell lines can be identified, for example, by mRNA-seq.

[0027] The cancer cell line of this embodiment may be a cancer cell line for use in in vitro and in vivo experiments. Conventionally known in vitro and in vivo experiments can be used as appropriate. For example, in vivo experiments include tests using animals. Tests using animals can be mainly used for testing and research to examine the development and metastasis of cancer tissue by transplanting the cancer cell line of this embodiment into animal tissue.

[0028] For in vitro and in vivo cancer experiments, markers can be used to examine the effects of drugs on cancer, for example. By measuring the concentrations of tumor markers such as serum AFP and GPC3, as well as the proliferation rate, migration ability, and invasion ability of cell lines, the effect of candidate cancer therapeutic drugs in inhibiting cancer cell proliferation can be determined.

[0029] For in vitro experiments, for example, a method for culturing a cell line that can be used for in vitro screening to screen drugs, and a method for contacting the cell line with a candidate substance for cancer treatment, a person skilled in the art can appropriately set optimal conditions according to the candidate substance. In addition, the above-mentioned method can be used for measuring tumor markers in in vitro screening.

[0030] In vivo experiments, for example, in vivo screening for screening drugs, can be used, for example, in vivo screening including a step of administering a candidate substance for cancer treatment to a non-human mammalian animal transplanted with the cancer cell line of this embodiment and testing the effect of inhibiting cancer cell proliferation. In this case, the effect of the candidate substance for cancer treatment to inhibit cancer cell proliferation can be determined using the tumor marker as an index or based on the histological findings of the tumor.

[0031] The administration method of the cancer therapeutic drug candidate substance in in vivo screening can be appropriately selected by those skilled in the art, such as intraarterial injection, intravenous injection, subcutaneous injection, or intranasal, transbronchial, intramuscular, transdermal, or oral administration. In addition, the tumor marker measurement in in vivo screening can be performed by the above-mentioned method. Histological findings can also be performed by the usual method.

[0032] The cancer cell line of this embodiment is particularly useful when it is capable of forming a tumor or mass subcutaneously, since the mechanism of the cancer cells can be easily confirmed by their development (growth, shrinkage, other changes).

[0033] The cancer cell line of this embodiment can preferably be used to form a central nervous system model, such as a model of a disease such as meningeal carcinomatosis or brain tumor.

[0034] In the cancer cell line of this embodiment, the tumor or mass is preferably reduced in size by administration of a RET inhibitor. Because RET inhibitors have the property of shrinking tumors or masses when administered, they can be useful in a wide range of tests, such as selecting cancer treatment drugs and diagnostic drugs, and examining drug efficacy. For example, the effect of a selective RET inhibitor in vitro can be confirmed using the MTT assay or the like.

[0035] (Use of the cancer cell line of this embodiment) The cancer cell line of this embodiment can be used to prepare models of various diseases using mice, etc. For example, cells obtained from the pleural effusion of the human RET fusion gene-positive lung cancer patient (e.g., PB302p) and cells transfected with luciferase (e.g., PB302 / luc) can be used to prepare central nervous system models such as brain metastasis models and meningeal carcinomatosis models. Cells capable of forming subcutaneous tumors (e.g., PB302 / luc ivs LM) and cells capable of forming subcutaneous masses (e.g., PB302 / luc ivs LM-SC) can be used to prepare models of subcutaneous tumor formation, thoracic cavity transplantation, and lung transplantation, in addition to the above models.

[0036] For example, as a central nervous system model, a mouse model of intracerebral transplantation of a cancer cell line can be created as follows. The mouse is anesthetized and held in place, and the skin between both ears is incised to expose the right skull. After removing the synovial membrane, a hole is made in the skull and the cell suspension is injected with a syringe or the like. The incised skin is sutured with a needle. The amount of cell suspension injected is 1 to 5 × 10 cells for a mouse of normal size. 5 The appropriate volume of the suspension is about 1 to 2 μL.

[0037] As a central nervous system model, a meningeal carcinomatosis model can be created by intrathecal administration of a mouse cancer cell line as follows. The mouse is anesthetized and restrained, and a needle is inserted into the gap between the bottom end of the skull and the top end of the spine, and the cell suspension is injected. The amount of cell suspension injected is 1 to 5 × 10 cells for a mouse of normal size. 5 A volume of approximately 20 to 100 μL is appropriate.

[0038] Furthermore, a mouse bone transplant model of a cancer cell line can be created as follows. The mouse is anesthetized after removing hair from the injection site, such as the leg. The knee is bent and a hole of about 5 mm is made in the white part of the knee joint with a syringe with a needle attached. The needle is removed and a microsyringe is inserted into the hole and the cell suspension is injected. The number of cells is 1 to 5 × 10 5 A volume of about 1 to 5 μL is appropriate.

[0039] (Effects of this embodiment) According to the cancer cell line of this embodiment, subcutaneous tumors can be easily formed, and in vitro and in vivo therapeutic experiments can be easily carried out. The cancer cell line of this embodiment includes a RET fusion gene-positive cancer cell line (PB302p) obtained from the pleural effusion of a patient with human RET fusion gene-positive lung cancer, and a cancer cell line (PB302 / luc ivs LM-SC or PB302) derived from the pleural effusion of a patient with human RET fusion gene-positive lung cancer, which has been induced and established therefrom, has a luciferase gene introduced therein, can be engrafted subcutaneously to form a subcutaneous tumor, and can easily form a subcutaneous mass when transplanted into an animal.

[0040] PB302p, which was obtained from the pleural effusion of a patient with human RET fusion gene-positive lung cancer, and the cell line containing PB302 derived from it, have the KIF5B and RET genes on chromosome 10. KIF5B is known as a fusion partner with the RET gene in cancer tissues, such as non-small cell lung cancer, with a frequency of 60-90%, making it an extremely frequent partner compared to other partners (e.g., EML4, TRIM33, etc.).

[0041] Previously, the LC-2 / Ad cell line, which was commercially available as a RET fusion gene lung cancer cell line, fused the RET gene with CCDC6, which is a relatively frequent partner gene, but it could not be engrafted subcutaneously, making it difficult to use in experiments, including subcutaneous tumor models. In addition, the effect of RET inhibitors in vitro was unstable, making it difficult to use it in experiments to verify cancer using RET inhibitors. Although cell lines have been found that have the EML4 and TRIM33 genes as fusion partners (CUTO42 and ECLC5B cell lines, respectively), these are less frequent fusion partners of RET. Cell lines (such as CUTO22) that contain the KIFB gene, which is a frequent fusion partner, have been established at the University of Colorado, but these do not engraft subcutaneously or are ineffective against RET inhibitors, making them difficult to use in experiments. Furthermore, none of these previously known RET fusion gene-positive lung cancer cell lines have been examined in other metastatic models.

[0042] In contrast, the cancer cell line (PB302) of this embodiment has the following characteristics: (1) the effect of RET inhibitors was confirmed in vitro, (2) it was KIF5B-RET fusion gene positive and subcutaneously engrafted in mice, and (3) a central nervous system model was successfully created. The only cell line that satisfies these three conditions is the one established by the inventors. Since the cancer cell line of this embodiment allows for reasonable confirmation of extracranial efficacy in in vitro and in vivo treatment experiments, it may attract attention in the pharmaceutical field as a cell line that can replace previously known RET fusion gene lung cancer cell lines such as LC-2 / ad. EXAMPLES

[0043] Examples will be given below, but the present invention is not limited to the Examples.

[0044] (Cell culture, storage conditions, etc.) The cell culture procedure was carried out by adding 5.0 × 10 3 cells / cm 2 After seeding, the cells were passaged about 5 to 8 days later. After washing twice with PBS, the cells were exposed to 0.05% trypsin-EDTA (25300054, Thermo Fisher) for 3 minutes at 37°C for passage. The cells were preserved by adding Cell Banker (catalog number CB021, lot number 220208) as a protective agent and freezing in a -160°C freezer. The preserved cells were thawed in a 37° C. water bath, suspended in 15 to 40 mL of the above-mentioned medium, and washed by centrifugation.

[0045] (Deposit conditions for human-derived cells) The human-derived cells used below meet the JCRB Cell Bank deposit conditions. JCRB Cell Bank research ethics clearance (deposit of human-derived cultured cell research resources) requires that the following two conditions must be met for human-derived cells to be deposited in a cell bank, based on the Science and Technology Agency's "Basic Principles for Human Genome Research (Apr. 11, 2000), Clause 9" and the Ministry of Health, Labor and Welfare's "Guidelines for Addressing Ethical Issues Associated with Genetic Analysis Research (Feb. 4, 2000), Clauses 6 and 7." (1) Cells must be anonymized and cannot identify the individual donor (unlinked) (Coded Unlinked Sample). (2) Written consent to research use must be obtained from the donor at the time of biological sample collection.

[0046] [Test Example 1] (Establishment of the PB302p cell line) With the consent of a RET fusion gene-positive lung cancer patient, cancer cells were isolated from the patient's pleural fluid using Lymphocyte Separation Medium (LMS) (MP biomedicals, LOT NO. S8658). Specifically, 4 ml of LMS was placed in a 15 ml tube, followed by 10 ml of pleural fluid. The tube was centrifuged at 400 g for 15 minutes. After centrifugation, the cell layer in the middle of the tube was aspirated using a 1000 μl pipette and placed in a 10 cm dish containing culture medium. The cancer cells were cultured and grown in 500 mL of RPMI1640 (Thermo Fisher, Catalog No. 11875093) medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were passaged multiple times to select a cell line that showed stable growth. This cell line was designated as the PB302p cell line.

[0047] We analyzed eukaryotic mRNA-seq for the PB302p cell line obtained by the present study. The analysis was performed in the following order: quality check of sequence reads using FastQC, trimming of sequence reads using Trimmomatic, mapping of sequence reads using STAR, and detection of fusion genes and creation of figures using arriba.

[0048] The results of mRNA-seq analysis of the PB302p cell line are shown in Figure 1. It was revealed that the KIF5B and RET genes are fused to chromosome 10. That is, the PB302p cell line has a fusion gene of KIF5B and RET, which is known as a fusion partner that fuses with the RET gene to become an oncogene.

[0049] [Test Example 2] (Establishment of PB302 / luc cell line) The PB302p cell line was infected with a viral vector to introduce a luciferase gene, thereby generating a PB302 / luc cell line.

[0050] As a reagent, Cancer cell line to be infected: PB302p Cells used in preliminary studies: 293T cell line (human fetal kidney cells) ·Lipofectamine(R)LTX & Plus Reagent (Invitrogen Cat 15338-100) Vector (MaRXTM IVf puro-EGFP-ELuc) ·Retrovirus Packaging Kit Ampho (TaKaRa #6161) ·8mg / mL Hexadimethrine bromide [also known as Polybrene] (SIGMA #9268) ·PEG-itTM virus precipitation solution (System Bioscience Cat LV810A-1) 10mg / mL Puromycin was used.

[0051] As a preliminary study using 293T cells, as described below, Sensitivity of infected cancer cells to Polybrene (24 h) (final conc. 0, 1, 2, 4, 8, 16 μg / mL) ·Sensitivity of infected cancer cells to Puromycin MTT assay (72~96 h) (final conc. 0, 0.03, 0.1, 0.3, 1, 3, 10μg / mL) was carried out.

[0052] The infection procedure was carried out as follows. Day 1: Cells were seeded onto two 10 cm dishes at 2.5x106 cells / 10mL (10% FBS-DMEM P / S[+]) and cultured overnight at 37℃. Days 2-3: The medium was removed and 9 mL of fresh medium (10% FBS-DMEM P / S[-]) was added. MaRXTM IVf puro-EGFP-ELuc (15 μg), pGP vector (7.5 μg) (included in the Retrovirus Packaging Kit Ampho), and pE-ampho vector (7.5 μg) (included in the Retrovirus Packaging Kit Ampho) were added to 2 mL of Opti-MEM I Reduced-Serum Medium. 30μL of PLUS reagent was added and stirred, and left at room temperature for 5 minutes, and then 75μL of Lipofectamine LTX was added. The mixture was pipetted several times and left at room temperature for 30 minutes. 1mL of the prepared solution was added to the dish on which the cells were seeded (1mL x 2 dishes) and cultured at 37℃. After 6-8 hours, the medium was replaced with 10mL of fresh medium (10%FBS-DMEM P / S[+]), and cultured at 37℃ for 48 hours. EGFP fluorescence was confirmed using a fluorescent microscope. Day 4: From this point on, all waste was treated as P2. Culture supernatant (20 mL) was collected in a 50 mL Falcon tube (cells were discarded). 20 mL of culture supernatant was passed through a 0.44 μm filter and collected in a new 50 mL tube. 5 mL of PEG-itTM virus precipitation solution was added. The mixture was stirred several times and left to stand overnight at 4°C. Day 5: Centrifuge at 1,500g x 30min, collect the precipitate, and discard the supernatant. The precipitate was suspended in 1mL of 10% FBS-RPMI P / S[+] and collected in an Eppendorf tube. This solution was used as a 20-fold concentrated virus solution and stored at -80℃.

[0053] The cancer cell infection experiment was carried out as follows. Day 1: Add 2 x 10 cells (PB302p) to 6 wells of a 24-well plate. 4 The cells were seeded at 1 mL and cultured overnight at 37°C. Day 2: The medium was removed, and 0.4 mL of fresh medium 10% FCS-RPMI P / S(+) was added to each well. The above virus solution was added to each well to make 1, 2.5, 5, and 10 times concentrated virus solution, and the above medium was added to the rest to make 600 μL. The concentration of Polybrene was determined in advance through preliminary investigations to determine the concentration that was not cytotoxic after 24 hours of culture, and 1 μL was added to each well except for the non-infected well. Culture was performed at 37°C overnight. Day 3: The supernatant was removed, 1 mL of fresh medium 10% FCS-RPMI P / S(+) was added, and the cells were cultured at 37°C for 24 to 48 hours. Day 4-5: The optimal well was selected under a microscope, and culture was started in a medium containing puromycin. The concentration of puromycin was determined in a preliminary study to be the concentration at which approximately 90% of the parent cell line's growth was inhibited.

[0054] In this example, the 20-fold concentrated virus solution was used in the above infection procedure. Cancer cells were infected with the virus at a 10-fold concentration, cultured in a medium containing Puromycin, and the introduction of the luciferase gene was confirmed. The PB302 / luc cell line, into which the luciferase gene was introduced by infection with a viral vector, was obtained.

[0055] The PB302 / luc cell line obtained did not form tumors subcutaneously, and did not engraft when transplanted into the thoracic cavity or lung. However, it had properties that allowed it to be used in brain metastasis and meningeal carcinomatosis models, similar to those of existing established cell lines.

[0056] [Test Example 3] (Establishment of PB302 / luc ivs LM cell line) Three mice with meningeal carcinomatosis model were generated using the PB302 / luc cell line through the following procedures.

[0057] Meningeal carcinomatosis model mice were generated from immunodeficient mice via the following procedures. The mouse was put to sleep by isoflurane inhalation anesthesia. The left hand was used to hold the mouse's nose and head in a straight line. There was a gap between the bottom of the skull and the top of the spine, so a 27G needle was inserted at a 45 degree angle and advanced so that the needle penetrated the spine. The mouse's body was raised by the needle. The concentration was 1×10 5 / 50 μL of PB302 / luc cells were injected in an injection volume of 50 μL.

[0058] In this mouse model of meningeal carcinomatosis, we confirmed that the cells had taken root in the spinal cavity (Figure 2). Figure 2 shows the progression of tumors over time in a mouse model of meningeal carcinomatosis (three mice in total, two mice on Day 28 are separate from the mouse in the box). The luminescence in the photograph is the luciferase signal of PB302 / luc, detected by administering luciferin intraperitoneally to the mouse using the IVIS Imaging System. Next, the mice were dissected, and the tumors were removed and cultured in RPMI on 10 cm dishes. We confirmed that a cell line (PB302 / luc ivs LM) had been established from the tumor, and also performed a subcutaneous tumor formation test using immunodeficient mice. Cells were cultured on multiple 15 cm dishes until they reached 80% confluence. The medium was removed and washed with PBS. 0.05% trypsin solution was added and the dishes were placed in a 37°C incubator until the cells detached. 10% FBS-RPMI was added to neutralize the trypsin, and the cell suspension was collected in a 50 mL tube. The dish was washed with 20 mL of medium, and the washing solution was collected. The cell pellet was centrifuged at 1,100 rpm for 5 min, suspended in 50 mL of HBSS (Hanks' balanced salt solution), and collected in a single 50 mL tube. Count the number of cells, centrifuge at 1,100 rpm for 5 min, and collect the cell pellet at 3–5 × 10 7 The cells were suspended in HBSS at a concentration of 0.1 mL / head (3–5 × 10 6 cells) were transplanted.

[0059] The mouse with the subcutaneous transplant is shown in Figure 3. It was confirmed that PB302 / luc ivs LM formed a subcutaneous tumor in the mouse. It took about two months for a subcutaneous tumor to form.

[0060] [Test Example 4] (Preparation of PB302 / luc ivs LM-SC cell line (PB302)) The cells were removed from the subcutaneous tumor formed in the mouse, and cultured and passaged under the same conditions as in Test Example 1 to establish a cell line, which was named the PB302 / luc ivs LM-SC cell line (also referred to simply as the PB302 cell line in this specification). In addition, from the infection of PB302p with the virus to the establishment of PB302, the cells were passaged a total of about 5 to 10 times, and it was considered that no virus remained in PB302.

[0061] The PB302 / luc ivs LM-SC cell line was subcutaneously transplanted into immunodeficient mice to form subcutaneous tumors. The conditions for forming subcutaneous tumors were the same as those in Test Example 3, and it was confirmed that a tumor had formed on the fourth day after transplantation. It was also confirmed that the size of the tumor had grown to 8.06 mm x 7.66 mm in two weeks.

[0062] Figure 4 shows a photograph of PB302 / luc ivs LM-SC cells observed on a dish. The observation conditions were a phase contrast microscope at 100x magnification. The PB302 / luc ivs LM-SC cells exhibited small round cells when in a confluent state.

[0063] This PB302 / luc ivs LM-SC cell line (PB302 cell line) was deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center (receipt date: July 3, 2023, accession number NITE P-03936, cell name "PB302").

[0064] The obtained PB302 / luc ivs LM-SC cells were further transplanted into another mouse to form a tumor. The conditions for forming a subcutaneous tumor were the same as those in Test Example 3. For mice subcutaneously transplanted with PB302 / luc ivs LM-SC cells, tumor formation was confirmed on the fourth day after transplantation. Subcutaneous tumors were formed in all three of the three mice transplanted with the cells. The size of the tumor was 8.06 mm x 7.66 mm at two weeks. These results demonstrated that the PB302 / luc ivs LM-SC cell line was able to reproducibly form subcutaneous tumors. Using the cell line of this example, subcutaneous tumors and subcutaneous masses can be easily formed with reproducibility, and therefore it is believed to be useful for carrying out cancer treatment experiments, including in vivo experiments.

[0065] [Test Example 5] (Verification of the effects of RET inhibitors) The effect of selective RET inhibitors on PB302 / luc ivs LM-SC (PB302) cells in vitro was examined using the MTT assay. Detach the confluent PB302 from the 10 cm dish and inoculate the cells (2–3 × 10 ) in 100 μl of medium onto a microplate (tissue culture grade, 96-well, flat bottom). 4 Cells were seeded in 100 wells (Day 1). The cell culture was incubated at 37°C, 5-6.5% CO2 for 24 hours, and on Day 2, 100 μl of medium containing a RET inhibitor (serpercatinib or pralsetinib) was injected into each well so that the drug concentration in each well was 0.001-10 μmol / L. On Day 5, after incubating the wells for 72 hours at 37°C, 5-6.5% CO2, 200 μl of medium in each well was aspirated and replaced with 100 μl of fresh medium, and 50 μl of MTT labeling reagent (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, final concentration 0.5 mg / ml) was added to each well. The wells were incubated for 1 hour at 5-6.5% CO2, and the medium in each well was aspirated and 100 μl of DMSO was injected. The plate was visually inspected to confirm that the purple formazan crystals were completely solubilized, and the absorbance of the sample was measured using a microplate (ELISA) reader. The wavelength for measuring the absorbance of the formazan product was 550-600 nm, and the reference wavelength was 650 nm or higher.

[0066] Figure 5 shows the results of the MTT assay. (a) shows the cell viability at each concentration of the RET inhibitors spelpercatinib and (b) shows the effect of the RET inhibitors. Both RET inhibitors showed a decrease in cell viability depending on the concentration, making the effect of the RET inhibitors in PB302 cells clear. Since the cell viability of PB302 is reduced by RET inhibitors in vitro, it is expected that they can be used in vivo for testing and diagnosis of the reduction of tumor and mass cells by PB302 cells.

[0067] [Reference example 1] (Cancer cell line transplantation model) Each cancer cell line of this embodiment can also be used to produce an intracerebral transplant model. An intracerebral transplant model was produced by the following procedure. 1 x 10 of cancer cell lines 8 A suspension of 1000 cells / mL in HBSS was prepared. Mice were put to sleep by isoflurane inhalation anesthesia. The mice were placed face down, both arms and legs were immobilized, and the skin between the ears was incised approximately 5 mm along the midline. The incision was widened to expose the right skull, the synovial membrane was scraped off with tweezers, and a hole was made in the skull with a microdrill. Care was taken not to damage the brain with the force of drilling the hole. A microsyringe (10 μL) was inserted 5 mm into the brain, and 1.5 μL (1.5 × 10 5 A cell suspension of 1000 cells was injected over 30 seconds. The needle was removed, and the wound was lightly pressed with absorbent cotton. The incised skin was sutured with 2-3 stitches using 6-0 vicryl.

[0068] [Reference example 2] (Creating a meningeal carcinomatosis model using cancer cell lines) Each cancer cell line of this embodiment can also be used to produce a meningeal carcinomatosis model. A meningeal carcinomatosis model was produced by intrathecal administration of the cancer cell line according to the following procedure. This procedure was performed with reference to the procedure used for the A925L / ELuc cell line. Cancer cell line 1×10 8 A suspension of 1000 cells / mL in HBSS was prepared. The mouse's left leg (especially the knee joint) was depilated with hair removal cream. The mouse was put to sleep with Avertin anesthesia. The left heel of the mouse was supported with the left hand, and the knee was bent at 90 degrees. A hole of about 5 mm was made in the tibia with a syringe with a 27G needle from the white part of the knee joint. If the syringe is released and stands upright at this time, it can be confirmed that it is firmly inserted into the tibia. Conversely, if the syringe falls over when the hand is released, it is not appropriate because it has been inserted into the muscle or skin rather than the tibia. The needle was removed, and a microsyringe (10 μL) was inserted 5 mm into the hole. At this time, care was taken not to widen the hole. 4 μL (4 × 105 cells) were injected.

[0069] Although several embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included in the scope of the invention and its equivalents described in the claims, as well as in the scope and spirit of the invention. [Industrial Applicability]

[0070] According to the present invention, a cancer cell line is obtained which is capable of easily forming subcutaneous tumors and which can easily be used for in vitro and in vivo therapeutic experiments. [Accession number]

[0071] NITE P-03936

Claims

1. A cancer cell line capable of forming subcutaneous tumors and positive for the RET fusion gene.

2. A cancer cell line derived from pleural effusion of a patient with human RET fusion gene-positive lung cancer, which is RET fusion gene-positive.

3. The cancer cell line according to claim 1 or 2, which is a cell line deposited under accession number NITE P-03936.

4. The cancer cell line according to claim 1 or 2, further comprising a luciferase gene introduced therein.

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