Cancer model animal and method for producing same

Abstract

Provided are: a cancer model animal in which a malignant tumor corresponding to a pathological image of human invasive cancer or metastasis has been formed or can be formed, despite the use of transplantation under the skin or into the abdominal cavity; and a method for producing the cancer model animal.　One aspect of the present invention is: a cancer model animal in which tumor cells that grow in a culture medium containing L-glucose are transplanted under the skin or into the abdominal cavity: a sarcoma model animal in which tumor cells that have been transplanted under the skin or into the abdominal cavity invade muscles; a cancer model animal in which tumor cells, such as adenocarcinoma cells, that have been transplanted under the skin or into the abdominal cavity show metastasis; or a cancer model animal in which tumor cells, such as adenocarcinoma cells, that have been transplanted under the skin or into the abdominal cavity show abnormality of function of orthotopic cells. Another aspect of the present invention is a method for producing a cancer model animal, the method including a step for transplanting a cell composition under the skin or into the abdominal cavity of an animal, the cell composition containing tumor cells that grow by culturing in a culture medium containing L-glucose and being used for producing a cancer model animal by transplanting the cell composition under the skin or into the abdominal cavity of the animal.

Description

Cancer model animals and methods for producing them

[0001] The present invention relates to a cancer model animal and a method for producing the same.

[0002] Once a newly discovered or developed drug is found to inhibit tumor growth in vitro, the next step is to verify whether it exhibits a similar tumor growth-inhibitory effect in vivo. In this case, it is common to first conduct a test using an animal model to determine whether the drug has a tumor-inhibitory effect, i.e., non-clinical trials, and then move on to human verification, i.e., clinical trials. Appropriate selection or creation of animal models to be used in animal experiments conducted prior to human trials is crucial for the development of novel antitumor substances. One such animal model is known as a lung cancer transplant animal model, which is prepared by injecting lung cancer cells into the trachea of ​​a non-human animal (Patent Document 1).

[0003] Japanese Patent Application Laid-Open No. 2007-274950

[0004] When cancer has deeply infiltrated tissues and even metastasized, the survival rate (patient outcome) of cancer patients significantly worsens. However, cancers with low survival rates are generally difficult to detect early, and because metastasis has already occurred at the time of the initial examination, surgical removal is impossible. Even if surgery is performed when no clear metastatic lesions are found, the cancer often recurs or metastases are discovered after surgery.

[0005] To address these difficulties, first, approaches are being developed to enable early diagnosis of cancer through the development of methods to detect cancer at an earlier stage, at the cellular level, or the development of biomarkers that correspond to cancer progression.Second, approaches are being developed to slow the progression of cancer that has already progressed, enabling patients to control their cancer and improving prognosis by developing chemotherapy, radiation therapy, and other treatment technologies.

[0006] However, taking chemotherapy as an example, in the case of cancers with low survival rates, even if aggressive therapeutic intervention using anticancer drugs recommended as first-line treatment appears to temporarily slow the disease, many patients will relapse and become resistant to treatment. New molecular targeted drugs and immune checkpoint inhibitors that target the immune system have also been put into practical use, but cases in which patients are targeted by these drugs and show significant efficacy are usually extremely limited. In particular, drugs that act on the immune system (immune-targeted agents) have a wide range of side effects that are difficult to deal with on-site, and can sometimes cause severe side effects that can be life-threatening. As there is currently no information available in advance as to whether they will be effective or not, they cannot be used as first-line drugs.

[0007] In fact, even in treatment guidelines for pancreatic and biliary tract cancers, which are known to have the lowest survival rates, the drugs recommended as first-line treatments for these cancers are still traditional anticancer drugs, such as gemcitabine, 5-FU, platinum-based drugs, and paclitaxel, used alone or in combination. These drugs have low cancer specificity and are associated with severe side effects that harm normal cells (Drug combinations used in pancreatic cancer. National Cancer Institute). Numerous combinations of traditional approaches have been attempted, and while some improvements in short-term survival rates have been observed, 10-year survival rates remain significantly low, and overcoming intractable cancers remains a challenging task (President Biden, "Fact Sheet: President Biden reignites cancer moonshot to end cancer as we know it." The White House. February 02, 2022).

[0008] In this sense, basic research is key to enabling groundbreaking technological innovations in all areas of cancer detection, diagnosis, and treatment. For example, if a newly discovered or developed drug is found to inhibit tumor growth in a test tube, the next step is to verify whether it exhibits similar tumor growth-inhibitory effects in vivo. In such cases, nonclinical studies are typically conducted first using model animals to determine whether the drug has tumor-inhibitory effects, before moving on to human testing, or clinical trials. Human testing is a complex process that involves stability and safety testing of the substance and several stages of clinical trials. If a drug's efficacy is not fully confirmed in humans, the development company suffers a significant setback. Therefore, the appropriate selection or creation of animal models for use in animal experiments conducted prior to human testing is crucial for the development of new antitumor drugs and other cancer therapies. The need for superior cancer model animals is also crucial for the development of groundbreaking cancer detection and diagnostic technologies. Thus, the development of effective cancer model animals plays a crucial role in advancing cancer detection, diagnostic, and treatment technologies. Conversely, it is no exaggeration to say that inappropriate animal models are a major factor in the early stages of research that hinders all technological innovation related to cancer detection, diagnosis, and treatment.

[0009] Cancer model animals, in which cultured cancer cells (tumor cell lines) are transplanted into laboratory animals, have long been used for the development of cancer detection technologies and for the early evaluation and screening of various treatments for tumor growth suppression. Among these, the method of subcutaneously transplanting cancer cells into mice to generate tumor-bearing mice remains widely used as a simple and easy method for generating model animals (Stribbling, S.M. et al., Nat. Protoc. 17: 2108-2128, 2022). However, although tumors generated in tumor-bearing animals by subcutaneously transplanting cultured cancer cells grow in the subcutaneous tissue, it is difficult to induce phenomena such as cancer invasion and metastasis. Furthermore, because the subcutaneous tissue is different from the organ from which the target cancer cells originate, so-called orthotopic transplantation, in which cancer cells are transplanted into the organ from which they originate, has also been performed. However, even with orthotopic transplantation, cultured cancer cells generally do not adhere well to tissues.

[0010] Therefore, in order to promote the establishment of transplanted cells, a mixture of extracellular matrices derived from tissues or species completely different from the organ from which the target cancer cells originate (a typical example is Matrigel) has been developed. ＴＭ It is common to add Matrigel (Matrigel, Thermo Fisher Scientific) at the time of transplantation (Stribbling, S. M. et al., Nat. Protoc. 17: 2108-2128, 2022). ＴＭ is a molecule extracted and adjusted from the basement membrane isolated and reconstituted from EHS mouse sarcoma cells. ＴＭ If it were necessary to add such molecules as cofactors during transplantation, it would be undeniable that an artificial microenvironment would be introduced, which is unnatural. Furthermore, orthotopic transplantation requires more complicated surgical procedures than subcutaneous or intraperitoneal transplantation, and the more precise the transplantation, such as the region and depth of the corresponding organ, the more difficult it becomes and the lower the success rate. Therefore, it is not suitable for simple use for the development of diagnostic methods or treatments, screening, etc.

[0011] In order to actually determine the tumor-suppressing effect of an antitumor substance, it is necessary to repeatedly conduct numerous non-clinical trials, including determining concentration dependency and administration schedule, verifying superiority by comparison with standard therapeutic drugs, and examining the relationship between drug efficacy and pathological evaluation of cancer. This requires the use of a large number of animals, a long experimental period, and, as a practical matter, a lot of work. Therefore, simpler animal models such as subcutaneous transplants continue to be used for initial screening, despite their drawbacks.

[0012] It is recognized that much attention should be paid not only to the transplant site but also to the cells themselves (Stribbling, SM. et al., Nat. Protoc. 2022). For example, it has been pointed out that commercially available tumor cell lines may retain the properties of cancer cells when initially extracted from the organs of cancer patients, but that over the years of passage in specific culture media and artificial culture environments, cells better adapted to the culture environment become dominant.

[0013] Therefore, mouse models using patient-derived xenografts (PDX) have been used, in which cells from tumor tissues, metastatic tissues, or blood samples from cancer patients are used directly without passaging or after a very limited number of passagings. Alternatively, these cells or tissues are subcutaneously transplanted into immunodeficient mice (Aparicio, S. et al., Nat. Rev. Cancer 15: 311-316, 2015). However, this method requires a long time to establish, is difficult to scale up and maintain, and can only be supplied to a limited number of researchers. Furthermore, it is recognized that even samples extracted from the same patient's cancer tissue may have different cellular characteristics depending on the location of the tissue. To put it in extreme terms, even if a drug is developed that is effective in suppressing a specific portion of a specific patient's tumor, it may not be effective against cancers in different locations of that patient or tumors in different patients, resulting in a lack of reproducibility. As described above, PDX and tumor-bearing mouse models using cultured cells (tumor cell lines) each have their own advantages and disadvantages, and at first glance they appear to be polar opposites.

[0014] However, cancers with low survival rates can sometimes share common features. For example, even if their origins are quite different, such as the digestive tract, pancreas, or prostate, they may exhibit common pathological cellular morphologies and characteristics, regardless of the site of origin. This includes neuroendocrine tumors, which are known to be highly resistant to treatment, and clear cell carcinoma, a type of gynecological cancer such as ovarian cancer and renal cancer known for its poor response to chemotherapy and poor prognosis. Furthermore, there are cancers, such as sarcomas, which originate from a variety of different origins but share the common characteristic of invading muscle, bone, etc.

[0015] In light of the above, if there were a cancer model animal that is easy to transplant and simple to use, and that exhibits the clinical symptoms of invasion and distant metastasis without the aid of cofactors such as extracellular matrix, it would be expected to be useful in developing and evaluating detection methods, diagnostic methods, and treatments for cancers with poor prognosis.

[0016] The object of the present invention is to provide a cancer model animal that has formed or is capable of forming malignant tumors comparable in pathology to human invasive cancer or metastasis, despite the use of subcutaneous or intraperitoneal transplantation, and a method for producing the same.

[0017] The present inventors have conducted extensive research to solve the above-mentioned problems. As a result, they have found that the above-mentioned problems can be solved by using a specific cancer model animal generated using L-glucose, and have completed the present invention. Specifically, the present invention provides the following.

[0018] (1) A cancer model animal in which tumor cells cultured and growing in a medium containing L-glucose are transplanted subcutaneously or intraperitoneally.

[0019] (2) Sarcoma model animals in which tumor cells transplanted subcutaneously or intraperitoneally infiltrate into the muscle.

[0020] (3) The sarcoma model animal according to (2), wherein the muscle is a skeletal muscle.

[0021] (4) A cancer model animal in which tumor cells transplanted subcutaneously or intraperitoneally show metastasis, wherein the tumor cells are selected from the group consisting of adenocarcinoma cells, neuroendocrine tumor cells, and sarcoma cells.

[0022] (5) The cancer model animal according to (4), wherein the tumor cells are adenocarcinoma cells, and the metastasis is metastasis to lymph nodes, lungs, or intraperitoneal organs, or accumulation in the renal hilus.

[0023] (6) The cancer model animal according to (5), wherein the adenocarcinoma cells are tumor cells derived from lung cancer.

[0024] (7) The cancer model animal according to (5), wherein the intraperitoneal organ is the liver or the spleen.

[0025] (8) The cancer model animal according to (4), wherein the tumor cells are neuroendocrine tumor cells, and the metastasis is to a lymph node, an intraperitoneal organ, or the peritoneum.

[0026] (9) The cancer model animal according to (8), wherein the neuroendocrine tumor cells are insulinoma cells.

[0027] (10) The cancer model animal according to (8), wherein the intraperitoneal organ is the liver.

[0028] (11) A cancer model animal in which tumor cells transplanted subcutaneously or intraperitoneally exhibit abnormalities in the functions of orthotopic cells, wherein the tumor cells are selected from the group consisting of adenocarcinoma cells, neuroendocrine tumor cells, and sarcoma cells.

[0029] (12) The cancer model animal according to (11), wherein the adenocarcinoma cells are tumor cells derived from lung cancer.

[0030] (13) The cancer model animal according to (11), wherein the neuroendocrine tumor cells are insulinoma cells.

[0031] (14) The cancer model animal according to (11), wherein the tumor cells exhibit abnormalities in the functions of orthotopic cells within 3 days after transplantation.

[0032] (15) The tumor cells are 5.0 × 10 ５ A cancer model animal according to (11), which exhibits abnormalities in the function of orthotopic cells with the following cell counts:

[0033] (16) A cell composition comprising tumor cells cultured and grown in a medium containing L-glucose, which is used to prepare a cancer model animal by subcutaneously or intraperitoneally transplanting the cell composition into an animal.

[0034] (17) A method for producing a cancer model animal, comprising the step of subcutaneously or intraperitoneally transplanting the cell composition according to (16) into an animal.

[0035] (18) A screening method for a substance having an anticancer effect, comprising: a step of administering a test substance to the model animal described in any one of (1) to (15); a step of evaluating tumor cell growth or tumor cell metastasis after the start of administration of the test substance; and a step of comparing the evaluation result in the evaluation step with the evaluation result of tumor cell growth or tumor cell metastasis for the model animal not administered the test substance.

[0036] According to the present invention, it is possible to provide a cancer model animal that has formed or is capable of forming a malignant tumor comparable to the pathological features of human invasive cancer or metastasis, despite the use of subcutaneous or intraperitoneal transplantation, and a method for producing the same.

[0037] Figure 1A is a photograph showing a low-magnification image of a tumor in which L-glucose-cultured human lung cancer-derived tumor cells A549 were intraperitoneally transplanted, showing clear infiltration into the abdominal muscle. Figure 1B is a photograph showing a high-magnification image of the tumor in Figure 1A. Figure 2A is a photograph showing liver metastasis of L-glucose-cultured human lung cancer-derived tumor cells A549. Figure 2B is a photograph showing lymph node metastasis of the tumor cells. Figure 2C is a photograph showing splenic metastasis of the tumor cells. Figure 2D is a photograph showing the infiltration of the tumor cells into each organ visualized from outside the body using a Whole Body Imager (IVIS Lumina Imaging System, Caliper) using luciferase-transfected A549 cells for transplantation. The image shows the tumor infiltrating not only the liver, spleen, and lymph nodes, but also the peritoneal cavity. Figure 3 is a photograph showing a tumor observed in the lung after intraperitoneal transplantation of L-glucose-cultured human lung cancer-derived tumor cells A549 into a mouse. The arrow indicates the tumor. Angiogenesis was also observed in the tumor, demonstrating its establishment in the lung. Figure 4A is a low-magnification photograph of a large thrombus observed in the renal hilar vein of the kidney, and Figure 4B is a magnified photograph of Figure 4A. Figure 5 is a photograph of a mouse 7 days after subcutaneous transplantation of L-glucose-cultured MIN6 cells into the right hind leg. The arrow indicates the subcutaneous transplantation site. The subcutaneous site into which the tumor cells were transplanted did not show the swelling typically observed in subcutaneous transplantation animal models. Figure 6A is a low-magnification photograph showing an HE-stained image of a sarcoma formed by infiltrating into the center of the muscle after tumor cells were subcutaneously transplanted into the hind leg of the mouse shown in Figure 5, without accumulating subcutaneously. Figure 6B is a high-magnification photograph of the sarcoma, revealing an extremely aggressive histopathological image. Figure 7 is a graph showing that the blood glucose levels of mice subcutaneously transplanted with insulinoma cells cultured in L-glucose (black triangles) were statistically significantly lower than the blood glucose levels of mice subcutaneously transplanted with insulinoma cells cultured using D-glucose as usual (black circles) and the blood glucose levels of mice in the normal control group (white circles).The blood glucose levels of mice subcutaneously implanted with insulinoma cells cultured in L-glucose showed a statistically significant decrease in blood glucose just one day after implantation compared to the blood glucose levels of mice subcutaneously implanted with insulinoma cells cultured in D-glucose, clearly demonstrating that the implanted tumor was connected to the bloodstream and functioning to secrete insulin. Figure 8A is a photograph showing an HE-stained image of a tumor that was completely engrafted into the peritoneal cavity and clearly connected to the bloodstream, and Figure 8B is a photograph showing a magnified image of the tumor in Figure 8A. Figure 8B shows that the tumor was highly atypia, had numerous mitotic figures, and was a fairly high-grade cancer. Figure 9A is a photograph showing an HE-stained image of a tumor that had infiltrated the abdominal muscle. The arrow indicates the tumor. Figure 9B is a photograph showing a magnified image of a highly atypia-marked high-grade malignant tumor. Fig. 10A is a photograph showing an HE-stained image showing lymph node metastasis of tumor cells transplanted into the abdominal cavity of a mouse, and Fig. 10B is a photograph showing a magnified image of the tumor in Fig. 10A. Fig. 11A is a photograph showing an HE-stained image showing liver metastasis of tumor cells transplanted into the abdominal cavity of a mouse, and Fig. 11B is a photograph showing a magnified image of the tumor in Fig. 11A. Fig. 12A is a photograph showing an HE-stained image showing smooth muscle metastasis of tumor cells transplanted into the abdominal cavity of a mouse, and Fig. 12B is a photograph showing a magnified image of the tumor in Fig. 12A.

[0038] Preferred embodiments of the present invention will be described in detail below, but the present invention is not limited to the following embodiments.

[0039] As described above, there is a strong need for cancer model animals and cancer cell lines that exhibit properties common to human cancers, are useful for evaluating the antitumor activity of drugs, and are as simple and easy to use as possible. Therefore, the inventors conducted research based on the following concept, which is completely different from PDX.

[0040] As described in the "Problem to be Solved by the Invention" section, the reason malignant tumors, i.e., cancer, are feared and require treatment is that malignant tumor cells infiltrate sites where they should not exist or metastasize to distant sites, causing abnormal proliferation, disrupting normal tissue function, and ultimately resulting in fatal consequences for the body. In contrast, benign tumors exhibit cell proliferation, but retain the original functional and morphological characteristics of the proliferating cells. Furthermore, one of the key characteristics that distinguishes benign tumors from malignant tumors is that tumor cells do not invade areas with histologically distinct functions. As long as tumor cells remain in their original locations while retaining their morphological and functional characteristics, they can receive nutrients through the bloodstream, even if the tumor is large. In contrast, tumor cells that invade areas with different functions from their own are expected to have difficulty receiving the nutrients necessary for growth, since they do not possess the characteristics of the cells in the invaded area.

[0041] Not only cancer cells, but all cells require nutrients (or carbon sources) to act as an energy source for proliferation. The most common nutrient (or carbon source) used by living organisms is D-glucose. Among cancer screening methods, the so-called PET scan is a technology that utilizes this property to enable cancer detection by imaging (Gambhir, SS Nat. Rev. Cancer 2: 683-693, 2002). Specifically, a radioactive label is attached to the 2-carbon position of D-glucose. １８FDG-PET is a highly useful imaging modality widely used worldwide for the purpose of detecting whether a wide range of cancer types are cancerous or where cancer has metastasized, by detecting the strong intracellular uptake of F-conjugated D-glucose derivative molecules (commonly known as FDG) from outside the body using positron emission tomography. However, in methods using D-glucose, D-glucose is a nutrient utilized not only by cancer cells but also by normal cells surrounding the cancer. For this reason, detection in the brain, which strongly uptakes D-glucose, can be difficult, muscle movement and fat can interfere with detection, and it is not uncommon for inflammation, a non-cancerous cellular abnormality, to be mistaken for cancer.

[0042] Furthermore, when cancer cells attempt to ingest D-glucose, they compete with normal cells for nutrients, giving normal cells, which have completed tissue construction and receive supplies from the bloodstream, an advantage. Therefore, if tumor cells can survive and proliferate even in an environment where nutrient intake is difficult, they can be said to be highly invasive tumor cells, i.e., cells with an important property for survival as cancer cells. The inventors hypothesized that tumor cells that can internalize "nutrients that normal cells cannot or hardly can utilize" and metabolize them to use as an energy source (or carbon source) for proliferation would survive without competing for nutrient intake with the majority of cells present at the invasive site, allowing tumor cells to proliferate and thus increase their likelihood of generating cancer. The inventors focused particularly on L-glucose as one such nutrient.

[0043] L-glucose is an enantiomer of D-glucose, the smallest building block of starch, and is rarely or never found in nature. Furthermore, normal cells are believed to be unable to internalize or metabolize L-glucose (Rudney, H. Science 1940, 92, 112-113). In fact, it is well known that L-glucose is not transported into cells by glucose transporters, the transmembrane proteins used by mammalian cells to internalize D-glucose (Ono, K. et al., Cancers 12: 850, 2020).

[0044] However, as described below, the inventors have demonstrated that various tumor cells exhibiting malignant characteristics take up L-glucose via a specific mechanism within the cells. First, they found that the L-glucose analog 2-NBDLG, which is conjugated with the green fluorescent group NBD, is taken up into many types of malignant tumor cells, causing the tumors to emit fluorescence (Sasaki, A. et al., Human Cell 29: 37-45, 2016). Next, they found that similar uptake into tumor cells was observed even when the fluorescent group was changed from NBD to the blue coumarin, and reported that L-glucose, which is common to both, plays an important role, and that this uptake is mediated by a channel-like protein that is inhibited by the apple polyphenol phloretin (Otsuka Y. et al., Org. Lett. 18: 1338-1341, 2016; Yamada, K. et al., US10001487B2; US10509041B2; EP3130596B1; Japanese Patent No. 6566348). Cells that exhibit similar L-glucose uptake have been found in cultured pancreatic tumor cells exhibiting malignant characteristics, cancer tissue from cholangiocarcinoma model hamsters, tumor tissue removed from gastric cancer patients, cells in ascites samples collected immediately after laparotomy or before organ removal from patients with uterine or ovarian cancer, urine from patients with urinary tract cancers such as bladder cancer and ureter cancer, and sarcomas (Yokoyama, H., et al., Human Cell 29: 111-121, 2016; Ogawa, T. et al., Human Cell 34: 634-643, 2021; Yamada, K. et al., US10551387B2; EP3199638B1; Japanese Patent No. 6670503; Japanese Patent No. 6406715).

[0045] To investigate whether tumor cells can grow using L-glucose as a nutrient, we cultured tumor cells by adding the same amount of L-glucose instead of D-glucose, which is normally added as a nutrient (hereinafter referred to as L-glucose culture). As a result, while many cells died or their condition worsened, a small percentage of cells survived and began to proliferate, spontaneously forming cell clusters called spheroids without the use of special low-adhesion containers or Matrigel (Patent No. 6721868).

[0046] L-glucose has the exact opposite configuration of its four asymmetric carbon atoms as D-glucose, making it unable to pass through the glucose transporter that transports glucose into cells via binding to the binding site. Conversely, if cells could internalize and metabolize L-glucose, they could potentially obtain an energy and carbon source for growth without competing with normal cells for nutrients. Conversely, L-glucose uptake into cells could be used as an indicator of survival, utilizing nutrients unavailable to normal cells even in harsh environments, and could potentially be used as a powerful indicator of highly metastatic cancer cells. Indeed, some cancer cells from invasive cancers and those with poor postoperative outcomes are able to take up L-glucose (Yamada, K. et al., US10551387B2; EP3199638B1; Japanese Patent No. 6670503; Japanese Patent No. 6406715).

[0047] However, there were no previous examples of transplanting L-glucose-cultured tumor cells into animals, and the properties of these cells in vivo were completely unknown. The inventors cultured tumor cells in a culture medium supplemented with an equal amount of L-glucose instead of D-glucose. Cells that survived under these culture conditions were suspended in physiological saline and transplanted subcutaneously into the hind limbs or intraperitoneally into immunodeficient mice without the use of any cofactors such as Matrigel. Prior to the experiment, the inventors had suspected that transplanting cultured cells without Matrigel would not establish themselves in the animals' bodies. In fact, cells cultured in L-glucose and transplanted subcutaneously did not grow subcutaneously, initially concluding that tumors had not taken root. However, upon noticing a drop in blood glucose in the animals, they suspected that a tumor must be present somewhere. They fixed the animals in formalin, divided their entire bodies into 5-mm sections, and used each section as a pathological specimen to search for cells. They then discovered the completely unexpected phenomenon of tumor cells deeply infiltrating into the muscle, forming a sarcoma. Furthermore, these sarcomas demonstrated function and metastatic potential comparable to the clinical picture. The phenomenon of subcutaneously implanted tumor cell lines infiltrating deeply into the muscle and forming sarcomas is unprecedented, and similar muscle invasion was observed when these tumor cells were implanted intraperitoneally. Furthermore, clear metastatic patterns were observed, including lymph node and liver metastasis.

[0048] [Cancer Model Animal] Examples of animals that can be used as cancer model animals according to the present invention include mice, as well as non-human animals such as rats, hamsters, guinea pigs, monkeys, cows, pigs, horses, rabbits, sheep, goats, cats, and dogs. The non-human animals are not particularly limited, and suitable examples include mildly immunodeficient mice such as nude mice lacking T cell function, such as BALB / c-nu / nu and F344 / N-rnu / rnu, or mice lacking T and B cells, such as NOD / SCID mice. Other examples include immunodeficient animals such as NSG mice, nude rats, and ALY mice, as well as improved strains thereof.

[0049] Tumor cells used in the cancer model animal according to the present invention are not particularly limited, and examples thereof include adenocarcinoma cells such as lung cancer-derived tumor cells and neuroendocrine tumor cells such as insulinoma cells. The cancer from which tumor cells are derived is not particularly limited, and examples thereof include adenocarcinomas such as lung cancer, pancreatic cancer, brain tumor, breast cancer, stomach cancer, gastroesophageal junction cancer, oral cancer, biliary tract cancer, ovarian cancer, and uterine cancer, as well as various cancers such as sarcoma and osteosarcoma. The animal species from which tumor cells are derived is not particularly limited, and examples thereof include mice and humans.

[0050] The cancer model animal according to the first embodiment of the present invention is a cancer model animal in which tumor cells cultured and grown in a medium containing L-glucose are subcutaneously or intraperitoneally transplanted.

[0051] A cancer model animal according to a second embodiment of the present invention is a sarcoma model animal in which tumor cells transplanted subcutaneously or intraperitoneally have infiltrated into the muscle. Examples of the muscle include skeletal muscle and smooth muscle. A sarcoma model animal in which the muscle is skeletal muscle is unprecedented and is particularly useful. In one aspect of the second embodiment of the present invention, the sarcoma model animal is a sarcoma model animal in which tumor cells transplanted subcutaneously do not swell in the same place but have infiltrated into the skeletal muscle.

[0052] A cancer model animal according to a third embodiment of the present invention is a cancer model animal in which tumor cells transplanted subcutaneously or intraperitoneally exhibit metastasis, the tumor cells being selected from the group consisting of adenocarcinoma cells, neuroendocrine tumor cells, and sarcoma cells. As used herein, metastasis refers to tumor cells infiltrating or proliferating in other sites rather than simply remaining at the site of transplantation. Examples of metastasis include tumor cells transplanted into the peritoneal cavity not only establishing themselves within the peritoneal cavity but also metastasizing to lymph nodes or peritoneal infiltration, metastasizing to skeletal muscles or smooth muscles such as abdominal muscles to form sarcomas, and metastasizing to intraperitoneal organs. As used herein, intraperitoneal organs refer to the liver, spleen, stomach, intestines, pancreas, gallbladder, kidneys, and adrenal glands.

[0053] In one aspect of the third embodiment of the present invention, the tumor cells are adenocarcinoma cells, and the metastasis is to lymph nodes, lungs, or intraperitoneal organs, or accumulation in the renal hilum. The adenocarcinoma cells are not particularly limited, and examples thereof include tumor cells derived from lung cancer. The intraperitoneal organs are not particularly limited, and examples thereof include the liver or spleen.

[0054] In another aspect of the third embodiment of the present invention, the tumor cells are neuroendocrine tumor cells, and the metastasis is to lymph nodes, intraperitoneal organs, or the peritoneum. The neuroendocrine tumor cells are not particularly limited, and examples thereof include insulinoma cells. The intraperitoneal organs are not particularly limited, and examples thereof include the liver.

[0055] A cancer model animal according to a fourth embodiment of the present invention is a cancer model animal in which tumor cells transplanted subcutaneously or intraperitoneally exhibit abnormalities in the functions of orthotopic cells, and the tumor cells are selected from the group consisting of adenocarcinoma cells, neuroendocrine tumor cells, and sarcoma cells. In one aspect of the fourth embodiment of the present invention, the adenocarcinoma cells are lung cancer-derived tumor cells. In another aspect of the fourth embodiment of the present invention, the neuroendocrine tumor cells are insulinoma cells. In yet another aspect of the fourth embodiment of the present invention, the tumor cells exhibit abnormalities in the functions of orthotopic cells within three days after transplantation, preferably within two days after transplantation, more preferably within one day after transplantation, and even more preferably one day after transplantation. In yet another aspect of the fourth embodiment of the present invention, the tumor cells are 5.0 x 10 ５ The number of cells is preferably 4.0 x 10 ５ The number of cells is preferably 3.0 x 10 ５ and even more preferably 2.0 x 10 ５ The lower limit of the number of tumor cells is not particularly limited, and the tumor cells are 1.0 × 10 ５ A cell count of this magnitude indicates an abnormality in the function of the orthotopic cells.

[0056] In the fourth embodiment of the present invention, for example, when the tumor cells are neuroendocrine tumor cells and the neuroendocrine tumor cells are insulinoma cells, a strong decrease in blood glucose due to excessive insulin secretion can be observed in a cancer model animal as early as one day after transplantation.

[0057] Human sarcomas are extremely diverse, and the cancer cells that form sarcomas originate from various organs. In the cancer model animal according to the fourth embodiment of the present invention, adenocarcinoma cells such as lung cancer-derived tumor cells or neuroendocrine tumor cells, typified by insulinoma cells, transplanted subcutaneously or intraperitoneally can deeply infiltrate into muscle to form sarcomas, and in the case of insulinoma cells, can cause a strong decrease in blood glucose, thereby exhibiting the functions of the organ from which the tumor cells originate.

[0058] In a preferred embodiment of the present invention, the cells may be modified so that a gene encoding a fluorescent protein or a luminescent protein is incorporated into the genome so that the protein can function within the cell, thereby stably expressing the protein. By using such cells to produce a model animal according to the production method of the present invention, it becomes possible to non-invasively and in real time observe the behavior of cancer cells, such as proliferation, migration, invasion, and metastasis, in vivo, using the fluorescence or luminescence as an indicator. Green fluorescent protein or any other protein commonly used in the technical field can be used as the fluorescent protein, and various luciferases and the like can be used as the luminescent protein.

[0059] In the present invention, cells are cultured using a medium supplemented with L-glucose rather than D-glucose as the glucose. Here, L-glucose medium refers to a medium to which L-glucose is added as glucose but D-glucose is not actively added, i.e., a glucose-containing medium in which the glucose is substantially composed of L-glucose. For example, when a serum-containing medium is used, small amounts of D-glucose are contained in the serum (typically, adding 10% of Hyclone's Defined Fetal Bovine Serum to the medium results in a D-glucose concentration of approximately 100 mg / L or less), and therefore small amounts of serum-derived D-glucose may be contained in the medium. However, even in these media, as long as the glucose in the medium is substantially composed of L-glucose, it is included in the L-glucose medium used in the present invention. The L-glucose medium used in the present invention refers to a glucose-containing medium in which the glucose is substantially composed of L-glucose, preferably 98% or more, more preferably 99% or more. The glucose concentration in the L-glucose medium used in the present invention is not particularly limited, and may be, for example, the glucose concentration when D-glucose is used as the glucose in a normal culture, such as, but not limited to, 0.5 mM to 50 mM, preferably 1.0 mM to 25 mM, and more preferably 5 mM to 25 mM.

[0060] As the L-glucose medium, any medium used for culturing conventional cells can be used as long as the glucose is substantially composed of L-glucose. Examples include BME medium, BGjB medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM medium, IMDM medium, Medium 199 medium, Eagles MEM medium, αMEM medium, DMEM medium, Ham's medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. However, any medium that can be used for culturing animal cells is not particularly limited. DMEM medium is preferred. These media are commercially available. As the serum, commercially available serum used in conventional cultures may be used. For example, good results can be obtained using serum manufactured by Hyclone.

[0061] In one embodiment of the present invention, D-glucose is removed from the medium and the cultured cells are cultured in L-glucose medium instead. When switching from D-glucose medium to L-glucose medium, it is important to start the replacement with L-glucose medium from an appropriate number of days of culture depending on the cells. The method of replacement with L-glucose medium can be appropriately modified depending on the cells, and examples include replacing D-glucose medium with L-glucose medium or gradually increasing the proportion of L-glucose in total glucose. Specifically, but not limited to, by performing half-exchanges with medium supplemented with L-glucose as glucose an appropriate number of times, the D-glucose concentration can be gradually reduced and the L-glucose concentration can be easily increased. Half-exchanges can be performed, but are not limited to, for example, on days 6 and 8 after the initiation of D-glucose culture. Furthermore, on day 10, the entire culture medium is discarded and washed with L-glucose medium to remove as much D-glucose as possible, ultimately resulting in a 100% L-glucose medium. After the medium becomes 100% L-glucose, half of the medium is replaced every 6 hours for, but not limited to, one or two days. Subsequent medium replacements are performed every few days if the cell number is low, and every day if the cell number is high.

[0062] In one embodiment of the present invention, cells are cultured for a certain number of days or more after replacing the medium with one containing 100% L-glucose (0% D-glucose) as glucose. The "certain number of days or more" is appropriately selected depending on the cells used, but can be, for example, 3 to 10 days. Generally, the cells are cultured for, for example, 3 days or more, preferably 10 days or more, more preferably 11 days or more, and even more preferably 13 days or more.

[0063] There are no particular limitations on the cells to which this culture method can be applied, and any cancer cells derived from animals or cancer patients can be used.

[0064] Based on the inventors' previous research, it is expected that this culture method can be performed in the same way even if L-fructose, an L-type sugar whose natural abundance is lower than that of D-type sugar, is used instead of L-glucose. That is, it is expected that this culture method will enable the production of not only tumor cells that grow when cultured in a medium containing L-glucose, but also tumor cells that grow when cultured in a medium containing L-fructose. Furthermore, it is expected that not only cancer model animals will be obtained by subcutaneously or intraperitoneally implanting tumor cells that grow when cultured in a medium containing L-glucose, but also cancer model animals will be obtained by subcutaneously or intraperitoneally implanting tumor cells that grow when cultured in a medium containing L-fructose.

[0065] The following description will be given using human lung cancer cell A549 as an example, but it will be clear to those skilled in the art that this method can also be applied to other cells. In such cases, the materials, reagents, and conditions to be used can be appropriately selected and used in accordance with publicly known information about the respective cells.

[0066] A cell suspension of human lung cancer cells A549 is prepared using a commercially available D-glucose-containing medium. The cells can be seeded onto a dish or a cover slip depending on the subsequent observation and analysis. For example, the number of cells to be seeded is 3.0 to 6.0 x 10 for a commercially available 90 mm diameter dish (Greiner TC Petri dish 664160). ５pcs / dish, and 1.0 x 10 for WillCo-Glass bottom dish (WillCo Wells 170 micron diameter 40 mm). ５ The cells can be seeded and used at an appropriate number depending on the purpose, such as 10 cells / dish.

[0067] Next, at a certain time point after the start of culture, the D-glucose medium is replaced with L-glucose medium. In the case of A549 cells, after culturing for one day using D-glucose medium, the medium is replaced with L-glucose medium from the second day onwards, and the culture is continued. Such medium replacement can be carried out by methods known to those skilled in the art. For example, it can be carried out by removing the D-glucose medium used for cell culture or by recovering the cells from the medium and adding L-glucose medium.

[0068] If a sudden change in medium composition is not desired, medium replacement can be carried out stepwise as follows. First, the medium is replaced so that the D-glucose concentration in the medium is 25% (6.25 mM) of the initial concentration. To achieve this, half of the medium can be replaced twice in succession to achieve a D-glucose concentration of 25%. The medium used for the medium replacement can be a D-glucose-free medium to which L-glucose has been appropriately added.

[0069] The appropriate time to reduce the D-glucose concentration to 25% of the initial concentration is between days 2 and 10 after the start of culture. After culturing for two days at a D-glucose concentration of 25%, half of the medium is replaced to reduce the D-glucose concentration to 12.5% ​​(3.125 mM) and culture for two more days. After that, the entire medium is discarded, washed twice with L-glucose-containing medium, and then L-glucose medium is added, thereby reducing the D-glucose concentration added to the medium to 0%.

[0070] When cultured in L-glucose medium, A-549 cells form a layer. After the cells have grown to a number sufficient for transplantation, they are trypsinized in the usual way to prepare a cell suspension in L-glucose medium.

[0071] When transplanted into a mouse, for example, 3 × 10 ６Tumors can be formed by transplanting cells at a concentration of 1.0 × 10 cells / (100 μL) / animal. Because transplantation without an extracellular matrix such as Matrigel results in a low rate of adhesion, a large number of cells must usually be transplanted. However, when tumor cells cultured in L-glucose medium are transplanted into animals, a concentration of 1.0 × 10 cells / (100 μL) / animal is required. ５ Even when a small number of cells, such as 20 μL / mouse, is transplanted, tumor formation is confirmed with a high probability, as will be described later (FIGS. 8 to 11).

[0072] Although the state varies depending on the cell, the following occurs, for example, when L-glucose medium is used. During culture, among the cells that have continued to grow, floating dead cells become noticeable at D-glucose concentrations of 12.5% ​​(3.125 mM) and above. Once the D-glucose concentration reaches 0%, floating cells become prominent, and the number of cells that had been increasing up to that point temporarily decreases. To remove metabolic products from floating dead cells, half of the medium is replaced with L-glucose-containing medium every 6 hours from the point at which the D-glucose concentration added to the medium reaches 0%. This minimizes the influence of metabolic products. It is desirable to replace the medium every 6 hours at least four times, preferably eight times or more, until floating cells are no longer noticeable. Regarding subsequent medium replacements, it is preferable to replace half of the medium every other day while there are few cells in the entire dish, and every day from day 19 onwards when the cells begin to increase sufficiently.

[0073] [Cell composition used to generate a cancer model animal] The cell composition of the present invention contains tumor cells that are cultured and grown in a medium containing L-glucose, and is used to generate a cancer model animal by transplanting the tumor cells subcutaneously or intraperitoneally into an animal. The animals, tumor cells, etc. are as described above. The cell composition of the present invention may contain the above-mentioned medium such as L-glucose medium, the above-mentioned serum, a solvent such as water, a physiologically acceptable salt, etc.

[0074] [Method for Producing a Cancer Model Animal] The method for producing a cancer model animal according to the present invention includes a step of subcutaneously or intraperitoneally transplanting the cell composition according to the present invention into an animal. Depending on the cancer model animal of interest, the cell compositions can be used singly or in combination of two or more types. The animal may be an adult, for example, a mouse aged 5 to 9 weeks or a rat aged 6 to 10 weeks. The method for subcutaneously or intraperitoneally transplanting the cell composition into an animal is not particularly limited, and examples include a method of subcutaneously or intraperitoneally transplanting the cell composition into an animal using a syringe equipped with a needle. Whether or not a cancer model animal has been produced can be confirmed by measuring various physiological functions, or by incorporating a gene encoding a fluorescent protein or a luminescent protein into the genome so that it can function in the cells and noninvasively observing the fluorescence or luminescence as an indicator, or by dissecting the animal.

[0075] [Method for screening for substances with anticancer activity] The method for screening for substances with anticancer activity according to the present invention comprises the steps of administering a test substance to a model animal according to the present invention, evaluating tumor cell growth or tumor cell metastasis after the start of administration of the test substance, and comparing the evaluation results from the evaluating step with the results of evaluating tumor cell growth or tumor cell metastasis in the model animal not administered the test substance. As used herein, a substance with anticancer activity refers to a substance that exhibits an inhibitory effect on tumor cell proliferation.

[0076] The test substance is not particularly limited, and examples thereof include sugars, nucleic acids, peptides, proteins, cell extracts, cell culture supernatants, plant extracts, mammalian tissue extracts, and plasma. The test substance may form a salt, and the salt of the test substance may be a salt with a physiologically acceptable acid or a salt with a physiologically acceptable base. The amount of the test substance is not particularly limited, and may be determined appropriately based on known literature, preliminary tests, etc. The administration route is selected appropriately depending on the test substance, and examples include known administration routes such as oral administration, intravenous administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, and intratumoral administration. The administration period, administration interval, etc. are not particularly limited, and may be selected appropriately depending on the test substance.

[0077] The timing of evaluating tumor cell growth or metastasis is not particularly limited, as long as it is after the start of administration of the test substance. When tumor cell growth can be observed visually, it may be confirmed by measuring the size, weight, etc., or by pathological autopsy. Furthermore, various fluorescent probes such as the fluorescent L-glucose derivative 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-L-glucose (2-NBDLG), in which the fluorescent group NBD has been introduced at the second position of L-glucose, or its enantiomer, the D-glucose derivative 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), may be used to confirm the presence of the fluorescent group NBD at the second position of L-glucose, using a whole-body imaging device, a fluorescent endoscope, a laparoscope, or the like. Alternatively, cells into which luciferase has been previously introduced may be transplanted, and the metastasis may be confirmed by luminescence imaging using various luminescent probes such as luciferin, or by animal X-ray CT, animal magnetic resonance imaging, or the like. Furthermore, the metastasis may be evaluated by positron emission tomography using a radioactively labeled probe such as 18F-2-deoxy-D-glucose (FDG). Similarly, metastasis of tumor cells may be evaluated by measuring dimensions, weight, etc., when visual observation is possible, or by pathological autopsy, or by using in vivo imaging methods such as fluorescence, luminescence, X-ray CT, MRI, and PET.

[0078] The evaluation results in the evaluation step are compared with the results of evaluating tumor cell growth or tumor cell metastasis in the model animal that has not been administered the test substance, and if the former evaluation results show significantly inferior tumor cell growth or tumor cell metastasis, the test substance is determined to have anticancer activity.

[0079] The present invention will be explained in more detail below based on examples, but the present invention is not limited to these examples.

[0080] [Cancer Model Animals Using Human Lung Cancer-Derived Tumor Cells] Human lung cancer-derived tumor cells A549 cultured in L-glucose were ectopically transplanted into the peritoneal cavity without Matrigel. BALB / c nu / nu (nude) mice were used. They were purchased and maintained at 5 to 7 weeks of age. After cell transplantation, tumor growth was confirmed using whole-body imaging in cases where luciferase-containing cells were transplanted, or other whole-body imaging methods such as CT or MRI in other cases. After anesthesia, the entire body was fixed in 10% neutral-buffered formalin. The hind limb muscles and organs from the cell-transplanted side and the contralateral side (control) were removed and fixed in 10% neutral-buffered formalin. After fixation, the entire body was embedded in 5 mm sections, and paraffin sections were prepared. These sections were then subjected to standard HE staining and immunostaining with antibodies, etc., for pathological examination. As a result, human lung cancer-derived tumor cells A549 invaded the abdominal muscle and formed a sarcoma (Figure 1A). As can be seen from the enlarged image, a clear invasive cancerous lesion is clearly visible, as if integrated into the abdominal muscle (Figure 1B). This result demonstrates the strong ability of tumor cells cultured in L-glucose medium to invade muscle, i.e., to form a sarcoma. To the inventors' knowledge, there is no other model in which a tumor cell line ectopically transplanted into the abdominal cavity can invade deeply into the abdominal muscle and form a highly atypical sarcoma.

[0081] Furthermore, in this model, L-glucose-cultured tumor cells A549 significantly infiltrated various organs in the abdominal cavity, including the liver (Fig. 2A), lymph node (Fig. 2B), and spleen (Fig. 2C), and clear cancer metastasis was observed (Fig. 2D).

[0082] A549 is a tumor cell line derived from human lung cancer, and it is important to investigate the effects of heterotopic transplantation on respiratory function and kidney function, which cooperates with the lungs in circulatory regulation, such as pH regulation. A pathological autopsy of mice in which L-glucose-cultured human lung cancer-derived tumor cell line A549 was transplanted intraperitoneally without the use of Matrigel revealed that the cancer had infiltrated the lungs, as shown in Figure 3.

[0083] After intraperitoneal transplantation of A549 human lung cancer-derived tumor cells obtained by culturing in L-glucose medium, the cancer cells that formed infiltrated into the lungs of mice took root in the lungs accompanied by angiogenesis, as is clear in Figure 3, and the lesions clearly showed signs of cancer. Therefore, from the standpoint of animal protection, the mice were euthanized in this case, but if left untreated, it would undoubtedly cause damage to the normal respiratory function of the lungs.

[0084] Furthermore, this model also induced remarkable functional changes in the kidneys, unprecedented, at least to our knowledge, in animal models. The lung is considered to be the most common primary site of cancer metastasis to the kidney in humans. Conversely, although several cases of lung cancer metastasis to the kidney have been reported in human lung cancer patients, most cases were not detected during premortem examinations and were only detected postmortem. In the kidneys of mice intraperitoneally implanted with A549 human lung cancer-derived tumor cells cultured on L-glucose, large tumor thrombi of unusual size formed in the renal hilum of both the left and right kidneys, as shown in Figures 4A and 4B.

[0085] Coagulation necrosis of nucleated cells distinct from red blood cells was observed within the thrombus. Imaging data suggested that these cells were tumor thrombi resulting from the high accumulation of transplanted human lung cancer cell lines A549 in the vasculature of the renal hilum. Generally, when malignant tumors invade blood vessels, they are prone to thrombus formation due to coagulation abnormalities. However, thrombi of this magnitude are not typically formed. For cancer to metastasize to other organs, many steps must be completed before it leaves the primary tumor, travels through blood vessels such as blood and lymphatic systems, and then establishes at the metastatic site. In particular, even if intravasation from the primary tumor is successful, travels through the bloodstream alive, and successfully reaches the blood vessels within the target organ, whether or not the tumor can then exit the blood vessels and invade the target organ tissue (extravasation) is a key issue in the distant cancer metastasis process, and remains a controversial and unresolved issue. The model animal of the present invention beautifully demonstrates an intermediate state in which a large number of human lung cancer tumor cells A549 have accumulated in the large vein at the renal hilum in the kidney but have not yet invaded the kidney tissue. This provides a valuable model that will be useful for research into the renal metastasis process of lung cancer cells (Stebbing, J. and Smith, IE Clinical Oncology 12: 326-327, 2000), the distant metastasis process of various other cancers to other organs, and the development of various anticancer drugs to prevent distant metastasis, which has an extremely low prognosis for life.

[0086] The kidneys are organs whose job is to constantly filter large amounts of blood, excrete toxins and unnecessary waste products in the urine, and reabsorb necessary nutrients and ions in the renal tubules and return them to the bloodstream.Since a huge blood clot has formed that appears to be blocking blood flow at the renal hilum, it is thought that chronic kidney disease has occurred, which significantly impairs the normal function of the kidney, as has been shown to occur in human malignant renal tumors.

[0087] [Cancer Model Animal Using Mouse Insulinoma Cells] Culturing using L-glucose medium can be performed not only with human lung cancer cells A549, but also with mouse insulinoma cells MIN6 cells. When the cells had sufficiently proliferated in D-glucose medium, they were replaced with L-glucose medium, and the medium was frequently changed for the next 2-3 days. After confirming that most of the dead cells had been removed, half of the medium was changed every other day. After one or two passages in L-glucose medium and 8-11 days of culture, a cell suspension was obtained by trypsinization, and 5 x 10 cells were injected subcutaneously into the right thigh of a mouse. ６ cells (100 μL) / animal, or 1.0 × 10 ５ Cells were transplanted without Matrigel at a ratio of 20 μL per mouse. As a control, MIN6 cells cultured in normal D-glucose medium were also transplanted. Because cells cultured in D-glucose medium proliferate rapidly, after 6 to 7 days of culture, the same number of cells as the L-glucose-cultured cells were transplanted subcutaneously into the right thigh of the mouse.

[0088] However, when MIN6 cells were cultured in L-glucose medium and then subcutaneously implanted into mice, tumors could not be detected by external observation, as is the case with conventional subcutaneous implantation models (Fig. 5). Therefore, pathological autopsy revealed that tumor cells, instead of growing subcutaneously, had infiltrated deeply into the central muscle layer of the underlying thigh skeletal muscle, forming a malignant tumor, i.e., a sarcoma, within the muscle (Figs. 6A and 6B). Standard HE staining and pathological examination by a pathologist revealed severe cellular atypia and marked angiogenesis, suggesting an aggressive malignant tumor, confirming the formation of a highly malignant sarcoma (Fig. 6B).

[0089] Numerous subcutaneous tumor cell line transplant models (the cell-line-derived subcutaneous tumor models) exist worldwide. However, a good animal model for sarcoma has been desperately needed but has not been readily available. In fact, to the inventors' knowledge, there is no known model in which a tumor cell line ectopically transplanted subcutaneously does not establish itself subcutaneously but instead infiltrates deeply into the underlying skeletal muscle, forming a highly atypical sarcoma. In particular, the present sarcoma cells not only infiltrated into the muscle but also exhibited the same functional characteristics as human insulinoma cells, namely, a clear hypoglycemia due to excessive insulin secretion in the animals (Figure 7). When MIN6 cells cultured using L-glucose medium were transplanted (black triangles in Figure 7), blood glucose levels showed a significant decrease from day 1 after transplantation compared to when MIN6 cells cultured using a conventional culture method using D-glucose medium were transplanted (black circles in Figure 7) and the negative control (white circles in Figure 7) (significant differences between Control vs. L and D vs. L; ANOVA and Bonferroni's multiple comparison test).

[0090] Furthermore, when the insulinoma cells cultured in L-glucose were transplanted into the abdominal cavity of mice, they not only colonized the abdominal cavity (Fig. 8A and Fig. 8B) but also deeply infiltrated into the abdominal muscle (Fig. 9A and Fig. 9B). These results demonstrate the strong ability of tumor cells cultured in L-glucose medium to infiltrate into muscle, i.e., to form sarcomas.

[0091] Even more surprisingly, mouse insulinoma cells transplanted intraperitoneally also metastasized to lymph nodes (Fig. 10A and Fig. 10B) and liver (Fig. 11A and Fig. 11B), which are common sites of metastasis for neuroendocrine tumors, including human insulinoma. The histopathological findings of the liver metastases were so similar to those of malignant tumors that they could be mistaken for human neuroendocrine tumors (Fig. 11B).

[0092] Insulinoma (also known as pancreatic neuroendocrine tumor, PNET) is a type of neuroendocrine tumor (NET). Among NETs, ​​it has the highest rate of distant metastasis at the time of diagnosis, reportedly at 40.3% in the United States, and the survival time for metastatic cases is only 12 months. Despite the extremely poor prognosis, reportedly with a 5-year survival rate of 22.7%, it is a rare disease, and much remains unknown about its actual condition. Therefore, the animal model of the present invention is a unique and valuable model that is expected to contribute to the medical elucidation and development of treatments for sarcomas derived from PNET, as well as the diagnosis and treatment of human malignant insulinoma, i.e., metastatic PNET.

[0093] [Study on screening method for substances with anticancer activity] A screening method for substances with anticancer activity was studied using 2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-L-glucose (hereinafter abbreviated as "CLG") (Japanese Patent No. 6566348), i.e., a molecule in which 7-OH-coumarin is bound to L-glucose via a nitrogen atom, as a test substance.

[0094] On day 0, L-glucose-cultured insulinoma cells MIN6 were transplanted intraperitoneally into four groups of eight mice (32 mice total). From day 1, the following groups were prepared: 1) Eight mice were intraperitoneally administered CLG (53 mg / kg, an expected safe concentration) in saline every day for a minimum of four days, up to day 4 (CLG group); 2) Eight mice were intraperitoneally administered saline alone without CLG every day for seven days, up to day 7 (saline group); 3) Eight mice were intraperitoneally administered 5-FU (25 mg / kg, a high concentration suitable for nude mice), a drug widely used in Japan and Europe as a standard treatment for pancreatic cancer and also used in the treatment of neuroendocrine tumors such as insulinoma, every day for seven days, up to day 7 (5-FU group). All mice were euthanized at 5 months, and pathological autopsies were performed.

[0095] In the CLG-treated group, blood glucose levels remained normal in four of the eight mice until final euthanasia at the fifth month. Necropsy of these four mice revealed no tumors. The remaining four mice began to show signs of hypoglycemia exactly one week after the eight mice in the saline-treated group began to show signs of hypoglycemia. After formalin fixation, the entire body was cut into rings at 5 mm intervals, and detailed pathological autopsies of each section revealed no tumors in one mouse. Another mouse had a small, well-differentiated tumor with firm cytoplasm, varying nuclei but not a high N / C ratio, which was hidden only by microscopy and covered by a fibrous capsule. The tumor was located subcutaneously outside the peritoneum within the fat, rather than within the peritoneal cavity. Another mouse had a small, well-differentiated, low-grade tumor surrounded by a capsule, with visible cytoplasm, no severe atypia, and uniform, regular nuclei. The tumor was well-differentiated and low-grade, but not within the peritoneal cavity. Based on their location and structural characteristics, the tumors in these two mice were considered to be completely resectable by surgery. Only one remaining mouse had a malignant tumor in the abdominal cavity that could be called neuroendocrine carcinoma (NEC), but more than 90% of this intraperitoneal tumor was necrotic. This mouse also had metastasis in the abdominal muscle, but the cells were compartmentalized, a feature that differed from the histological appearance of cancer in the other groups.

[0096] That is, of the eight mice in the CLG-administered group, four mice (50%) had normal blood sugar and no tumors. Of the remaining four mice, one had a drop in blood sugar but no tumor was found, another had a benign tumor subcutaneously outside the transplanted peritoneum, another had a low-grade tumor subcutaneously outside the peritoneum, and the remaining one had a malignant tumor accompanied by necrosis.

[0097] That is, CLG showed a remarkable effect in vivo even after only 4 days of administration. That is, the results expected from the separately conducted in vitro experiments on the anticancer activity of CLG were also demonstrated in vivo.

[0098] In contrast, in the saline-treated group, all mice were analyzed except for one that withdrew due to infection after treatment. Of the remaining seven mice, four were found to have obvious cancer. Specifically, one mouse had a high-grade sarcoma with significant atypia that had infiltrated the abdominal wall muscle, another had clear abdominal muscle infiltration, and yet another mouse exhibited hypoglycemia and a highly malignant tumor in the peritoneal cavity. Another mouse also exhibited hypoglycemia and a large, highly atypia-like, clearly malignant neuroendocrine tumor that had metastasized to the lymph nodes. Furthermore, the remaining mouse had a lesion suspected to be a neuroendocrine tumor in the peritoneal cavity. Specifically, of the seven mice that were evaluated, four had metastatic malignant tumors, and one had a tumor. The remaining two mice showed no tumors.

[0099] Furthermore, one of the eight mice in the 5-FU group subsequently developed hypoglycemia after day 84 and was found to have large, highly atypia-like, and highly malignant liver metastases, with the primary tumor attached to the surface of the seminal vesicle. Liver metastases from neuroendocrine tumors are similar to those observed in humans, making this a useful model. One mouse had invasive carcinoma (neuroendocrine tumor) in the smooth muscle surrounding the transverse colon (Figures 12A and 12B). Three other mice were dissected on day 10 due to suspected deterioration. Of these three mice, one had a tumor on the retroperitoneal surface, one had a tumor on the peritoneal surface, and one had an unusual tumor in the peritoneal cavity between the retroperitoneum and the intestine. Immunostaining with chromogranin, synaptophysin, and insulin antibodies confirmed that all tumors were neuroendocrine tumors, and the tumors were established despite the short time after MIN6 transplantation.

[0100] The above results confirm that the cancer model animal of the present invention is extremely useful for screening substances with anticancer activity. Furthermore, because the cancer model animal of the present invention shows functional expression early after transplantation, it is expected to be useful for developing various methods that contribute to the early detection of cancer.

Claims

1. A model animal in which tumor cells cultured and grown in a medium containing L-glucose were transplanted subcutaneously or intraperitoneally.

2. A model animal as described in claim 1, wherein the tumor cells exhibit metastasis to muscle.

3. A model animal as described in claim 2, wherein the muscle is a skeletal muscle.

4. A model animal in which tumor cells transplanted subcutaneously or intraperitoneally have infiltrated into the muscle, The tumor cells are tumor cells that have been cultured and grown in a medium containing L-glucose, and the model animal is derived from a cancer other than sarcoma.

5. A model animal as described in claim 4, wherein the muscle is a skeletal muscle.

6. A model animal in which tumor cells transplanted subcutaneously or intraperitoneally show metastasis, The model animal is a tumor cell that has been cultured and grown in a medium containing L-glucose, and is selected from the group consisting of adenocarcinoma cells and neuroendocrine tumor cells.

7. A model animal as described in claim 6, wherein the metastasis is to muscle.

8. A model animal as described in claim 7, wherein the muscle is a skeletal muscle.

9. The model animal described in claim 6, wherein the tumor cells are adenocarcinoma cells, and the metastasis is metastasis to lymph nodes, lungs, or intraperitoneal organs, or accumulation in the renal hilum.

10. The model animal described in Claim 9, wherein the adenocarcinoma cells are tumor cells derived from lung cancer.

11. A model animal as described in claim 9, wherein the intraperitoneal organ is the liver or spleen.

12. The model animal described in claim 6, wherein the tumor cells are neuroendocrine tumor cells and the metastasis is to lymph nodes, intraperitoneal organs, or the peritoneum.

13. The model animal described in claim 12, wherein the neuroendocrine tumor cells are insulinoma cells.

14. A model animal as described in claim 12, wherein the intraperitoneal organ is the liver.

15. A model animal in which tumor cells cultured and grown in a medium containing L-glucose are subcutaneously or intraperitoneally transplanted, The tumor cells of the model animal exhibit abnormalities in the functions of orthotopic cells and are selected from the group consisting of adenocarcinoma cells and neuroendocrine tumor cells.

16. The model animal described in claim 15, wherein the adenocarcinoma cells are tumor cells derived from lung cancer.

17. The model animal described in claim 15, wherein the neuroendocrine tumor cells are insulinoma cells.

18. A model animal as described in claim 15, in which the tumor cells exhibit abnormalities in the functions of orthotopic cells within three days after transplantation.

19. The model animal according to claim 15, wherein the tumor cells exhibit abnormalities in the functions of orthotopic cells at a cell number of 5.0 x 10 5 or less.

20. A tumor cell cultured and grown in a medium containing L-glucose, A cell composition used to produce a model animal by transplanting it subcutaneously or intraperitoneally into an animal.

21. A method for producing a model animal, comprising the step of transplanting the cell composition described in claim 20 subcutaneously or intraperitoneally into an animal.

22. A method for screening a substance having an anticancer effect, comprising: A step of administering a test substance to the model animal according to any one of claims 1 to 19; assessing tumor cell growth or tumor cell metastasis after initiation of administration of the test substance; a step of comparing the evaluation result in the step of evaluating with the result of evaluating tumor cell growth or metastasis in the model animal to which a test substance has not been administered; A screening method comprising: