Patient-derived xenograft model of cancer, preparation method thereof and an apparatus and a method for analyzing drug responsiveness using the same

Abstract

The present invention relates to a method for efficiently inducing sarcoma cancer in mammals and an animal model of patient-derived xenograft sarcoma cancer produced using such a method. The animal model of the present invention applies the optimal volume and optimal insertion subcutaneous depth of sarcoma tissue to be isolated from the patient and xenotransplanted into the animal, thereby reflecting various biological characteristics such as tumor proliferation, metastasis, and drug responsiveness in the human body with high reliability. Accordingly, the present invention can be usefully used as an efficient analytical means for molecular mechanism research, therapeutic agent screening, and in vivo drug evaluation of sarcoma cancer with unclear pathogenesis and frequent recurrence and metastasis. Furthermore, the present invention relates to a kit for drug responsiveness analysis comprising cancer tissue analogues and the same. The cancer tissue analogue of the present invention efficiently simulates the tumor microenvironment in vivo by solidifying the cell aggregates obtained by three-dimensional culture of cancer cells, especially sarcoma cells, into a Matrigel Matrix, and then forming a vascular endothelial cell channel. Accordingly, the cancer tissue analogue of the present invention can be used as a highly reliable drug screening system by reproducing the drug responsiveness of tumors in vivo with high accuracy, and when established using patient-derived autologous sarcoma cells, the patient's treatment responsiveness to the drug, the response dose, and the optimal combination of drugs can be quickly analyzed in vitro to establish a patient-tailored treatment strategy at an early stage.

Description

[0001] PATIENT-DERIVED XENOGRAFT MODEL OF CANCER, PREPARATION METHOD THEREOF AND AN APPARATUS AND A METHOD FOR ANALYZING DRUG RESPONSIVENESS USING THE SAME

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority from U.S. Provisional Application Serial No. 63 / 520,924, filed August 21, 2023, the contents of which are incorporated herein by reference.

[0004] GOVERNMENT LICENSE RIGHTS STATEMENT

[0005] The present disclosure was made with the support of the 2021 Bio-Medical Technology Commercialization Support Project (BT210043) of the Seoul Business Agency (SBA) of Seoul, Republic of Korea, titled "Establishment Of A Clinical Linkage Precision Medicine Demonstration Base For Intractable Rare Cancers Through The Use Of An Expanded Platform For In Physio™ Drug Evaluation Service". The government of City of Seoul may have certain rights in the invention.

[0006] FIELD OF THE DISCLOSURE

[0007] The invention relates to an animal model of patient-derived xenograft sarcoma cancer and a device for containing a cancer tissue analogue, specifically a sarcoma cancer tissue analogue, in a fractionated plurality of culture spaces to simultaneously perform an anticancer drug reaction test under the same conditions.

[0008] BACKGROUND OF THE DISCLOSURE

[0009] For the development of anti-cancer target therapies, preclinical trials that can accurately predict the degree of tumor response to therapeutic agents are essential, but the lack of a reliable preclinical evaluation system makes it difficult to efficiently discover promising therapeutic candidates. In general, in cancer research, immortalized tumor cell lines or early tumor tissue isolated from cancer patients are used as a vitro evaluation system. This makes it relatively easy to observe the effects of anticancer drugs, but not only does tumor tissue change its molecular properties and phenotype depending on environmental factors, but tumor cells change their characteristics for survival and actively use the vivo environment, so this in-vitro evaluation system is difficult to reflect tumor characteristics in vivo, so it is not suitable for discovering new anticancer drugs.

[0010] To solve this problem, a patient-derived xenograft (PDX) model has recently been actively developed to evaluate the sensitivity of therapeutic agents by transplanting patient-derived tumor tissue into animals. This model not only has the advantage of being a preclinical model that can select the most suitable treatment for each patient among various anticancer drugs, but can also be useful as an animal model to understand the progression and metastasis process of cancer.

[0011] Sarcoma refers to tumors that occur in non-epithelial connective tissue such as bone, cartilage, muscle, fat, nerves, and blood vessels, and has different characteristics from solid cancers that occur in tissues originating from ectoderm, such as breast cancer and colon cancer. Sarcoma can be broadly divided into malignant bone tumors that occur in bones and soft tissue sarcomas that occur in soft tissues such as skin, fat, nerves, blood vessels, and muscles, but apart from the simple division according to these sites of occurrence, there are various types and subtypes, and they are characterized by different genetic, molecular, and pathological characteristics.

[0012] In most sarcomas, the causal relationship is not clear with smoking, dietary habits, genetic factors, etc., which are commonly known to be the causes of cancer, and it is very difficult to treat and prevent them as the cause of the disease is not clearly identified. Currently, surgical surgery, chemotherapy, and radiotherapy are mainly used for the treatment of sarcoma, but despite the high recurrence and metastasis rates of various sarcomas, it has been used for a long time and no new therapeutic approaches beyond these standard treatments have not been proposed. In addition, there is a growing demand for an efficient analysis system that enables not only reliable screening of candidate drugs but also the selection of drugs optimized for patients by completely reproducing the phenotype and drug responsiveness of human sarcoma tissue in vivo.

[0013] Throughout this specification, a number of papers and patent documents are referenced and cited. The disclosure contents of the cited papers and patent documents are inserted as a reference in the present specification as a whole, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

[0014] SUMMARY OF THE DISCLOSURE

[0015] One embodiment of the present disclosure provides a xenograft animal model for sarcoma and uses thereof.

[0016] One embodiment of the present disclosure provides an efficient animal model that mimics the characteristics of sarcoma carcinoma (sarcoma), a non-epithelial connective tissue tumor with unclear pathogenesis and frequent recurrence and metastasis, with high accuracy. The present disclosure also provides an optimal size and optimal subcutaneous depth of the sarcoma tissue inserted to produce a patient-derived xenograft (PDX) breeding cancer animal model, wherein the corresponding size and insertion subcutaneous depth were applied.

[0017] Another embodiment of the present disclosure provides a method for inducing sarcoma cancer in mammals and a sarcoma cancer animal model produced using the same.

[0018] Yet another embodiment of the present disclosure provides a method of screening a composition for the prevention or treatment of sarcoma cancer using the sarcoma cancer animal model.

[0019] Another embodiment of the present disclosure provides an apparatus for analyzing drug responsiveness comprising cancer tissue analogues.

[0020] An embodiment of the present disclosure provides an efficient three-dimensional organotypic culture that mimic the in vivo characteristics of cancer tissue, especially sarcoma, a non-epithelial connective tissue tumor with an unclear pathogenesis and frequent recurrence and metastasis, wherein, when the cell aggregates obtained by three-dimensional culture of sarcoma cells are solidified into a Matrigel Matrix in the culture container, and then the vascular endothelial cells are dispensed through the inlet connected to the culture container, the tumor microenvironment in vivo is not only reproduced with high accuracy, but also the vascular endothelial cells at the injection port serve as non-tumor control cells. Another embodiment of the present disclosure provides a method for preparing cancer tissue analogues and a kit for drug responsiveness analysis comprising the same.

[0021] Other embodiment and benefits of the present disclosure are further clarified by the detailed description, claims and drawings of the invention below.

[0022] BRIEF DESCRIPTIONS OF THE DRAWINGS

[0023] Figure 1 shows the state of sarcoma tissue obtained from a surgical patient through surgical resection (FIG. 1A) and sarcoma tissue pretreated for PDX transplantation (FIG. IB), respectively.

[0024] Figure 2 shows the results of evaluating the degree of tumor growth by measuring tumor volume for 63 days after transplanting tumor tissue derived from sarcoma 1 patient into NOG mice.

[0025] Figure 3 shows the results of evaluating the degree of tumor growth by measuring tumor volume for 32 days after transplanting tumor tissue derived from sarcoma 2 patients into NOG mice.

[0026] Figure 4 shows the results of evaluating the degree of tumor growth by measuring tumor volume for 12 days after transplanting tumor tissue derived from sarcoma patient No. 3 into NOG mice.

[0027] Figure 5 shows the results of evaluating the degree of tumor growth by measuring tumor volume for 18 days after transplanting tumor tissue derived from sarcoma patient No. 4 into NOG mice.

[0028] Figure 6 shows the results of evaluating the degree of tumor growth by measuring the tumor volume for 12 days after transplanting tumor tissue derived from a patient with sarcoma 5 into NOG mice.

[0029] Figure 7 is a figure showing the concentration adjustment process of the staining reagent for each cancer cell and normal cell to be accommodated in the culture container of the present invention, and the result of staining sarcoma cancer cells and vascular endothelial cells with an unstained control group (FIG. 7A) and PKH26 (red) and PKH67 (green), respectively (FIG. 7B). By adjusting the concentration of each dye, we sought to explore the optimal concentration at which the two cells were clearly visualized before drug administration.

[0030] Figure 8 shows the results of comparing the survival rate of cancer cells and normal cells after treatment with doxorubicin at each concentration.

[0031] DETAILED DESCRIPTION OF THE DISCLOSURE

[0032] One embodiment of the present disclosure provides a xenograft animal model for sarcoma and uses thereof.

[0033] The present disclosure provides an efficient animal model that mimics the characteristics of sarcoma carcinoma (sarcoma), a non-epithelial connective tissue tumor with unclear pathogenesis and frequent recurrence and metastasis, with high accuracy. The present disclosure also provides an optimal size and optimal subcutaneous depth of the sarcoma tissue inserted to produce a patient-derived xenograft (PDX) breeding cancer animal model, wherein the corresponding size and insertion subcutaneous depth were applied.

[0034] Another embodiment of the present disclosure provides a method for inducing sarcoma cancer in mammals and a sarcoma cancer animal model produced using the same.

[0035] Yet another embodiment of the present disclosure provides a method of screening a composition for the prevention or treatment of sarcoma cancer using the sarcoma cancer animal model.

[0036] In one embodiment, the present disclosure provides a method of producing a cancer- induced animal comprising:

[0037] (a) fragmenting the sarcoma tissue separated from the subject into a section;

[0038] (b) making an incision on the surface of the animal's skin; and

[0039] (c) Injecting the fragmented sarcoma tissue through the incision to a subcutaneous depth, alternatively, the present disclosure provides a method of producing a cancer-induced animal comprising:

[0040] (a) fragmenting the sarcoma tissue separated from the subject into a section of 25 mm3- 35 mm3volume;

[0041] (b) making a 3 mm to 7 mm incision on the surface of the animal's skin; and

[0042] (c) Injecting the fragmented sarcoma tissue through the incision to a subcutaneous depth of 0.001 pm - 3.0 mm, alternatively, the present disclosure provides a method of producing a cancer-induced animal comprising:

[0043] (a) fragmenting the sarcoma tissue separated from the subject into a section of 20 mm3- 30 mm3volume;

[0044] (b) making a 3 mm to 7 mm incision on the surface of the animal's skin; and

[0045] (c) Injecting the fragmented sarcoma tissue through the incision to a subcutaneous depth of 0.001 pm - 3.0 mm. alternatively, the present disclosure provides a method of producing a cancer-induced animal comprising:

[0046] (a) fragmenting the sarcoma tissue separated from the subject into a section of 30 mm3- 40 mm3volume;

[0047] (b) making a 3 mm to 7 mm incision on the surface of the animal's skin; and (c) Injecting the fragmented sarcoma tissue through the incision to a subcutaneous depth of 0.001 pm - 3.0 mm.

[0048] The present disclosure provides an efficient animal model that accurately replicates the characteristics of sarcoma cancer with unclear pathogenesis and frequent recurrence and metastasis, wherein the optimal size and optimal depth of insertion of patient-derived sarcoma cancer tissue inserted to produce an animal model of PDX sarcoma cancer are provided, and when the size and depth of insertion subcutaneously are applied, tumor proliferation and metastasis in the human body and reactivity to drugs are successfully reproduced in animals.

[0049] The advantages of the present invention includes but not limited to:

[0050] (a) The present invention relates to a method for efficiently inducing sarcoma cancer in mammals and an animal model of patient-derived xenograft sarcoma cancer produced using such a method.

[0051] (b) The animal model of the present invention applies the optimal volume and optimal subcutaneous depth of sarcoma tissue to be isolated from the patient and transplanted (xenotransplant) into the animal, thereby reflecting various biological characteristics such as tumor proliferation, metastasis, and drug responsiveness in the human body with high confidence.

[0052] (c) The present invention can be usefully used as an efficient analytical tool for molecular mechanism research, therapeutic screening, and evaluation of in vivo drugs in sarcoma cancers with unclear pathogenesis and frequent recurrence and metastasis.

[0053] In the present specification, the term "subject" means a human subject providing sarcoma cancer tissue to be transplanted into the animal model of the present invention, and hereby means a sarcoma cancer patient.

[0054] The subject of the present disclosure may be, for example, a patient with stage 1 or stage 2 sarcoma cancer, specifically a patient with stage 1 sarcoma cancer, and more specifically, stage 1 fibrosarcoma, melanoma, synovial sarcoma, liposarcoma or angiosarcoma. The subject of the present invention may be a sarcoma cancer patient who has undergone surgical resection, in which case the tumor tissue incised during surgery can be used as a graft section without additional invasive tissue collection. When the subject of the present invention is a sarcoma cancer patient who has undergone surgical resection, it may be a patient who has undergone surgical resection and radiotherapy, or a patient in which only surgical resection has been performed. More specifically, the subject of the present invention is a patient in which only surgical resection has been performed.

[0055] In this specification, the term "sarcoma" refers to a primary malignant tumor arising from non-epithelial connective tissue such as bone, cartilage, muscle, fat, and blood vessels. There are several reports that sarcoma cancer may be caused by genetic factors, irradiation, viral infection, and immune deficiency, but the exact etiology and fundamental therapeutic composition targeting it have not yet been developed. Therefore, the animal model of the present invention can be useful for the study of the in vivo phenotype and pathogenesis of sarcoma cancer and the search for therapeutics.

[0056] In this specification, the term "transplant" refers to the process of delivering a living tissue, organ or a combined artificial support separated from the donor to the recipient for the purpose of maintaining the biological properties and functional integrity of the tissue or organ transplanted into the recipient.

[0057] In this specification, the term "drug responsiveness" refers to the degree to which the survival and proliferation of the tumor is reduced at the tissue and cell level by contact with anticancer drugs, and is used in the same sense as "treatment responsiveness".

[0058] In this specification, the term "animal" means an animal other than a human and, more specifically, a mammal other than a human. The term "mammal" refers to all animals belonging to the mammalian family other than humans, including, but not limited to, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons or rhesus monkeys. Specifically, the mammal is a rodent, more specifically a mouse, and most specifically, a mouse with reduced or missing immune cell function or an immunodeficiency mouse.

[0059] In this specification, the term "immune cell" refers to any cell involved in the initiation or promotion process of an immune response, and more specifically an immune effector cell. Immune cells include, but are not limited to, for example, T cells, B cells, natural killer T (NKT) cells, natural killer T (NKT) cells, and monocytes and mast cells.

[0060] The immunodeficiency mice that can be used in the present invention include nude mice in which the function of T cells is deleted, SCID mice in which the functions of T cells and B cells are deleted, NOD / SCID mice in which monocyte function deletion is additionally induced by crossing NOD mice and SCID mice, and NOG mice in which the functions of T cells, B cells, NK cells, dendritic cells, monocytes and complement systems are all deleted by crossing NOD / SCID mice with IL-2Rynul1mice. This is not limited to this. Specifically, the immunodeficiency mice used in the present invention may be NOG mice.

[0061] In this specification, the term "animal model" refers to a non-human animal in which sarcoma cancer has been artificially induced and modified to have similar traits, phenotypes, symptoms, and drug responsiveness of sarcoma cancer as in humans, and specifically refers to an animal that reproduces the overall biological process that proceeds from the development of human sarcoma cancer by xenotransplantation of human patient-derived sarcoma cancer tissue.

[0062] According to a specific embodiment of the present invention, step (a) is performed by sectioning the sarcoma tissue separated from the object to a volume of 25 mm3- 35 mm3, and more specifically, by sectioning to a volume of about 30 mm3.

[0063] According to a specific embodiment of the present invention, step (b) is performed by making an incision of 4 mm to 6 mm on the skin surface of the animal, and more specifically, by making an incision of about 5 mm.

[0064] According to a specific embodiment of the present invention, the method of the present invention further comprises stirring washing the sarcoma tissue separated from the object with isotonic saline prior to step (a).

[0065] In the present specification, the term "isotonic saline" means a saline solution that does not show osmotic differences from the surrounding environment and does not cause significant substance transfer, and may specifically be a phosphate-buffered saline (PBS).

[0066] In the present specification, the term "agitation washing" refers to the process of making a form suitable for xenotransplantation by applying an appropriate stirring force using a cleaning buffer such as PBS to remove blood and impurities from the sarcoma tissue separated from the subject. Agitation may be performed manually (i.e., by shaking the container containing the sarcoma rock sample directly by the experimenter) or may be performed using a stirring device in which a certain stirring force and stirring time are set by rotation of an impeller, etc.

[0067] According to specific embodiments of the present invention, the sarcoma cancer is fibrosarcoma, melanoma, synovial sarcoma, liposarcoma or angiosarcoma, and more specifically, fibrosarcoma.

[0068] According to another aspect of the present invention, the present invention provides cancer induced animals prepared by the method of the present invention described above.

[0069] Since the sarcoma cancer induction method in animals used in the present invention and the sarcoma cancer animal model prepared thereby have already been described in detail, the description is omitted to avoid excessive duplication. Another embodiment of the present disclosure provides an apparatus for analyzing drug responsiveness comprising cancer tissue analogues.

[0070] An embodiment of the present disclosure provides an efficient three-dimensional organotypic culture that mimic the in vivo characteristics of cancer tissue, especially sarcoma, a non-epithelial connective tissue tumor with an unclear pathogenesis and frequent recurrence and metastasis, wherein, when the cell aggregates obtained by three-dimensional culture of sarcoma cells are solidified into a Matrigel Matrix in the culture container, and then the vascular endothelial cells are dispensed through the inlet connected to the culture container, the tumor microenvironment in vivo is not only reproduced with high accuracy, but also the vascular endothelial cells at the injection port serve as non-tumor control cells.

[0071] Another embodiment of the present disclosure provides a method for preparing cancer tissue analogues and a kit for drug responsiveness analysis comprising the same.

[0072] According to another aspect of the invention, the present invention provides a screening method for a composition for the prevention or treatment of sarcoma cancer comprising the following steps:

[0073] (a) administering the test substance to the sarcoma cancer-induced animal of the present invention described above;

[0074] (b) measuring one or more indicators selected from the group consisting of the animal's mortality, survival, and tumor volume; wherein, the mortality rate or tumor volume of the animal decreases or the survival time increases, the test substance is determined to be a composition for the prevention or treatment of sarcoma cancer.

[0075] The term "test substance" used while referring to the screening method of the present invention refers to an unknown substance administered to an animal model of sarcoma cancer of the present invention and used in screening to examine whether it affects tumor proliferation, activity, or animal survival. The test materials include, but are not limited to, compounds, nucleotides, peptides and natural extracts. Changes in mortality, survival time and tumor volume in animals administered with test substances can be analyzed by various measurement methods known in the art. As a result of the measurement, if the mortality rate or tumor volume of the animal decreases or the survival time increases compared to the control group, the test substance can be determined as a composition for the prevention or treatment of sarcoma cancer.

[0076] In the present specification, the terms "mortality reduction" and "tumor volume reduction" mean that the mortality rate and tumor volume of animals induced by sarcoma cancer by the anticancer action of the test substance screened in the present invention are significantly reduced to the extent that they are measurable compared to the control group, and specifically, the mortality rate and tumor volume are reduced by 20% or more compared to the control group, more specifically, a state in which the mortality rate and tumor volume are reduced by more than 40%, and more specifically, a state in which it is reduced by 60% or more.

[0077] In the present specification, the term "increase in survival" means that the overall survival or recurrence-free survival of animals induced by sarcoma cancer by the anticancer action of the test substance screened in the present invention is significantly increased to the extent that it is measurable compared to the control group, and specifically, the overall survival time or recurrence-free survival is increased by 20% or more, more specifically, it may mean an increase of 40% or more, and more specifically, an increase of 60% or more.

[0078] In this specification, the term "prevention" means to suppress the occurrence of a disease or disease in an object that has not been diagnosed as having a disease or disease, but is likely to suffer from such a disease or disease.

[0079] In this specification, the term "treatment" means (a) the inhibition of the development of a disease, disease or symptom; (b) alleviation of disease, illness or symptoms; or (c) to eliminate any disease, disease, or symptom. The compositions screened by the method of the present invention serve to inhibit, eliminate or alleviate the development of symptoms in individuals with sarcoma cancer by removing or reducing sarcoma cancer tissue, inhibiting proliferation, or delaying progression. Thus, the composition screened through the present invention may itself be a composition for the treatment of these diseases, or it may be administered together with other pharmacological components having a therapeutic effect on sarcoma cancer and applied as a therapeutic adjuvant for the disease. Herein, the term "treatment" or "therapeutic agent" in this specification includes the meaning of "therapeutic adjuvant" or "therapeutic adjuvant".

[0080] According to specific embodiments of the present invention, the sarcoma cancer is fibrosarcoma, melanoma, synovial sarcoma, liposarcoma or angiosarcoma, and more specifically, fibrosarcoma.

[0081] According to another aspect of the invention, the present invention provides a method for preparing cancer tissue analogues including:

[0082] (a) forming a cancer cell aggregate by three-dimensional culture of cancer cells isolated from the subject into a first culture medium;

[0083] (b) three-dimensional culture of the cancer cell aggregate into a second culture medium comprising an extracellular matrix or an analogue thereof in a culture vessel comprising a culture space of a certain volume and an inlet connected to the culture space; and

[0084] (c) Injecting vascular endothelial cells through a passage connected to the inlet.

[0085] The present disclosure provide an efficient three-dimensional organotypic culture that mimic the in vivo characteristics of cancer tissue, especially sarcoma, a non-epithelial connective tissue tumor with an unclear pathogenesis and frequent recurrence and metastasis, wherein when the cell aggregates obtained by three-dimensional culture of sarcoma cells are solidified into a Matrigel Matrix in the culture container, and the vascular endothelial cells are dispensed through the inlet connected to the culture container, the tumor microenvironment in vivo is not only reproduced with high accuracy, but also the vascular endothelial cells at the injection port serve as non-tumor control cells.

[0086] In this specification, the term "tissue analogues" refers to an in vitro three-dimensional cellular cluster composed of a primary tissue, a tissue subunit, or a single cell (e.g., cancer cells). When tissue analogues have the ability to self-renew and self-organize, the cells that make up the tissue analogue reproduce phenotypic and biological functions similar to the tissue from which they originate, so that tissue analogues can also be named "organ analogues", "organ analogues", or "organoids".

[0087] In the present specification, the term "three-dimensional culture" is a concept relative to two-dimensional culture, and refers to culturing the culture target cells in a floating state in the culture medium without fixing them in a substrate. Therefore, the term "three-dimensional culture" is used interchangeably with "suspension culture". Adhesion-dependent cancer cells cause cell aggregation during flotation culture, and cells that are not included in such aggregation and float alone cause apoptosis. In the present specification, the term "cell aggregation" refers to the formation of a cell agglomeration mass of a three-dimensional structure while selfassembling cells cultured in an environment such as a floating culture in which three-dimensional growth is allowed, rather than a monolayer. The cell aggregates resulting from three-dimensional culture may be sphere or non-spherical depending on their size and the number of selfassembled cells. Spherical cell aggregates are called spheroids, but spheroids do not have to be geometrically perfect spherical.

[0088] The cell aggregates formed in this way provide an environment similar to tissues in vivo, and in particular, in the case of cancer cell aggregates, conditions similar to the tumor microenvironment are formed. The term "tumor microenvironment" refers to the surrounding space of tumor cells in vivo, including non-cancerous cells such as stromal cells that surround and nourish tumor cells, extracellular matrix including proteins, signaling molecules, and blood vessels. Tumor cells can change the composition and composition of the microenvironment, and the microenvironment can affect the growth and spread of tumors, so tumor cells and the microenvironment affect each other.

[0089] In the present specification, the term "culture medium" refers to a mixture for cell growth and proliferation in an invitro comprising elements essential for cell growth and proliferation, such as sugars, amino acids, various nutrients, minerals, and the like. Additional components that may be included in the cell culture medium are, for example, glycerin, L-alanine, L-arginine hydrochloride, L-cysteine hydrochloride-monohydrate, L-glutamine, L-histidine hydrochloridemonohydrate, L-lysine hydrochloride, L-methionine, L-proline, L-serine, L-threonine, L-valine, L- asparagine-monohydrate, L-aspartic acid, L-cystine 2HCI, L-glutamic acid, L-isoleucine, L-leucine, L-phenylalanine, L-tryptophan, L-tyrosine disodium salt dihydrate, i-inositol, thiamine hydrochloride, niacinamide, pyridoxine hydrochloride, biotin, D-calcium pantothenate, folic acid, riboflavin, vitamin B12, sodium chloride (NaCI), sodium bicarbonate (NaHCOs), potassium chloride (KCI), calcium chloride (CaCh), Sodium hydrogen phosphate monohydrate (NaFhPC - H2O), copper sulfate pentahydrate (CUSO4-5H2O), ferric sulfate heptahydrate (FeSO4-7H2O), magnesium chloride (anhydrous) (MgCI2), magnesium sulfate (MgS04), hydrogen phosphate (Na2HPO 4), zinc sulfate heptahydrate (ZnSO4-7H2O), D-glucose (dextrose), sodium pyruvate, hypoxanthine sodium, linolenic acid, lipoic acid, putrescine, 2HCI and thymidine.

[0090] The cell culture medium according to the present invention can be artificially manufactured and used, or commercially marketed and purchased. Examples of commercially available culture media include, but are not limited to, Iscove's Modified Dulbecco's Medium (IMDM), Alpha Modification of Eagle's Medium (ot-MEM), Nutrient Mixture F-12 (F12) and DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) and EMEM (Eagle's Minimum Essential Medium).

[0091] According to the present invention, the present invention uses a first culture medium for three-dimensional culture of cancer cells to form cancer cell aggregates and a second culture medium for culturingthe formed cancer cell aggregates again in a culture space including an inlet In the present specification, the term "analogue of an extracellular matrix" refers to a mixture having a similar composition to an extracellular matrix consisting of a three-dimensional network of extracellular macromolecules, and an artificial or natural protein mixture having the same or similar biological function as a natural extracellular matrix.

[0092] According to a specific embodiment of the present invention, the extracellular matrix or its analogue is a gelatinous protein mixture, more specifically a Matrigel Matrix.

[0093] In the present specification, the term "inlet" means an inlet formed in a compartmentalized culture space having a certain volume and through which an external substance enters the culture space, and "a passage connected to the inlet" means a channel connected to the inlet to provide a material inflow path from the outside. The injection port and the passage connected thereto may be an inlet channel for culture medium or vascular endothelial cells, and may be used as an inlet channel for a test drug for contacting a cancer tissue analogue of the present invention with a test drug, as described below. The inlet and the passage connected thereto may be one or more forms in the culture space, specifically two to four, and more specifically, two.

[0094] The inlet of the present invention may have, for example, a cross-section diameter of 0.25 mm - 1.30 mm.

[0095] According to specific embodiments of the present invention, the culture container includes a plurality of the culture spaces, and the plurality of culture spaces are spatially separated from each other.

[0096] According to the present invention, the culture container of the present invention comprising a plurality of compartmentalized culture spaces can accommodate a plurality of cancer tissue analogues to perform simultaneous mass analysis of drug responsiveness. Each culture space may contain tissue analogues of different carcinomas at the same time, normal tissue analogues may be accepted as a control group to perform a more objective analysis of drug responsiveness, and a large number of tissue analogues of the same carcinoma may be accepted so that the responsiveness to each different drug may be analyzed at the same time.

[0097] "Spatially separated" in this specification includes cases in which compartmentalized culture spaces are completely spatially closed off from each other by bulkheads, as well as cases in which they are partially blocked to such an extent that substantial material transfer cannot take place.

[0098] More specifically, the plurality of culture spaces have a volume of 1 to 10 mm 3 per each culture space.

[0099] According to a specific embodiment of the present invention, step (a) is performed by three-dimensional culture of cancer cells of 0.5 x 105to 1.5 x 105cells / mL. More specifically, 0.7 x 105to 1.3 x 105cells / mL, and most specifically, 0.9 x 105to 1.1 x 105cells / mL of cancer cells are cultured in three dimensions.

[0100] According to a specific embodiment of the present invention, the cancer cell aggregate is a cancer cell spheroid. In the present specification, the term "spheroid" refers to a spherical microtissue composed of a plurality of aggregated cells that grows or differentiates while interacting with an artificially created environment in all three-dimensional spaces. Spheroids may not be geometrically perfect spherical, and may be a group of allogeneic cells or a group of heterologous cells. Compared to conventional 2D culture, spheroids provide an environment similar to that of in vivo, which more accurately reproduces the cytological and physiological properties of cancer tissues and drug responsiveness.

[0101] More specifically, the cancer cell spheroid has an average particle size of 40 to 100 pm.

[0102] According to a specific embodiment of the present invention, the cancer cells used in the present invention are sarcoma cancer cells.

[0103] In this specification, the term "sarcoma" refers to a primary malignant tumor arising from non-epithelial connective tissue such as bone, cartilage, muscle, fat, and blood vessels. There are several reports that sarcoma cancer may be caused by genetic factors, irradiation, viral infection, and immune deficiency, but the exact etiology and fundamental therapeutic composition targeting it have not yet been developed. Therefore, the sarcoma cancer tissue analogues of the present invention can be useful for the study of in vivo phenotypes and pathogenesis mechanisms of sarcoma cancer and the search for therapeutics.

[0104] According to a specific embodiment of the present invention, the method of the present invention further comprises solidifying the second culture medium between steps (b) and step (c).

[0105] In the present specification, the term "solidification" refers to a phase transition from a liquid state in which the molecular structure is disordered to a solid state in which the degree of disorder is reduced. Solidification includes, for example, when the second culture medium is completely solidified, as well as when the viscosity and / or strength is increased to such an extent that it can maintain its shape.

[0106] According to another aspect of the invention, the present invention provides a kit for drug responsiveness analysis including:

[0107] (aa) cancer tissue analogues prepared by the methods of the foregoing invention; and

[0108] (bb) a culture vessel comprising a plurality of culture spaces accommodating the cancer tissue analogue and an inlet formed in the culture space; and

[0109] (cc) A material exchange channel connected to the inlet.

[0110] Since the cancer tissue analogues used in the present invention and the culture container accommodating the same have already been described in detail, the description is omitted to avoid excessive duplication.

[0111] In this specification, the term "drug responsiveness" refers to the degree to which the survival and proliferation of the tumor is reduced at the tissue and cell level by contact with anticancer drugs, and is used in the same sense as "treatment responsiveness".

[0112] The features and benefits of the present invention includes the following, but not limited to:

[0113] (aa) The present invention provides a kit for drug responsiveness analysis comprising cancer tissue analogues and the same.

[0114] (bb) The cancer tissue analogue of the present invention efficiently simulates the tumor microenvironment in vivo by solidifying the cell aggregates obtained by three-dimensional culture of cancer cells, especially sarcoma cells, into a matriegel mixture, and then forming a vascular endothelial cell channel.

[0115] (cc) Accordingly, the cancer tissue analogues of the present invention can be used as a reliable drug screening system by reproducing the drug responsiveness of tumors in vivo with high accuracy, and when established using patient-derived autologous sarcoma cells, the patient's treatment response to the drug, the response dose, and the optimal combination of drugs can be quickly analyzed in vitro to establish a patient-tailored treatment strategy at an early stage.

[0116] EXAMPLES

[0117] Hereinafter, the present invention will be described in more detail through an embodiment. These embodiments are only for the purpose of describing the present invention in more detail, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these embodiments according to the gist of the present invention.

[0118] Preparation of sarcoma tissue for subcutaneous transplantation

[0119] Sarcoma tissue and normal tissue obtained from the surgical patient through surgical resection were placed in a 90 mm petridish each. Refrigerated PBS was dispensed into 50 mL conical tubes of 25 mL each, then each sample tissue was added, the lid was closed, and it was shaken to the point of foaming to remove blood and impurities. It was then poured into a 90 mm petridish, picked up the tissue with forceps and transferred to a 90 mm petridish containing the new PBS. A 90 mm petridish containing tissue and PBS was placed on graph paper and photographed. Afterwards, the sarcoma tissue was made into 8-10 pieces with a size of 3 x 3 mm for PDX implantation. The table below is sarcoma cancer sample information written in the embodiment.

[0120] Table 1.

[0121] Fabrication of the Fl PDX model

[0122] The quarantine and circuit period of the mouse was 1 week, and the mice with the circuit period completed were used. NOG mice were anesthetized by dissolving 30 mg / kg of Zoletil and 10 mg / kg of Xylazine in normal saline solution and injecting into the abdominal cavity of mice with a 1 cc syringe. Subsequently, the skin of the right flank area was disinfected with povidone, and an incision of about 0.5 cm was made using scissors, and then the incision was held with forceps, the tissue for PDX implantation was placed subcutaneously, and the skin was sutured with a stapler or suture, and then sterilized with povidone. Place the implanted mouse in a cage with the implant facing the ceiling and see if it wakes up from anesthesia.

[0123] Tumor size measurement

[0124] The size of the tumor was measured once a week using a vernier caliper, but the longest and shortest sides of the tumor were measured, and the volume was calculated by the following formula.

[0125] Volume (mm3) - (length x width x depth) / 2

[0126] Sarcoma cancer cell extraction and culture

[0127] HT1080 (ATCC, USA) cell line, a fibrosarcoma cancer cell, was cultured in EMEM (Eagle's Minimum Essential Medium) medium (SH3002401, cytiva, MA, USA) with a culture mixture of 10% fetal bovine serum (16000-044, Gibco, NY, USA) and 1% antibiotics. The culture medium was replaced at intervals of 2 - 3 days, and sarcoma cancer cells were treated with TrypLE express (12605-010, Gibco, NY, USA) at intervals of 7 days and subcultured.

[0128] Three-dimensional culture for in vivo environmental simulation

[0129] Sarcoma cancer cells were formed spheroids by three-dimensional culture using a 3D culture chip (Aimbiotech, DAX01, Singapore). In summary, sarcoma cancer cells were washed with PBS once, and then treated with TrypLE express, peeled, and centrifuged at 1,300 RPM, for 3 minutes to produce cell pellets. The culture medium was treated and pipetted into a cell pellet and diluted to 1 x 105 cells / mL, and then dispensed in a petridish without surface treatment to induce spheroids to be produced naturally.

[0130] The produced spheroids were filtered and separated with a 100 pm strainer (93100, SPL, Republic of Korea) and a 40 pm strainer (93040, SPL, Republic of Korea) to select only spheroids with a diameter of 40 ~ 100 pm. Matrigel (356237, Corning, NY, USA): The selected spheroid was treated with a matrigel mixture mixed with the culture medium in a 1: 1 ratio, and then 10 pL was dispensed into the gel channel of the three-dimensional culture chip. Afterwards, it was stored at 37 °C. for 15 minutes to allow the gel to solidify completely.

[0131] After the gel dispensed in each gel channel was solidified, the culture medium was dispensed into two cultures channels located on either side of the gel channel to hydrate the culture medium channel. Thereafter, vascular endothelial cells were dispensed with 1 x 105cells / mL at 10 pL on both sides of each culture medium channel and cultured at 37 °C. and 5% CO2 environment to simulate the vascular channel. Subsequently, the reactivity to the anticancer drug was analyzed by treating each anticancer drug candidate in the culture medium channel of the fabricated three-dimensional culture chip and measuring the cell viability.

[0132] Screening for effective concentrations through treatment of anticancer drugs by concentration

[0133] In two cultures channels of the prepared three-dimensional culture chip were treated with Doxorubicin (D1515, Sigm-Aldrich, MO, USA) 0, 500, and 1,500 nM, respectively, and incubated at 37 °C under 5% CO2 environment for 24 hours. After 24 hours, cell viability was observed through Live & Dead assays. In summary, Live & Dead assay kit (L3224, Invitrogen, MA, USA) was diluted in the culture medium to 1 / 2000, and the culture medium was removed from the two culture media channels in the three-dimensional culture chip, and then washed once with PBS. Afterwards, the culture medium diluted with the Live & Dead assay kit was treated with each channel and reacted in a 370C. and 5% CO2 environment for 10 minutes. Afterwards, after one wash with PBS, the color of fluorescence expressed in sarcoma cancer cells and vascular endothelial cells was checked through a 4-magnification fluorescence microscope (1X73, Olympus), and the fluorescence intensity was compared between each group. As a result of confirming the cell survival rate after 24 hours after treatment with doxorubicin through a three- dimensional culture chip, it was confirmed that there was almost no difference in cell survival rate in both spheroids and vascular endothelial cells of sarcoma cancer cells when doxorubicin treatment up to 1500 nM (FIG. 2). Accordingly, it was confirmed that doxorubicin at a concentration of less than 1500 nM in vascular endothelial cells and sarcoma cancer cell-based spheroids has no anticancer efficacy and cytotoxicity, and it was confirmed that doxorubicin higher than 1500 nM should be treated to evaluate the anticancer efficacy that shows toxicity to sarcoma cancer cells without reducing the cell viability of vascular endothelial cells.

[0134] The present invention allows the anticancer drug candidate to simultaneously confirm the death effect on sarcoma cancer cells in hydrogel including vascular endothelial cells, control normal cells, and extracellular matrix, etc.

[0135] Having described in detail a specific part of the present invention above, it is clear that for a person skilled in the art, such a specific technique is only a desirable embodiment, and the scope of the present invention is not limited thereby. Thus, the substantive scope of the present invention will be defined by the attached claims and their equivalents.

Claims

CLAIMS1. A method for preparing a sarcoma cancer-induced animal comprising:(a) Fragmenting the sarcoma tissue separated from the subject into a section of 20 mm3- 40 mm3volume;(b) making a 3 mm to 7 mm incision on the surface of the animal's skin; and(c) Injecting the fragmented sarcoma tissue through the incision to a subcutaneous depth of 0.001 pm to 3.0 mm.

2. The method of claim 1, the subject is a patient with stage 1 fibrosarcoma, melanoma, synovial sarcoma, liposarcoma or angiosarcoma.

3. The method of claim 1, wherein the animal is a rodent animal.

4. The method of claim 3, wherein the rodent animal is a mouse with reduced or missing immune cell function or immunodeficiency.

5. The method of claim 1, the method of the method further comprises stirring and washing the sarcoma tissue separated from the subject prior to step (a) with isotonic saline.

6. The method of claim 1, wherein the sarcoma cancer is fibrosarcoma cancer.

7. An animal model induced with sarcoma cancer manufactured by the method of claim 1.

8. A screening method of composition for the prevention or treatment of sarcoma cancer, comprising:(a) administering the test substance to an animal induced with the sarcoma cancer referred to in paragraph (7);(b) measuring one or more indicators selected from the group consisting of the animal's mortality, survival, and tumor volume;When the mortality rate or tumor volume of the animal decreases or the survival time increases, the test substance is determined to be a composition for the prevention or treatment of sarcoma cancer.

9. The method of claim 8, wherein the sarcoma cancer is fibrosarcoma, melanoma, synovial sarcoma, liposarcoma or angiosarcoma.

10. A method for preparing cancer tissue analogues comprising:(aa) forming a cancer cell aggregate by three-dimensional culture of cancer cells isolated from the subject into a first culture medium;(bb) three-dimensional culture of the cancer cell aggregate into a second culture medium comprising an extracellular matrix or an analogue thereof in a culture vessel comprising a culture space of a certain volume and an inlet connected to the culture space; and(cc) Injecting vascular endothelial cells through a passage connected to the inlet.

11. The method of claim 10, wherein the culture container includes a plurality of the culture spaces, and the plurality of culture spaces are spatially separated from each other.

12. The method of claim 11, wherein the plurality of culture spaces have a volume of 1-10 mm3per each culture space.

13. The method of claim 10, in which step (aa) is performed by three-dimensional culture of cancer cells of 0.5 x 105to 1.5 x 105cells / mL.

14. The method of claim 10, wherein the cancer cell aggregate is a cancer cell spheroid.

15. The method of claim 14, wherein the cancer cell spheroid has an average particle diameter of 40-100 pm.

16. The method of claim 10, wherein the extracellular matrix or its analogue is a gelatinous protein mixture.

17. The method of claim 16, wherein the gelatinous protein mixture is a Matrigel Matrix.

18. The method of claim 10, wherein the cancer is a sarcoma cancer.

19. The method of claim 10, the method further comprises a step of solidifying the second culture medium between steps (bb) and step (cc).

20. A drug responsiveness analysis kit comprising:(i) cancer tissue analogues prepared by the method referred to in paragraph (1);(ii) a culture vessel comprising a plurality of culture spaces accommodating the cancer tissue analogue and an inlet formed in the culture space; and(iii) a material exchange channel connected to the inlet;21. The kit of claim 20, wherein the plurality of culture spaces have a volume of 1-10 mm3per each culture space.

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