Method for generating human cancer in mice

Abstract

The present invention relates to a method for generating human cancer in mice using a methodology that affects biology of normal mice, which comprises generating stress by altering the circadian rhythm of the mice, administering immunosuppressants or antirejection agents temporarily, and grafting cells that promote cancer growth and cells that generate cancer in order to create the microenvironment necessary for the development thereof. The invention also relates to a kit comprising parts for modelling the generation of human cancer in mice.

Description

[0001] METHOD FOR GENERATING HUMAN CANCER IN MICE

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to a method for generating human cancer in mice through a methodology that affects the biology of normal mice by generating stress through the alteration of their circadian cycle, the temporary administration of immunosuppressive or anti-rejection agents, and the grafting of cancer-promoting cells and cancer-generating cells to create a microenvironment necessary for their development. The present disclosure also allows for personalized modeling of cells of patient origin and study models of more economical anticancer agents.

[0004] DESCRIPTION OF THE STATE OF THE ART

[0005] Cancer is the second leading cause of death worldwide, behind cardiovascular disease (Barba et al., 2020; Bray, Laversanne, Weiderpass, & Soerjomataram, 2021). This represents a serious public health problem and a challenge for research and clinical practice. Over the past 30 years, funding for understanding the disease and developing drugs has increased. However, the clinical validity of drug approvals for cancer treatment remains modest (Ireson, Alavijeh, Palmer, Fowler, & Jones, 2019).

[0006] Multiple factors may contribute to the lack of new therapeutic options, one of the most important being the modeling of human cancer in mice for the discovery and development of effective anticancer drugs. Rodents, primarily mice, have been widely used to understand the etiology and pathophysiology of human cancer, as well as to evaluate potential new drugs. However, the success of these models largely depends on the degree to which data from mouse and other models predict clinical efficacy. The low predictive value of conventional preclinical models is one of the main reasons why 95% of agents entering Phase 1 clinical development fail to obtain market approval (Ireson et al., 2019).Therefore, it is crucial to develop animal models that mimic human cancer and thus conduct trials that have a high probability of success in the clinic.

[0007] Preclinical mouse models are valuable throughout the drug discovery and development process. Simple models, such as subcutaneous models with cancer cells injected into the skin of immunosuppressed mice, are crucial in the discovery phase. This ensures that the molecules have the appropriate pharmacology and activity in a biological system. Once drug candidates have been selected, more complex preclinical models become essential, providing an efficacy signal of sufficient magnitude to allow progression to clinical evaluation. These models are generated by injecting patient cells into the tumor's site of origin; for example, in the case of a mammary tumor, cells are injected into the mouse's breast.Other models include the use of mice humanized by genetic modification or previous cell transplants, which can develop cancer (Day, Merlino, & Van Dyke, 2015).

[0008] The development of new drugs to treat cancer is expensive. These costs include the preclinical, in vitro and in vivo phases, and clinical research. Preclinical in vivo research includes infrastructure maintenance costs, personnel, mouse models, among others. The price of transgenic or immunosuppressed mouse models can cost between $3,700 and $000 USD, depending on the technique used to generate them (“Cost Estimates & Fees | Transgenic Animal Model - UM Biomedical Research Core Facilities,” n.d.). These costs do not include the platform and related experiments. Such is the cost of this development that, for countries with emerging economies, it is observed and recommended that this preclinical research phase not be considered, and that resources be directed toward patient access to new therapies (Pramesh et al., 2022).

[0009] The development of a mouse model that can support the development of human cancer using a normal mouse is key to reducing the cost of developing oncology drugs for both academia and industry. Based on the fundamental characteristics of tumorigenesis toward cancer, our research team proposes a methodology that affects the biology of normal mice through stress generated by the alteration of their circadian cycle, the temporary administration of immunosuppressive or anti-rejection agents, and the grafting of cancer-promoting cells and cancer-generating cells to create the microenvironment necessary for their development. This method has been defined as OncoMix. The method of the invention allows for the discovery and development of new drugs, with the flexibility of a simple screening phase through a subcutaneous injection of cancer cells or an injection at the site of origin of the tumor.It also allows for personalized modeling of cells of the patient's origin in order to define management protocols and help people reach remission more quickly and improve their lifestyle.

[0010] A normal cell undergoes a series of changes that transform it into a cancerous cell. These processes are genetic, epigenetic, metabolic, and other factors (Hanahan, 2022). However, the relationship between cancer cells and their microenvironment and the body is crucial for their development.

[0011] Patent CN115299405 relates to the manufacture of animal models, in particular to a method for manufacturing a transplanted tumor model in nude mice. The nude mouse tumor xenograft model is controlled through multiple processes, which can not only expand the range of inoculated tumor types, but also improve the success rate of inoculated tumor bodies. The method comprises the following steps: Step 1: Selecting 4-6 week-old nude mice and inoculating them with tumor cells under sterile conditions, wherein the inoculation amount of tumor cells is (0.1-2)xl 07 / 200 µl / only; Step 2. Immunosuppressants are used to accelerate the growth of tumors after the tumors are planted, and the amount of immunosuppressants added is (1-2) mg / day / monitor; Step 3: Continue the culture for at least 5 days to obtain a nude mouse xenograft tumor model.

[0012] Document CN102599106A relates to a method for constructing an animal model of oral cancer and lymphatic metastasis, in particular to a method for constructing a model of oral cancer and lymphatic metastasis in mice, comprising the steps of: (1) Selection and grouping of animals: 6-week-old male Balb / c mice as experimental mice; (2) Preparation of reagent: 4-nitroquinoline-l -oxide (4-nitroquinoline-oxide, 4-NQO) was used to prepare a concentration of 0.1% with sterile distilled water and stored in a refrigerator at 4 ° C in the dark. (3) Animal feeding with the reagents of the previous step; (4) Experimental observation: Within 9-40 weeks after feeding the mice in the experimental group and the control group, 4 mice in the experimental group were randomly euthanized every 2 weeks, and 2 mice in the control group were randomly euthanized every 2 weeks.Observation was performed by hematoxylin-eosin staining (hematoxylin-eosin staining, referred to as HE staining) staining observation, broad-spectrum immunohistochemical cytokeratin (pan-cytokeratin, pan-ck) staining of lymph nodes observation of metastatic cancer cells.

[0013] US Patent 1,112,773 provides a cancer stem cell mass from which cells incapable of forming cancer are substantially removed and which has the characteristic property of reproducing a layered structure of cancerous tissue; a process for producing the cancer stem cell mass and the use of the cancer stem cell mass. The document describes repeatedly culturing human cancer tissue in a NOG mouse, separating cancer cells from the developed cancer tissue, and comparing various cancer cell culture processes with each other.As a result, as a result, they were able to successively produce a cancer stem cell composition that is homogeneous and substantially free from the coexistence of cells capable of forming cancer and cells incapable of forming cancer in a mixed state by employing an attached culture process using a serum-free stem cell culture medium, instead of a generally employed floating culture process.

[0014] However, despite the ongoing development and research in this field, there remains a need to develop methods that allow for the generation of human cancer using normal mice, to develop reproducible models, and thus reduce the cost of generating oncology drugs for both academia and industry.

[0015] BRIEF DESCRIPTION

[0016] This disclosure relates to a method for generating human cancer in mice. One of the main problems in this technical field is the modeling of human cancer in mice for the development of effective anticancer drugs. The success of the models depends largely on the degree to which data from tests performed in mice and other models are predictive of clinical efficacy. The low predictive value of conventional preclinical models is one of the main reasons why 95% of agents that enter Phase 1 clinical development do not obtain market authorization (Ireson et al., 2019). Therefore, it is crucial to develop animal models that resemble human cancer and thus conduct tests that have a high probability of success in the clinic.

[0017] The proposed method is a method that allows the generation of human cancer at low cost and that allows reproducible models, comprising: (a) altering the circadian cycle of mice; (b) administering to said mice a determined dose of one or more immunosuppressive or anti-rejection agents; (c) grafting said mouse with cancer-promoting cells; (d) grafting said mouse with cancerous, deregulated or cancer-generating human cells; (e) suspending or decreasing the administration of immunosuppressive agents in step b).

[0018] BRIEF DESCRIPTION OF THE FIGURES

[0019] FIG. 1. The two photographs show the subcutaneous administration of the combination of cancer and human cells in different areas of the mouse.

[0020] FIG. 2. Mass formation at the subcutaneous administration sites shown in Fig. 1. The masses are evident as bumps protruding from the normal surface of the mouse.

[0021] FIG. 3. Images showing the formation of tumor masses at the three administration sites. Blood vessel formation around the tumor masses is also observed.

[0022] FIG. 4. Graph showing changes in mouse weight during the method of generating human cancer in mice.

[0023] FIG. 5. Diameters of the tumor masses resulting from the method for generating human cancer in mice at the three administration sites. FIG. 6. Presence of mitochondrial DNA from human cells in the tumor masses found.

[0024] FIG. 7. Presence of mitochondrial DNA from murine cells in the tumor masses found.

[0025] FIG. 8. Histological sections of tumor masses where filtration of cancer cells into the surrounding tissue is observed, resembling an invasion. (A) Histological sections of tumor masses where filtration of cancer cells into the surrounding tissue is observed, resembling an invasion. Hematoxylin & Eosin 4x: Tumor lesion implant with defined edges and serpeginous necrosis in approximately 20% of its structure. (B) Histological sections of tumor masses where filtration of cancer cells into the surrounding tissue is observed, resembling an invasion. Hematoxylin & Eosin 10x: Poorly differentiated malignant tumor lesion, with cellular distribution in sheets without ductal formation, accompanied by tumor necrosis. (C) Histological sections of tumor masses where filtration of cancer cells into the surrounding tissue is observed, resembling an invasion.Hematoxylin & Eosin 40x: Poorly differentiated malignant neoplasm showing a combination of epithelioid and sarcomatoid spindle cells, with intense cellular pleomorphism exhibiting nuclear gigantism, multinucleation, very prominent nucleoli and atypical mitotic activity.

[0026] DETAILED DESCRIPTION

[0027] For the purposes of interpreting the terms used throughout this document, their usual meaning in the technical field should be taken into account, unless a specific definition is incorporated or the context clearly indicates otherwise. Additionally, terms used in the singular form also include the plural form.

[0028] The object of the present invention is a method that allows the generation of human cancer at low cost and that allows reproducible models, comprising: (a) altering the circadian cycle of mice; (b) administering to said mice a determined dose of one or more immunosuppressive or anti-rejection agents; (c) grafting said mouse with cancer-promoting cells; (d) grafting said mouse with cancerous, deregulated or cancer-generating human cells; (e) suspending or decreasing the administration of immunosuppressive agents in step b).

[0029] For the purposes of the present invention, “cancer” typically refers to a physiological condition in mammals characterized by uncontrolled cell proliferation or a related physiological condition. Cancer types in the present invention include, but are not limited to, carcinomas (epithelial cancer) such as pancreatic cancer, prostate cancer, breast cancer, skin cancer, gastrointestinal cancer, lung cancer, hepatocellular carcinoma, cervical cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, vaginal cancer, liver cancer, bile duct cancer, bladder cancer, urinary tract cancer, thyroid cancer, adrenal cancer, kidney cancer, among other glandular tissue cancers.Sarcomas, (non-epithelial cancer) includes liposarcomas, leiomysarcoma, rhabdosarcoma, synovial sarcoma, angiosarcoma, fibrosarcoma, malignant peripheral nerve tumor, gastrointestinal stromal tumor, desmoid tumor, Ewing's sarcoma, osteosarcoma, chondrosarcoma, leukemia, lymphoma, melanoma, and other solid tumors, e.g., melanoma or brain tumor.

[0030] In accordance with the present invention, “cancer-promoting cells” refers to the ability of i) two divided fixed cells to self-replicate to produce cells that maintain the ability and degree of differentiation equivalent to that of the parental cells, according to the cell lineage, ii) the ability to differentiate into multiple types of cancer cells that constitute a cancer cell mass. The ability to form cancer refers to the cells or cell population transplanted into non-human individuals forming a cancer cell mass, preferably a mass having a hierarchical structure. The hierarchical structure refers to a portion of a unique structure characteristic of a normal tissue that is detected histopathologically in the structures of a tumor originating from a normal tissue. In general, this structure is highly reproducible in highly differentiated cancers.Cancer-promoting cells also include those whose original phenotype or function changes after interaction with cancer cells. That is, cancer-promoting cells can include fibroblasts, mesenchymal stem cells (MSCs), immune cells, stromal cells, hematopoietic progenitors, and any other cells that can support tumor growth and induce immunoregulation, exchange subcellular material that enhances cancer cell growth, or favor the selection of cancer cells in the tumor microenvironment.

[0031] Other abilities of these cells include maintaining proliferation and inducing it through proliferative signaling pathways, evading growth inhibitors, resisting cell death, enabling replicative immortality, inducing angiogenesis and activating tissue invasion and metastasis mechanisms, and evading the immune system, among others. Within cancer cells they are found, but are not limited to, adrenal cancer lines (NCI-H295R), bladder (5637, HT-1376, J82, SW 780, T24T24-Luc-Neo), bone (MG-6, Saos-2, SJSA-1, SW 1353), brain (D54-Luc, DBTRG-05M, GGli36-DsRed, R-Luc (rescued), LN-18, LN-229, LN-827 LucNeo, M059K, SF-295, SF-539, SF-767, SNB-19, U-251, U-251-Luc-mCh-Puro, U-87 MG, U-87 MG-Luc), cervical (HeL, aKB), colon (C2BBel, Caco-2, COLO 205, COLO 205-Luc #2, DLD-1, HCC2998, HCT-116, HCT-116-Luc, HCT-15, HCT-8, HT-29, HT-29-Luc, LoVo, LS 174T, LS411N, NCI- H508, SW-480, SW-620), endometrial (KLE, KLE-Luc-mCh-Puro), epidermoid (A- 431),epithelial (HEKn, HEKn-2), esophageal (OE33), Ewig sarcoma (A- 673, RD-ES), fibrosarcoma (HT-1080), gastric (GIST-T1, NCI-N87, NUGC-4, MKN-45, NCI-N87, head and neck (SCAL), 27 FaDu), lymphoma (KARPAS 299), leukemia (EOL-1, Kasumi-1, Kasumi-3, Kasumi-3-Luc-mCh-Puro, KG-l-Luc-mCh-Puro, MOLM-13, MV-4-11, MV-4-11-Luc-mCh-Puro, NAHP-1, NAMO-1, NO NALM6-Luc- MChPuro, Reh, Reh Luc-Neo, RS4;11, K-562, K-562-Luc2, HEL, HEL 92.1.7, HEL 92.1.7-Luc-Neo, HEL-Luc-Neo, ARH-77, CCRF-CEM, DPND-41-Luc-Ch-Ch-Cr Jurkat-Clone E6-1, MOLT-4, MOLT-4-Luc-MCh-Pure), liposarcoma (SW 872), liver (Hep 3B2.1-7, Hep G2), lung (Calu-6 , HCC2935, NCI-H1703 , NCI-H170-Luc-Luc-Pure NCI-H2030, NCI-H2110 , NCI-H2122 , NCI-H3122 , NCI-H322, MNCI- H82, A549, A549-Luc-C8, Calu-1, Calu-3, HCC827, HCC827-Luc-Puro-HCC, HCI-H25 NCI-H125-Luc, NCI-H1299, NCI-H1650, NCI-H1975, NCI-H1975-Luc, NCI- H23, NCI-H292, NCI-H441, NCI-H460, NCI-H460-Luc2, NCI-H52, PC-Luc-92 mCh-pure, DMS 114, NCI-H446,NCI-H69, SHP-77, EBC-1, SK-MES-1), lymphoma (DB, DBM2), B-JAB, David, David-Luc-mCh-Puro, NAMALWA, Raji, Raji-Luc, Ramos, Ramos-Luc, HuT 78, HT-6m, SU-P-DHL-6 GRANT-519, OCI, Lyl LN, OCI-Lyl R10-Luc-mCh-Pure , OCLLyl R7-Luc-mCh-Pure , OCI-Lyl9-Luc, Neo, OCI-Ly3-Luc-mCh-Pure, OCI-Ly7,SU,P-Dfei-Neo SU-DHL-10, LN-High, SU-DHL-16, SU-DHL-4-Luc-mCh-Puro, SU-DHL-8, TMD8, Toledo-Luc- Neo, WSU-DLCL2, WSU-FSCCL, NK-92MI, KARPAS 291399), mamario ( ,AU-565,BT-20,BT-474,HCC70,Hs 578Bst,Hs 578T,MCF 10, a,MCF-7„ MCF7-Luc-mCh-Pure, MDA-MB-231, MDA-MB-231 -2LMP, MDA-MB-231- D3H2LN, MDA-MB-231 -Luc-D3H1, MDA-MB-231 -Luc-31-M4DAH-MBDA2N MDA-MB-468, MX-l-Luc, SK-BR-3, T47D, ZR-75-1, melanoma (A2058, A375, COLO 829, G-361, LOX-IMVI, M14, MDA-MB-435S, OCM-1, Oc-m-m, SKh,1-PUL SK-MEL-28, SK-MEL-28-Luc-mCh-Puro, UACC-62, WM-115, WM-266-4, myeloma (JJN-3-Luc, KMS-11, KMS-26, KMS-34, MM.1S, (pM9,-NCI-Luc NCI-H929-Luc-mCh-Pure, OPM-2, RPMI 8226, U266B1), Neuroblastoma (SK-N-AS, SK-N-FI, SK-N-SH), neuroendocrine skin (MKL- 1), ovary (A2780, A2780-Luc IGROVl-Luc-Mch-Puro, NIH:OVCAR-3 , NIH:OVCAR-3-Luc-mCh-Puro , OV-90 , OVCAR-4, OVCAR-5, OVCAR-5-Luc- mCh-Puro, OVCAR-8, SKAR-mCO-8-Vuc-Luc (Subcutaneous), SK-OV-3-Luc-D3 (Intraperitoneal)), pancreatic (AsPC-1, Bx-PC-3, BxPC-3-Luc2„ Sheath- 1, Sheath-2, KP4, MIA PaCa-2, MIA PaCa-2-Luc, PANC-18, PANC.-SU6, PANC-186, SW 1990), pharyngeal carcinoma (Detroit 562), placental choriocarcinoma (BeWo), prostate (22Rvl, CWR-22-R, DU 145, DU 145-Luc, LnCap clone FGC, PC-3, PC-3-Luc, PC-Luc-Luc-Ca-VCP), renal (293, 293-Luc-mCh-Puro, 769-P, 786-0, 786-O-Luc-Neo (rescued), A-498, ACHN, Caki-1, TK-10, RPTEC), thyroid (MB-1, TT), vulva (SK- LMS-1).

[0032] For the purposes of the present invention, MSCs are understood to mean multipotent stem cells or by their English term “Mesenchymal Stem / Stromal Cells (MSC), which are found in the bone marrow, umbilical cord, adipose tissue, among other sources of the mouse, are a cell population with demonstrable progenitor functionality, self-renewal and differentiation capacity, which may also possess notable capacities for secreting immunoregulatory factors and “homing”. These can express proteins similar in mice to CD73, CD90 and CD 105, and lack the expression of the hematopoietic and endothelial markers CD1 lb, CD14, CD19, CD34, CD45, CD79a and HLA-DR. These cells are capable of differentiating in vitro into the lines of adipocytes, chondrocytes and osteoblasts, and are important for manufacturing and repairing skeletal tissue, such as cartilage, bone and bone marrow fat.In one embodiment of the present invention, the ratios of cancer cells to MSCs are 1:1, 2:2, 4:4, 2:1, 4:1, and 4:2, where 1 equals 125,000 cells.

[0033] In one embodiment of the invention, grafting cancer cells refers to grafting from 10,000 cancer cells to 1,000,000 cancer cells to achieve a significant tumor mass.

[0034] In one embodiment of the invention, the cancer cell / MSC ratios used are, but are not limited to, 1:1, 1:2, 1:4, and 4:1. In one embodiment of the invention, the cell ratios are adjusted depending on the type of cancer being grafted. In one embodiment, for cancers with high levels of aggressiveness, fewer cells are grafted than for a cancer with low levels of aggressiveness. In one embodiment, grafting high-aggressiveness cancer cells includes, but is not limited to, grafting from about 10,000 cells. In another embodiment, grafting low-aggressiveness cancer cells includes, but is not limited to, grafting from about 1,000,000 cells.

[0035] An animal can be used as a non-human individual, this animal can be a mouse without a modified genetic background or of a particular line (Wild Type), such as Swiss mice. The following lines can be used, and are not limited to BALB / cJ, C57BL / 6, DBA / 2J, B6.129P2-ApoetmlUnc / J, B6.SJL TRADITIONAL CONGENIO, C57BL / 6Boy, CBA / J, Swiss Webster TRADITIONAL OUTBRED, NMRI TRADITIONAL OUTBRED mice. For the purposes of the present invention, mice from 3 weeks to 2 years of age with weights between 10 to 30 g can be used.

[0036] For the purposes of the present invention, “altering the organism” means aa) altering the circadian cycle of mice, and b) administering to said mice a certain dose of one or more immunosuppressive or anti-rejection agents.

[0037] The "generation of the tumor microenvironment" in the present invention has the steps of c) grafting cancer-promoting cells into mice; d) grafting human cancerous, deregulated, or cancer-causing cells into mice; e) suspending or decreasing the administration of immunosuppressive agents from step b). For the purposes of the present invention, "alteration of the circadian cycle" includes a series of cellular adaptation mechanisms to periodic and largely light-dependent changes in activity, rest, regeneration, and growth. These cyclic mechanisms control metabolism, gene expression, and the physiological regulation of the organism towards health. Numerous studies indicate that changes in the circadian cycle in humans are associated with a higher probability of developing breast cancer in women or prostate cancer in men.Circadian cycle dysregulation involves hormonal changes, with one of the most affected hormones being melatonin. Melatonin is secreted at night in diurnal animals. Melatonin has antioxidant functions and regulates proper mitochondrial function. If this fails, free radicals and metabolic alterations are produced, which can lead to a lack of efficiency of the immune system in recognizing cancer cells or infectious agents. Cycle dysregulation also leads to a change in nutritional balance and makes the tumor microenvironment thrive (Masri & Sassone-Corsi, 2018). The absence of light causes a disturbance in the circadian cycle in rodents, causing changes in their temperament such as depression and anxiety (Imamura & Takumi, 2022). For the purposes of the present invention, this alteration can occur from 3 weeks to 2 years of age.Mice can be isolated in pairs, or their number can be adjusted according to the size of the box, in a completely dark area.

[0038] According to the present invention, "immunosuppressive or anti-rejection agents" are understood to be those agents that inhibit the activity of the immune system. These agents include glucocorticoids, cytostatics, antibodies, drugs that act on immunophilins, among other agents. Glucocorticoids include prednisone, prednisolone, methylprednisolone, beclomethasone, among others. Cyclostatics, for the purposes of the invention, include cyclophosphamides, chlorambucil, carmustine or busulfan, methotrexate or 5-fluorouracil, triamcinolone acetonide, among others.

[0039] In one embodiment of the invention, the immunosuppressive agents are methylprednisolone, dexamethasone, azathioprine, or cyclophosphamide and methotrexate. In another embodiment, the dose of the immunosuppressants is between 0.01 mg / kg to 500 mg / kg. In one embodiment, the dose of methylprednisolone is in a range between 30-500 mg / kg, or in a range between 30-400 mg / kg, or between 30-300 mg / kg, or between 30-200 mg / kg, or between 30-100 mg / kg. In another embodiment, the dose of dexamethasone is within 0.01-5 mg / kg, or between 0.01-4 mg / kg, or between 0.01-3 mg / kg, or between 0.01-2 mg / kg. In another embodiment, the dose of azathioprine is between 2 and 3 mg / kg. In another embodiment, the dose of myclophosphamide and methotrexate is between 250 and 400 mg / kg, or 250 and 300 mg / kg.

[0040] For the purposes of the present invention, "suspending or decreasing the administration of immunosuppressive agents" implies that it does not generate total or lasting immunosuppression in the mouse, generating a better model of human cancer in mice where the immune response resumes after the tumor mass is generated or established. In this way, a better simulation of what the tumor faces and its progression can be generated, this suspension or decrease can be modulated according to the required model. In the case of testing with agents that generate a disruption of proliferation or induce the death of cancer cells, the dose of immunosuppressants can be maintained, continuing the application after the 1st, 2nd and 3rd doses every 48 hours, and this can be decreased.

[0041] In one embodiment of the invention, doses of immunosuppressants such as 6% triamcinolone acetonide from 30 mg / kg to 15 mg / kg.

[0042] In another embodiment, if the agents being tested promote immune system activity, the immunosuppressant doses would be maintained for the first, second, and third doses, with a fourth dose being considered for tumor mass establishment. Its use can then be discontinued and the agent that promotes immune system activity administered later to eliminate the tumor more quickly and continue this process with the model.

[0043] The present invention includes in one embodiment a kit of parts comprising: a) one or more immunosuppressive agents; b) a set of cancer cells and cancer-promoting cells. In one embodiment of the invention, the kit may contain a) as a glucocorticoid suppressor agent, cytostatics, antibodies, drugs that act on immunophilins, among other agents, glucocorticoids (prednisone, prednisolone, methylprednisolone, beclomethasone, among others), cyclostatics (cyclophosphamide, chlorambucil, carmustine or busulfan, methotrexate or 5-fluorouracil, triamcinolone acetonide, among others), b) cancer cells of the preference to be studied, frozen or in a culture dish or other support used for their proliferation, cancer-promoting cells. In one embodiment of the invention, the cells are MSCs of murine origin.

[0044] In a further embodiment, the kit of the invention has a circadian cycle disturbance guide.

[0045] In another embodiment, the kit includes the mouse to be modified, specified in advance. In yet another embodiment, the kit includes the mouse already modified to be injected with the cancer cells.

[0046] EXAMPLES

[0047] Mice:

[0048] Mice without a modified genetic background or from a particular line are used; the term is "wild-type" mice. In this case, Swiss mice bred in the laboratory were used, without a special diet, "ad libitum," and exposed during their development and before the experiment to physiological cycles of light and darkness. Females were used for the tests. The mice used in the tests were 3 to 6 weeks old and weighed 10 to 30 g.

[0049] Circadian cycle disturbance:

[0050] The isolated mice underwent circadian disruption in individual cages 24 hours before the use of the immunosuppressive agent and 48 hours before grafting with mesenchymal and cancer or tumor cells. Mice were also subjected to circadian disruption throughout the entire process before the immunosuppressive agent was applied, and until after grafting with mesenchymal and cancer or tumor cells. Disruption of the circadian cycle can lead to metabolic dysregulation, gene expression, and mouse health. Hormonal changes are expected, with melatonin being one of the most affected hormones, generating free radicals and others that can lead to a lack of efficiency of the immune system in recognizing cancer cells or infectious agents. A change in nutritional balance is also expected, which would allow the tumor microenvironment to thrive.It can be seen that disruption of the circadian rhythm induces changes in temperament, such as depression and anxiety, leading to negative impacts on immune system activity and energy balance. Immunosuppression:

[0051] A first injection of 6% triamcinolone acetonide (TA) at a fixed dose of 30 mg / kg was performed intraperitoneally (IP) 24 hours prior to the grafting of cancer and mesenchymal cells in the right iliac fossa region. The second and third doses of TA were administered on the fifth and eighth days after the grafting of cancer and mesenchymal cells, respectively. The dose of the immunosuppressant was calculated based on the mouse weight just before the injection. The dose is established according to the mouse weight, that is, if we use a 10 g mouse, with a ratio of 30 mg / kg, 0.3 mg will be applied, making a dilution in a final volume of 10 pl to 1.5 ml. Similarly, if the mouse weighs 20 g, at a dose of 30 mg / kg, 0.6 mg will be administered, diluting the final volume to 10 µl to 1.5 ml. The agent can also be administered intramuscularly, subcutaneously, intravenously, or intradermally.IP injections must be administered correctly, avoiding inadvertent injection into abdominal organs and subcutaneous, retroperitoneal, or intramuscular spaces. The injection should be made in the lower right abdominal quadrant of the mouse or rat to avoid hitting the liver and intestinal organs in the upper quadrants, the bladder in the lower midline, and the cecum in the lower left abdominal quadrant. The injected material must be sterile, isotonic, and non-irritating. Injection of large volumes may cause pain, peritonitis, fibrous tissue formation, perforation of abdominal organs, hemorrhage, and respiratory distress. Failure to administer the injection according to veterinary standards may result in tissue damage within the abdominal cavity, cumulative irritant effects, and needle-induced damage to the peritoneum.The recommended volume for a mouse is 20 ml / kg (maximum: 40 ml / kg) of body weight; this recommendation should not be exceeded, nor should a total volume of 10 ml. For other routes of administration, the veterinary clinical standard should be followed.

[0052] Immunosuppression can also be carried out with the following agents in particular concentrations:

[0053] Grafting of cancer and mesenchymal cells

[0054] The cancer cells used were MDA-MB-231. These cells are a human epithelial breast cancer cell line established from the pleural effusion of a 51-year-old Caucasian woman with metastatic mammary adenocarcinoma. Experiments were also conducted with SK-MEL-28 melanoma cells, which correspond to melanocytes isolated from the skin tissue of a 51-year-old male patient with malignant melanoma.

[0055] The mesenchymal cells used were isolated from the bone marrow of C57 / 6 mice and characterized as stem cells according to the criteria defined by the International Society for Cell and Gene Therapy (ISCT).

[0056] Cancer cells are cultured in a medium enriched with fetal bovine serum, glutamine, antibiotic agents, as recommended within the state of the art for MDA-MB-23 1 and SK-MEL-28.

[0057] The combinations used with different ratios of cancer cells to MSCs are 1:1, 2:2, 4:4, 2:1, 4:1, and 4:2, where 1 equals 125,000 cells. The following ratios were used:

[0058] Controls of 1,000,000 mesenchymal, tumor, and fibroblast cells, in PBS alone, are also prepared. Cell dosage calculations take into account the dead volume of the syringes used to make the combinations. The syringes are 1 ml, 27G x 1" 0.4 x 13 mm. A total of 100 pl were injected, and a residual "dead" volume of 50 pl is estimated; a total of 150 pl are loaded.

[0059] The syringes are loaded with the solution of cancer cells and MSCs, and they are kept refrigerated until 1h while the mice are inoculated. It is important that the cells spend as little time as possible outside the mouse's body, up to 48 hours, but in this case, the cells are likely to no longer be viable. At the time of grafting, the mice are prepared with weights of around 20-30 grams. They are sedated by inhalation, and the mice are kept supine (See FIG. 1).

[0060] The hair is shaved to a depth of 2 mm, and three key regions are located, sufficiently distant from each other to allow independent growth of the tumor mass, thus preventing one mass from coming into contact with another. The regions are the left hypochondria, the right flank, and the left iliac fossa; we also avoid the right iliac fossa, the fixed site of TA injections (See FIG. 1).

[0061] Subdermal injections are performed by lifting the skin with a tent-like grip. To access the area, the needle is positioned at a 20-30° angle to the abdominal plane. Once the mixture begins to penetrate, the skin can be released, and the formation of a subdermal fluid vesicle should be evident (See FIG. 1).

[0062] The three vesicles in the cell mixture are placed under the same conditions in the same mouse. After the cancer cells (MSCs) have been grafted, the sedative flow is stopped, and the mouse is placed in a warm, controlled environment so it can wake up. After this, clinical characteristics and anthropometric body mass are monitored daily.

[0063] Results:

[0064] Subcutaneous administration of the combination of cancer and human cells, according to the inventive methodology, allowed observation of the formation of a lump after application. The formation of the lump demonstrates a concentration of both cells for the successful generation of a tumor mass (See FIG. 1).

[0065] After 3 weeks of applying the mixture, it is observed that at the site where the cancer cells and MSC were applied, a mass is formed that is seen as a lump that protrudes from the normal surface of the individual, a hard consistency is felt and different from that of the skin (See FIG. 2).

[0066] After 3 weeks, the mice were euthanized and dissected, revealing the formation of tumor masses in all three applications. Blood vessel formation was also observed around the tumor mass, which can be correlated with angiogenesis (See FIG. 3).

[0067] After the first 15 days of graft application, a highly significant decrease in mouse weight was observed among mice under normal conditions (Normal Growth), controls with immunosuppressive treatment, changes in the circadian cycle (CC Control), and those to which the combination of cancer and mesenchymal cells was added (CC+MSC). The decrease in weight may be related to treatment with immunosuppressants, the change in the circadian cycle, and in part to the addition of the cell combination. The addition of cells causes a decrease in weight given the metabolic and nutrient needs of cancer cells. (See FIG. 4)

[0068] Measurements of the tumor masses were taken in the analyzed mice (3 mice per condition with three applications A, B, C, 4 biological replicates, the application sites are in figure 2). It could be observed that the tumor masses of the 500,000 CMM / 125,000 MDA MB 231 condition had an approximate diameter less than 6 mm. Then, for the 500,000 CMM / 250,000 MDA MB 231 condition, a diameter of 6 mm was estimated and for the masses formed by 500,000 CMM / 250,000 SK-MEL-28 melanoma, a diameter less than 4 mm (see FIG. 5).

[0069] After dissection, a sample was taken for the detection of human mitochondrial DNA in the tumor mass. DNA was extracted from the mass with a kit and human mitochondrial DNA was detected by real-time PCR using specific primers. The presence of human mitochondrial DNA from the cancer cells is observed significantly in relation to the control with CSM cells (mesenchymal stem cells) from mice. The detection of human mitochondrial DNA after 3 weeks is an indicator of the proliferation of cancer cells that, under conditions different from those of the method of the invention, do not survive and are destroyed by the immune system and assimilated by the mouse body (see FIG. 6).

[0070] We also observed that, when a sample was taken for human mitochondrial DNA detection, mouse mitochondrial DNA was found, as expected, in proportions similar to its human counterpart. This would imply that both cell types proliferate together, distributing homogeneously throughout the tumor (see FIG. 7).

[0071] Histological sections of the tumor mass reveal proliferation and invasion of tumor cells into the surrounding tissue. These characteristics resemble those found in a solid breast cancer (see FIG. 8).

[0072]

Claims

CLAIMS 1. A method for generating human cancer in mice comprising: a) altering the circadian cycle of mice; b) administering to said mice a determined dose of one or more immunosuppressive or anti-rejection agents; c) grafting said mouse with cancer-promoting cells; d) grafting said mouse with human cancerous, deregulated, or cancer-generating cells; e) suspending or decreasing the administration of immunosuppressive agents of step b); wherein the cancer-promoting cells of step c) generate a microenvironment that allows tumor progression; wherein the cancer-promoting cells of step c) are cells with modified stem cell or fibroblast characteristics; and wherein the alteration to the circadian cycle of the mice of step a) is carried out throughout the method.

2. The method of Claim 1, wherein the alteration of the circadian cycle is selected from: a modification of the light and dark cycles in periods from a total absence of light to a total presence of light or in successive intervals.

3. The method of Claim 1, wherein steps c) and d) are carried out on day D+l or D+2.

4. The method of Claim 1, wherein the immunosuppressive or anti-rejection agents of step b) are selected from: depot agents, long-acting (long-acting), non-depot agents with frequent doses (immediate action).

5. The method of Claim 1, wherein step b) is carried out between at least 1 day before and 12 months after its inoculation of c) and d).

6. The method of Claim 1, wherein the cells of step c) are mouse or human mesenchymal stem cells.

7. The method of Claim 6, wherein the ratio of said mouse or human mesenchymal stem cells versus said human cancerous, deregulated, or cancer-causing cells is between 1:1 to 1000:

1.

8. The method of Claim 1, further comprising a step f) of a second administration of one or more immunosuppressive agents, after step e).

9. The method of Claim 1, wherein the mice are three weeks old to 24 months old.

10. A kit of parts comprising: a) one or more immunosuppressive agents; b) an array of cancer and cancer-promoting cells.

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