Renal cancer assembloid and producing method thereof

Abstract

The present invention relates to a renal cancer assembloid and a producing method thereof. The renal cancer assembloid according to the present invention is produced by co-culturing RCC, endothelial cells, and fibroblasts to produce an RCC spheroid, and then assembling the RCC spheroid with a renal organoid. It was confirmed that the renal cancer assembloid expresses not only a tumor-related gene but also a renal gene, and thus, the renal cancer assembloid according to the present invention has the effect of simultaneously reproducing a renal tumor and a tumor microenvironment (TME). Therefore, the renal cancer assembloid according to the present invention is expected to be useful for drug screening and biomarker evaluation for renal cancer.

Description

Kidney cancer assemble and method for producing the same

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

[0002] This invention claims priority to Republic of Korea Patent Application No. 10-2023-0171547, filed on November 30, 2023, the entire disclosure of which is incorporated herein by reference.

[0003]

[0004] Kidney cancer accounts for approximately 3% of all solid tumors. Approximately 85% of kidney tumors are classified as renal cell carcinoma (RCC). Approximately 80% of diagnosed RCCs arise from the epithelial cells lining the adjacent portions of the kidney's urine-forming tubules, the urinary tracts. Due to their microscopic appearance, these cancer types are known as renal clear cell carcinoma (RCCC (ccRCC), 65%) or renal papillary cell carcinoma (RPCC, 15%). RCCC and RPCC account for 80% of diagnosed RCCs and are more closely related to the 100% death rate attributed to renal cell carcinoma.

[0005] Renal cell carcinoma (RCC) is the sixth most commonly diagnosed cancer in men and the tenth most commonly diagnosed cancer in women. The incidence of RCC is increasing due to increased risk factors, including smoking, obesity, and hypertension, as well as improved diagnostic imaging, leading to increased detection. The annual financial burden of RCC treatment exceeds $4 billion in the United States alone, and continues to rise, with more than 60,000 new cases diagnosed each year.

[0006] In vitro models, such as cell lines, are fundamental for further investigating the mechanisms of tumor development, tumor invasion, and metastasis, as well as their translation into new therapeutic approaches. However, significant differences exist between in vitro system modeling and in vivo conditions in patients, leading to inaccurate predictive capabilities of many preclinical models.

[0007] Current standards for RCC culture involve primary or immortalized cells grown on conventional two-dimensional (2D) tissue culture plastic. In many cases, the phenotype of the parent tumor from which the 2D cell line was established is unknown, or the culture does not maintain its basal phenotype over time. Validity issues in 2D in vitro studies are not limited to RCC; they also pose challenges in predicting the success or failure of new drug candidates and in predicting renal toxicity.

[0008] While novel 3D culture methods require a scaffold capable of supporting growth and initial phenotype, this technology will enhance the ability to model tumor behavior in culture by providing a supportive environment. Tumor spheroids, a 3D cancer model, are playing an increasingly important role in cancer research. Their relative simplicity and convenience, coupled with their ability to recapitulate key features of tumor biology missing from traditional 2D models, have made them the most widely studied 3D tumor model. Their applications as therapeutic tools or as a means of studying tumor-host interactions are well established in various cancers, and RCC spheroids hold promise for this field.

[0009] Meanwhile, the tumor microenvironment (TME) is complex and continuously evolving, containing not only tumor cells but also various supporting cell types, such as activated fibroblasts, blood vessels, infiltrating immune cells, and the extracellular matrix. The TME is regulated by various cells, hormones, and inflammatory responses and is crucial for understanding tumor formation, as well as tumor progression and metastasis. Therefore, it plays a key role in tumor prevention, diagnosis, and prognosis and has long been considered an important aspect of oncology research.

[0010] In therapeutic approaches using RCC spheroid models, tumor cells serve as direct targets of treatment. However, RCC spheroid models have limitations in recapitulating the TME of RCC, which may hinder their use in immunotherapy, an innovative cancer treatment. Therefore, a novel in vitro RCC model that simultaneously recapitulates both the RCC tumor and TME is needed.

[0011] Accordingly, the inventors of the present invention studied to manufacture an RCC model capable of simultaneously implementing RCC tumor and TME, and developed a protocol for manufacturing an RCC kidney assembler to provide a powerful preclinical tool for drug screening and biomarker evaluation in alternative in vitro methods such as tumor tissue slice culture or in vivo xenograft models.

[0012]

[0013] The present inventors conducted research to solve the problems of the conventional RCC spheroid model and to produce a new RCC model that can simultaneously reproduce RCC tumors and TME. As a result, they produced RCC spheroids by co-culturing RCC, endothelial cells, and fibroblasts, and then assembled them with renal organoids to produce RCC assembles, and based on this, completed the present invention.

[0014] Accordingly, the purpose of the present invention is to provide a method for manufacturing a renal cancer assemblage and a renal cancer assemblage that implements a tumor microenvironment manufactured therefrom.

[0015]

[0016] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0017]

[0018] To achieve the above-described purpose, the present invention provides a method for manufacturing a renal cancer assembloid, comprising the following steps:

[0019] (a) a step of manufacturing and differentiating renal organoids;

[0020] (b) a step of producing a spheroid by co-culturing renal cancer cells with at least one selected from the group consisting of fibroblasts and endothelial cells; and

[0021] (c) A step of producing a renal cancer assembler by adding the differentiated renal organoid to the spheroid produced in step (b).

[0022] As one embodiment of the present invention, the kidney organoid can be produced using induced pluripotent stem cells (iPSCs), but is not limited thereto.

[0023] As another embodiment of the present invention, the renal organoids and spheroids may be cultured in a medium containing geltrex, but are not limited thereto.

[0024] As another embodiment of the present invention, the renal cancer cells may be, but are not limited to, clear cell renal cell carcinoma (ccRCC) cells.

[0025] As another embodiment of the present invention, the fibroblasts may be, but are not limited to, lung fibroblasts.

[0026] As another embodiment of the present invention, the endothelial cells may be, but are not limited to, human umbilical vein endothelial cells (HUVEC).

[0027] As another embodiment of the present invention, the renal organoids can be cultured in mTeSR1 or RPMI medium, but are not limited thereto.

[0028] As another embodiment of the present invention, the manufactured kidney assemble can embody a tumor microenvironment, but is not limited thereto.

[0029] In addition, the present invention provides a renal cancer assembly that implements a tumor microenvironment, characterized by being manufactured by the above method.

[0030] In addition, the present invention provides a composition for producing a renal cancer assembler, comprising a first composition, a second composition, and a third composition, wherein the first composition is a medium composition for producing and differentiating renal organoids, the second composition is a medium composition for producing spheroids, and the third composition is a medium composition for producing renal cancer assembler, wherein the first composition, the second composition, and the third composition are each used sequentially.

[0031] In addition, the present invention provides a kit for producing a renal cancer assembly comprising a composition and a description of a method for producing a renal cancer assembly according to the present invention.

[0032] In addition, the present invention provides a use of a renal cancer assembly manufactured by the above method for screening a renal cancer treatment agent.

[0033] The present invention also provides a use of the composition or the kit for producing a renal cancer assembly.

[0034]

[0035] The renal cancer spheroid according to the present invention is produced by co-culturing RCC, endothelial cells, and fibroblasts to produce RCC spheroids, which are then assembled with renal organoids. It was confirmed that the renal cancer spheroid expresses not only tumor-related genes but also renal genes, and thus the renal cancer spheroid according to the present invention has the effect of simultaneously reproducing a renal tumor and the tumor microenvironment (TME). Therefore, the renal cancer spheroid according to the present invention is expected to be useful for drug screening and biomarker evaluation for renal cancer.

[0036]

[0037] FIG. 1A is a diagram illustrating a protocol for producing renal cell carcinoma spheroids by culturing renal organoids with various combinations of renal cell carcinoma (RCC) spheroids, lung fibroblasts (LF), or human umbilical vein endothelial cells (HUVEC) together with geltrex according to one embodiment of the present invention.

[0038] FIG. 1b is a diagram illustrating a protocol for producing renal cancer assembles by culturing renal organoids with RCC spheroids, LF, and HUVECs together with geltrex according to one embodiment of the present invention.

[0039] FIG. 1c is a drawing showing the results of observing renal organoids, RCC spheroids+LF+EC, and renal cancer assembles using a brightfield microscope according to one embodiment of the present invention.

[0040] FIGS. 2A and 2B are drawings showing immunofluorescence images of renal cancer assembles according to one embodiment of the present invention, wherein FIG. 2A shows the immunofluorescence staining results of NPHS1, LTL, and CAIX (scale bar = 200 μm), and FIG. 2B shows the immunofluorescence staining results of NPHS1, LTL, and PECAM1 (scale bar = 200 μm).

[0041] FIG. 3 is a drawing showing the results of confirming the mRNA expression of various genes in a renal cancer assembler according to one embodiment of the present invention.

[0042]

[0043] The present invention provides a method for manufacturing a renal cancer assembloid, comprising the following steps:

[0044] (a) a step of manufacturing and differentiating renal organoids;

[0045] (b) a step of producing a spheroid by co-culturing renal cancer cells with at least one selected from the group consisting of fibroblasts and endothelial cells; and

[0046] (c) A step of producing a renal cancer assembler by adding the differentiated renal organoid to the spheroid produced in step (b).

[0047] In the present invention, “organoid” refers to a three-dimensional aggregate of one or more cell types that mimics the surface appearance or actual structure or function of a tissue or organ, which is a culture produced by recombining cells isolated from stem cells or organ cells in vitro, and can be interpreted as having the same meaning as an artificial organ, bio-organ, mini-organ, spheroid, or embryoid body. The origin of the cells that constitute the organoid is not limited, and for example, the organoid can be derived from tissue, embryonic stem cells, or induced pluripotent stem cells, and can be cultured in three dimensions due to its self-renewal and differentiation capabilities. In the present invention, the organoid may be a kidney organoid, and may be produced using induced pluripotent stem cells (iPSCs), but is not limited thereto.

[0048] In the present invention, “induced pluripotent stem cell” means a cell that has pluripotency similar to embryonic stem cell that can differentiate into cells of all organs that make up our body by artificially stimulating already differentiated somatic cells.

[0049] In the present invention, a "spheroid" is a small sphere created through three-dimensional cell culture. A spheroid is a type of small tissue that has distinct exterior and interior, divided functions, and a form that mimics the functional unit of an tissue. Spheroid culture not only resembles the inherent form and properties of animal or human tissue, but is also being developed as a platform that can be applied to research. Spheroids created using cancer cells have a form that is more similar to tumor tissue, and thus show more similar results in response to anticancer drugs than cancer cells cultured on a flat surface.

[0050] In the present invention, the term "renal cancer assembleoid" refers to an assembled cell system resulting from the combination of renal organoids and renal cell carcinoma spheroids. The renal cancer assembleoid is a combination of renal organoid compartments and renal cell carcinoma spheroid compartments, each of which is distinct. The assembleoid accurately mimics human tissue, pathological, and physiological characteristics by reconstituting tissue stem cells and various cells present in human organs through cellular reconstitution of stem cells. Furthermore, by additionally combining renal organoids and renal cell carcinoma spheroids with fibroblasts and vascular endothelial cells, the system can precisely recreate the microenvironment surrounding actual human renal cancer. This system can more precisely mimic the microenvironment surrounding renal cancer and the tumor than existing renal cancer spheroids and renal cancer organoids. Assembroids are advanced through in vitro organ culture and can reproduce the structure and function of mature human organs better than existing organoids, enabling modeling of complex and diverse diseases. They can serve as next-generation human disease development models for elucidating the pathogenesis of these diseases or developing treatments.

[0051] In the present invention, the renal cell carcinoma spheroid exhibits vascularization within the renal cell carcinoma spheroid compartment, which is similar to the phenotype of actual human renal cell carcinoma. Furthermore, the renal cell carcinoma spheroid was confirmed to express not only tumor-related genes but also renal genes, indicating that the renal cell carcinoma spheroid according to the present invention can simultaneously embody renal cell carcinoma and a tumor microenvironment.

[0052] In the present invention, the “tumor microenvironment (TME)” refers to the environment in which a tumor exists, which is an acellular area within the tumor and an area that extends directly from the tumor tissue, but which does not belong to the intracellular compartment of the cancer cells themselves. It is a comprehensive concept that collectively refers to not only the constituent cell populations such as vascular cells, stromal cells, and immune cells present within the tumor, but also their environments (slightly acidified and hypoxic). The tumor and the tumor microenvironment are closely related and constantly interact with each other. A tumor can change its microenvironment, and the microenvironment can affect how the tumor grows and spreads. The tumor microenvironment exhibits a slightly acidic pH, lower concentrations of glucose and other nutrients compared to plasma, higher concentrations of lactic acid, hypoxia, and a temperature 0.3 to 1 °C higher than the normal physiological temperature.

[0053] In the present invention, “differentiation” means the progression of a cell from an earlier or initial stage of the developmental pathway to a later or more mature stage of the developmental pathway.

[0054] In the present invention, "kidney cancer" refers to cancer that occurs in the kidney, and includes both primary cancer that occurs in the kidney and cancer that has metastasized to the kidney from other organs. 85-90% of kidney cancers are classified as malignant renal cell carcinoma. In the present invention, the renal cancer cells may be, but are not limited to, clear cell renal cell carcinoma (ccRCC) cells.

[0055] In the present invention, "fibroblasts" are a type of cell that synthesizes extracellular matrix and collagen, creates the structural framework (stroma) of animal tissues, and plays an important role in wound healing. Fibroblasts are the most common cells in animal connective tissues. Fibroblasts include, for example, lung fibroblasts, liver fibroblasts, kidney fibroblasts, and skin fibroblasts. In the present invention, the fibroblasts may be lung fibroblasts, but are not limited thereto.

[0056] In the present invention, “endothelial cell” refers to a cell forming the endothelium, and the endothelium refers to the epithelial tissue that contacts the lumen of animal blood vessels. The endothelium, the basement membrane of the endothelium, and the connective tissue beyond it (subendothelial layer) form the intima, the innermost layer of the blood vessel. Morphologically, it belongs to the simple squamous epithelium, and functionally, it performs the role of a barrier that selectively permeates substances. In addition, the role of the endothelium is very diverse, such as a site for metabolism and secretion of various substances such as hormones, a factor regulating blood flow and blood coagulation, and a factor regulating immune response. In the present invention, the endothelial cell may be, but is not limited to, human umbilical vein endothelial cells (HUVEC).

[0057] In the present invention, the medium is a liquid or gel-like nutrient source designed to proliferate microorganisms, cells, or small plants such as moss. Different types of medium are used depending on the type of cells to be cultured. In the present invention, the renal organoids and spheroids may be cultured in a medium containing Geltrex, but are not limited thereto.

[0058] In the present invention, the renal organoids may be cultured in mTeSR1 or RPMI medium, but are not limited thereto. The mTeSR1 medium may further contain Y27632, but is not limited thereto. Furthermore, the RPMI medium may further contain CHIR99021 or B27 supplement, but is not limited thereto.

[0059] In the present invention, the step (a) may include a step of plating human induced pluripotent stem cells on mTeSR1 medium; a step of replacing the medium with mTeSR1 medium or RPMI medium containing geltrex; and a step of differentiating kidney organoids by replacing the medium with RPMI medium every 2 to 3 days until day 16, and specifically a step of plating human induced pluripotent stem cells on mTeSR1 medium containing Y27632; 0.5 to 2%, 0.5 to 1.8%, 0.5 to 1.6%, 0.5 to 1.5%, 0.7 to 2%, 0.7 to 1.8%, 0.7 to 1.6%, 0.7 to 1.5%, 1 to 2%, 1 to 1.8%, 1 to 1.6%, 1 to 1.5%, 1.2 to 2%, 1.2 to 1.8%, 1.2 to 1.6%, 1.2 to 1.5%, 1.4 to 2%, 1.4 to 1.8%, 1.4 to 1.6%, 1.4 to 1.5%, 1.5 to 2%, 1.5 to 1.8%, 1.5 to 1.6%, or 1.5% A step of replacing the mTeSR1 medium and RPMI medium containing geltrex, wherein the step of replacing the mTeSR1 medium on days 1 and 3, the step of replacing the RPMI medium containing CHIR99021 on day 4, and the step of replacing the RPMI medium containing B27 supplement on day 5; and the step of differentiating the renal organoids by replacing the RPMI medium containing B27 supplement every 2 to 3 days until day 16, may include, but is not limited to, this.

[0060] In the present invention, the concentration of CHIR99021 may be, but is not limited to, 8 to 16 μM, 8 to 14 μM, 8 to 12 μM, 10 to 16 μM, 10 to 14 μM, 10 to 12 μM, 12 to 16 μM, 12 to 14 μM, or 12 μM.

[0061] In the present invention, the step (b) is a step of producing a spheroid by co-culturing renal cancer cells with at least one selected from the group consisting of fibroblasts and endothelial cells. For example, the step may be a step of producing a spheroid by co-culturing renal cancer cells and fibroblasts; renal cancer cells and endothelial cells; or renal cancer cells, fibroblasts, and endothelial cells, but is not limited thereto. According to one embodiment of the present invention, when producing a spheroid by co-culturing renal cancer cells with fibroblasts and endothelial cells, a tumor and a tumor microenvironment can be best implemented, but is not limited thereto.

[0062] In the present invention, the co-cultivation may be performed for, but is not limited to, 18 to 30 hours, 18 to 28 hours, 18 to 26 hours, 18 to 24 hours, 20 to 30 hours, 20 to 28 hours, 20 to 26 hours, 20 to 24 hours, 22 to 30 hours, 22 to 28 hours, 22 to 26 hours, 22 to 24 hours, 24 to 30 hours, 24 to 28 hours, 24 to 26 hours, or 24 hours.

[0063] In the present invention, the ratio of renal cancer cells: fibroblasts: endothelial cells for producing the spheroids is 2:0.5 to 1.5:3 to 5, 2:0.5 to 1:3 to 5, 2:0.5 to 1:3 to 4.5, 2:0.5 to 1:3 to 4, 2:0.5 to 1:3.5 to 5, 2:0.5 to 1:3.5 to 4.5, 2:0.5 to 1:3.5 to 4, 2:0.5 to 1:4 to 5, 2:0.5 to 1:4 to 4.5, 2:0.5 to 1:4, 2:1 to 1.5:3 to 5, 2:1 to 1.5:3 to 4.5, It may be, but is not limited to, 2:1 to 1.5:3 to 4, 2:1 to 1.5:3.5 to 5, 2:1 to 1.5:3.5 to 4.5, 2:1 to 1.5:3.5 to 4, 2:1 to 1.5:4 to 5, 2:1 to 1.5:4 to 4.5, 2:1 to 1.5:4, 2:1:3 to 5, 2:1:3 to 4.5, 2:1:3 to 4, 2:1:4 to 5, 2:1:4 to 4.5, or 2:1:4.

[0064] In the present invention, step (c) may include, but is not limited to, a step of adding differentiated renal organoids to the spheroids prepared in step (b) and then culturing them to produce a renal cancer assembler. The culturing may be performed for, but is not limited to, 1 to 3 days, 1 day, 2 days, or 3 days.

[0065] In the present invention, the step (c) may be a step of producing a renal cancer spheroid by adding the differentiated renal organoid to the spheroid produced in the step (b) and culturing the differentiated renal organoid in a medium containing EGM™-2 medium and Rb medium containing B27 at a 1:1 ratio for 1 to 3 days.

[0066] In addition, the present invention provides a renal cancer assembly that implements a tumor microenvironment, characterized by being manufactured by the above method.

[0067] In addition, the present invention provides a composition for producing a renal cancer assembler, comprising a first composition, a second composition, and a third composition, wherein the first composition is a medium composition for producing and differentiating renal organoids, the second composition is a medium composition for producing spheroids, and the third composition is a medium composition for producing renal cancer assembler, wherein the first composition, the second composition, and the third composition are each used sequentially.

[0068] The above medium composition may have the same composition as the medium used in the method for producing a renal cancer assemble of the present invention, and may further include any component known in the art to improve the production efficiency of the renal cancer assemble, but is not limited thereto.

[0069] The term "assembly production" as used in the present invention includes all acts capable of creating or maintaining an assembler.

[0070] In addition, the present invention provides a kit for producing a renal cancer assembly comprising a composition and a description of a method for producing a renal cancer assembly according to the present invention.

[0071] In the present invention, a "kit" may mean, but is not limited to, a tool including a composition for producing a renal cancer assemble, a preparation that enables production of a renal cancer assemble, and an instruction manual describing a method for producing the same.

[0072] In addition to the composition and instructions according to the present invention, the kit of the present invention may include other components, compositions, solutions, devices, etc. that are typically required for a method of producing a renal cancer assembly. There is no limitation on the order in which the above materials are applied, and the application of each material may be performed simultaneously or in microseconds.

[0073] In the present invention, the kit may further include, but is not limited to, a container. The container may serve to package the material, and may also serve to store and secure the material. The material of the container may be, but is not limited to, plastic, glass, or the like.

[0074] In addition, the present invention provides a method for screening a renal cancer treatment agent, comprising the following steps: a step of producing a renal cancer assemble using the above method; and a step of treating the renal cancer assemble with a renal cancer treatment candidate substance and then confirming the therapeutic effect.

[0075] In one embodiment of the present invention, the method may further include, but is not limited to, a step of selecting a therapeutic agent for kidney cancer when a therapeutic effect is shown after treatment with the therapeutic candidate substance.

[0076] In the present invention, “screening” may mean selecting a substance having a specific desired property from a candidate group consisting of various substances through a specific manipulation or evaluation method.

[0077] For the purpose of the present invention, the screening method of the present invention may mean a series of processes including, but not limited to, a step of treating a candidate substance to a renal cancer assemble of the present invention to identify a renal cancer treatment agent that produces the best therapeutic effect in a renal cancer patient, a step of confirming the therapeutic response and effect of the renal cancer assemble of the present invention, and a step of determining the renal cancer treatment candidate agent that produces the best therapeutic effect as a renal cancer treatment agent.

[0078] The above steps for confirming the therapeutic response and effectiveness may be repeated several times depending on the therapeutic candidate substance, and may additionally include steps used in the art as a general screening method, such as adding additional substances or steps to confirm the therapeutic response and effectiveness, but are not limited thereto.

[0079] In the present invention, the term "confirmation" may correspond to, but is not limited to, the meaning of "analysis." In the present invention, "analysis" may preferably mean "measurement," qualitative analysis may mean measuring and confirming the presence or absence of a target substance for determining a therapeutic response, and quantitative analysis may mean measuring and confirming changes in the presence level (expression level) or amount of a target substance. In the present invention, analysis or measurement may be performed without limitation, including both qualitative and quantitative methods.

[0080] In the present invention, the above-mentioned renal cancer treatment candidate includes all formulations and methods that can be used to treat various types of renal cancer. For example, it may be an anticancer agent used to treat renal cancer, but is not limited thereto.

[0081] The therapeutic candidate substances to which the screening method according to the present invention is applied may include not only specific drug preparations such as the anticancer agent, but also all anticancer therapies such as radiation therapy, electricity, electromagnetic waves, etc., or chemotherapy.

[0082] The present invention provides a method for treating kidney cancer, which further comprises, in the above screening method, a step of administering the therapeutic agent to an individual in need thereof.

[0083] In addition, the present invention provides a use of a renal cancer assembly manufactured by the above method for screening a renal cancer treatment agent.

[0084] The present invention also provides a use of the composition or the kit for producing a renal cancer assembly.

[0085] When the term “comprising” is used throughout the present invention, it means that other components may be included rather than excluding other components unless specifically stated to the contrary.

[0086]

[0087] Hereinafter, preferred examples and experimental examples are presented to aid in understanding the present invention. However, the following examples and experimental examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples and experimental examples.

[0088]

[0089] [Example]

[0090] Example 1. Renal organoid differentiation

[0091] The WTC11 human iPSC cell line obtained from Catholic University of Korea was used, and cells were used between passages 30 and 60. Six-well plates were prepared by coating DMEM F-12 medium with 1% Geltrex (A14133-02, Thermo Fisher Scientific). Then, hiPSCs were plated on the geltrex-coated 6-well plates in mTeSR1 medium (85850, STEMCELL Technologies) containing 10 μM Y27632 (1254, Tocris) at a density of 15,000 cells / well (day 0). The medium was replaced with mTeSR1 containing 1.5% geltrex (day 1), mTeSR1 (day 3), RPMI containing 12 μM CHIR99021 (Tocris) (12633012, Thermo Fisher Scientific) (day 4), or RPMI containing B27 supplement (Thermo Fisher Scientific) (day 5), and RPMI containing B27 supplement was replaced every 2–3 days to promote renal organoid differentiation.

[0092]

[0093] Example 2. ccRCC, LF, and HUVEC cell culture

[0094] The human renal cell carcinoma cell line SNU-349 (00349, KCLB) was purchased from the Korea Cell Line Bank (KCLB), and lung fibroblasts (LF) (CC-2512, LONZA) and human umbilical vein endothelial cells (HUVEC) (CC-2519, LONZA) were purchased from LONZA.

[0095] The SNU-349 cell line was cultured in RPMI 1640 medium (22400089, Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (35-015-CV, CORNING) and 1% penicillin / streptomycin (Pen / Strep) (Thermo Fisher Scientific). LFs were cultured in Fibroblast Growth Medium-2 BulletKit™ (FGM™-2) (CC-3132, LONZA) containing 1% Pen / Strep. HUVECs were cultured in Endothelial Cell Growth Medium-2 BulletKit™ (EGM™-2) (CC-3162, LONZA) containing 1% Pen / Strep.

[0096]

[0097] Example 3. Assembled (in vitro)

[0098] For dissociation, 0.05% trypsin-EDTA (25-051-CI, CORNING) was used for SNU-349 cells, LF, and HUVEC. Cells were counted, and a total of 2,000 SNU-349 cells, 1,000 LF cells, and 4,000 HUVEC cells were co-seeded in each well of a 96-well round-bottom plate (34896, SPL Life Sciences) containing RPMI 1640 medium containing 1.5% Geltrex. The cells were then centrifuged at 1,000 rpm for 30 s and cultured at 37°C in 5% CO2. After 24 h, spheroids were formed, and the selected renal organoids were differentiated using a renal organoid differentiation protocol and transferred one by one to each well containing spheroids. Then, the cells were cultured for 1 to 3 days in a medium containing EGM™-2 medium and Rb medium containing B27 in a 1:1 ratio.

[0099]

[0100] Example 4. Immunofluorescence analysis

[0101] For immunofluorescence, assembles were fixed with 4% paraformaldehyde (BPP-9004, Tech&Innovation), and the samples were washed three times with PBS. Fixed assembles were blocked in 5% donkey serum (017-000-121, Jackson ImmunoResearch Labs) + 0.3% Triton-X-100 / PBS, incubated overnight in PBS containing 1% bovine serum albumin (A-3294, Sigma-Aldrich) + primary antibody, washed, and incubated with Alexa Fluor-conjugated secondary antibodies (Invitrogen), washed, and stained with DAPI or mounted in VECTASHIELD Antifade Mounting Medium (H-1000, Vector Labs). Images were then acquired using a Zeiss LSM 700 confocal microscope (Carl Zeiss) and ZEN 3.1 software.

[0102] The primary antibodies used in the present invention are as follows: anti-NPHS1 (R&D Systems, AF4269, 1:200 dilution), anti-LTL (Vector Labs, FL-1321, 1:200), anti-CAIX (Santa Cruz, sc-365900, 1:200), and anti-PECAM1 (Abcam ab9498, 1:200).

[0103]

[0104] Example 5. Real-time quantitative PCR analysis

[0105] Samples were collected and total RNA was isolated using TRIzol Reagent (Life Technologies). RNA was quantified spectrophotometrically and analyzed using SuperScript ®cDNA synthesis was performed using 3 μg of RNA with Reverse Transcriptase III (Life Technologies). For qPCR, 100 ng of cDNA was used as a template for PCR amplification using Brilliant SYBR green QPCR master mix according to the manufacturer's instructions (FastStart DNA Master SYBR Green I, Roche Molecular Biochemicals, Mannheim, Germany). In addition, serial dilutions of cDNA (1 ng / μL to 1 fg / μL) were used as templates to generate a standard curve. Standards and cDNA samples were amplified using the overlapping primers shown in Table 1 below, and standards and unknown samples were amplified in duplicate in 96-well plates.

[0106]

[0107]

[0108]

[0109] LightCycler ® The thermal profile of the device (Roche Molecular Biochemicals) was optimized using an initial denaturation at 95°C for 5 min, followed by 45 amplification cycles consisting of 95°C for 10 s, 60°C for 45 s, and 72°C for 10 s, respectively. The comparative Ct method was used to determine the expressed target mRNA level for each sample as a relative amount to the percentage of GAPDH mRNA level. The Ct ratio was measured using a LightCycler ® The analysis was performed using software (version 4.05), and specificity was verified through melting curve analysis after execution.

[0110]

[0111] Example 6. Statistical Analysis

[0112] Data are expressed as mean ± standard deviation (SD). The Kruskal-Wallis nonparametric test was used to evaluate differences between groups, and the Mann-Whitney U test (GraphPad Software, La Jolla, CA) was used to compare the control group with the treatment group and the treatment group with the combination therapy group. A P value < 0.05 was considered statistically significant.

[0113]

[0114] [Experimental Example]

[0115] Experimental Example 1. Formation of ccRCC 3D assembler using hiPSC-derived kidney organoids.

[0116] The protocol according to the present invention describes step-by-step methods for establishing ccRCC spheroids using renal organoids, growing them into 3D assemble-like structures under co-culture with ccRCC cells together with human lung fibroblasts and endothelial cells, and characterizing the ccRCC 3D assemble-like structures together with renal organoids. The current standard for mimicking the in vivo microenvironment more closely constructs in vitro models utilizing 3D culture systems. Here, we describe the use of ccRCC spheroids, hiPSC-derived renal organoids, and the assemble-like system, which offers several key advantages over existing technologies. The final protocol 4 platform contains 2K RCC, 1K LF, and 4K HUVEC with geltrex in each well of a low-attachment 96-well U-bottom plate with layered compartments for extracellular matrix (ECM). These four protocols are schematically represented in Figure 1A.

[0117] As a result, when the assembleoid was made using only RCC (Protocol 1), the cancer microenvironment was not implemented, so LF, HUVEC, and geltex were added. In addition, when the assembleoid was made with the same number of RCC, LF, and HUVEC cells (2K:2K:2K) (Protocol 2), blood vessel formation was poor, so the cell number was adjusted. When making the assembleoid, activated T cells were added (Protocol 3) to see the responsiveness to immunotherapy, but the T cells did not work well. Therefore, when the assembleoid was made using RCC (2K), LF (1K), HUVEC (4K), and geltrex (Protocol 4), it was confirmed that it best represented the cancer microenvironment, so the assembleoid was made under the conditions of Protocol 4.

[0118] To generate renal organoids, human iPSCs were differentiated into renal organoids using an adherent cell culture protocol. Specifically, each well of a 6-well plate was coated with diluted Geltrex, then separated, and undifferentiated human iPSCs were evenly distributed. After 24 hours, 1.5% Geltrex was added, and hiPSCs were intercalated. The sandwiched hiPSCs formed dense, ball-shaped colonies within 48 hours, with an internal cavity developing. On day 3, the cells were treated with CHIR and then supplied with appropriate media every 2–3 days to promote renal organoid differentiation, as shown in Figure 1B. Preliminary studies were then conducted to establish heterogeneous 3D cocultures of ccRCC cells with human lung fibroblasts (LF) and endothelial cells (EC), as well as spheroid cultures of distinct human clear cell renal carcinoma (ccRCC) cell lines. To further enhance the complexity of spheroid models that more realistically mimic the tumor microenvironment, we generated assembles of renal organoids with ccRCC lineages, including lung fibroblasts and endothelial cells, directly onto the spheroids. This process is illustrated in Figure 1b. As a result, a gel was formed surrounding and encapsulating the spheroids with well-defined boundaries as early as day 1. Brightfield microscopy revealed that the assembles displayed a combination of ccRCC spheroid compartments and renal compartments, as shown in Figure 1c, confirming that the assembles generated by coculture of ccRCC spheroids with renal organoids and LF+ECs constituted a 3D tumor model.

[0119]

[0120] Experimental Example 2. 3D Tumor Microenvironment Model of Assembled Cells

[0121] Kidney organoids previously demonstrated the presence of nascent vascular endothelial cells lining glomerular structures, but lacked appropriate vascular patterning. To confirm that assembleoids were formed from serial passages of kidney organoids and ccRCC spheroids and that they could be cultured and grown during reconstitution, confocal imaging was used for NPHS1, LTL structures in kidney organoids, and the tumor marker CAIX, as well as PECAM1-positive vascular infiltration into the assembleoids. NPHS1 (red) represents podocytes, LTL (white) represents proximal tubules, CAIX (green) represents clear cell renal cell carcinoma, and PECAM1 (green) represents endothelial cells.

[0122] Carbonic anhydrase IX (CAIX) is a well-characterized enzyme in renal cell carcinoma. Its expression is regulated by hypoxia-inducible factor 1 alpha, known to interfere with hypoxia processes. As shown in Figure 2a, immunostaining of assembleoids revealed that CAIX expression in assembleoids was rarely observed in renal organoids, whereas tumorigenesis was observed around ccRCC spheroids. Furthermore, as shown in Figure 2b, vascularization was observed around ccRCC, but PECAM1-positive vascular infiltration into assembleoids was not observed in renal organoids. Figure 2b shows the results of PECAM1 vascular infiltration into assembleoids, with PECAM1 observed in some parts of renal organoids. These findings suggest that renal organoids interact with ccRCC spheroids to create a 3D tumor microenvironment model.

[0123]

[0124] Experimental Example 3. Confirmation of Gene Expression in ccRCC Tumor Assemblies

[0125] Each ccRCC and renal organoid specimen was screened using a panel of markers that identified relative mRNA gene expression levels. As shown in Figure 3 , consistent with the changes observed in confocal images, mRNA expression of NPHS1 (podocyte gene), ECAD (distal tubule gene), GGT1 (proximal tubule gene), and CDH16 (distal tubule gene) was virtually absent in ccRCC spheroids with or without LF+EC.

[0126] However, when tumor-associated genes were identified, we found that expression of mRNAs of ccRCC standards, including CA9, PECAM1, ENPP3, FABP7, KISS1R, OCT4, and SIX1, but not LOX and EGLN3, was significantly increased in the assemblers compared to ccRCC spheroids with or without LF+EC (n=3, *, vs. renal organoids; #, vs. RCC spheroids; †, vs. RCC spheroids+LF+EC, post hoc Mann-Whitney U test).

[0127] The above results showed that ccRCC assemblages express not only RCC genes but also renal genes, indicating that ccRCC assemblages simultaneously recapitulate RCC tumors and the tumor microenvironment (TME).

[0128]

[0129] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0130]

[0131] The renal cancer spheroid according to the present invention is produced by co-culturing RCC, endothelial cells, and fibroblasts to produce RCC spheroids, which are then assembled with renal organoids. It was confirmed that the renal cancer spheroid expresses not only tumor-related genes but also renal genes, and thus the renal cancer spheroid according to the present invention has the effect of simultaneously reproducing a renal tumor and a tumor microenvironment (TME). Therefore, the renal cancer spheroid according to the present invention can be usefully used for drug screening and biomarker evaluation for renal cancer, and thus its industrial applicability is recognized.

Claims

1. A method for manufacturing a renal cancer assemble, comprising the following steps: (a) a step of preparing and differentiating renal organoids; (b) a step of producing a spheroid by co-culturing renal cancer cells with at least one selected from the group consisting of fibroblasts and endothelial cells; and (c) a step of adding the differentiated renal organoid to the spheroid manufactured in step (b) to manufacture a renal cancer assembler.

2. In paragraph 1, A method for producing a renal cancer assembler, characterized in that the renal organoid is produced using induced pluripotent stem cells (iPSCs).

3. In paragraph 1, A method for producing a renal cancer spheroid, characterized in that the renal organoids and spheroids are cultured in a medium containing geltrex.

4. In paragraph 1, A method for producing a renal cancer assembler, characterized in that the renal cancer cells are clear cell renal cell carcinoma (ccRCC) cells.

5. In paragraph 1, A method for producing a renal cancer assembler, characterized in that the fibroblasts are lung fibroblasts.

6. In paragraph 1, A method for producing a renal cancer assembler, characterized in that the endothelial cells are human umbilical vein endothelial cells (HUVEC).

7. In paragraph 1, A method for producing a renal cancer assembler, characterized in that the renal organoid is cultured in mTeSR1 or RPMI medium.

8. A method for manufacturing a renal cancer assembler, characterized in that the manufactured renal assembler of claim 1 implements a tumor microenvironment.

9. A renal cancer assembler implementing a tumor microenvironment, characterized by being manufactured by the method of any one of claims 1 to 8.

10. Comprising a first composition, a second composition, and a third composition, The above first composition is a medium composition for producing and differentiating the renal organoids of the first claim, The above second composition is a medium composition for producing the spheroid of the first clause, The third composition is a medium composition for manufacturing the kidney cancer assembly of the first clause, A composition for producing a renal cancer assembly, wherein the first composition, the second composition, and the third composition are each used sequentially.

11. A kit for producing a renal cancer assemble, comprising a description of the method of Article 1 and a composition of Article 10.

12. Use of the renal cancer assembly manufactured by the method of Article 1 for screening renal cancer therapeutic agents.

13. Use of the composition of claim 10 or the kit of claim 11 for producing a renal cancer assemble.

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