Structure and method for transplanting tumor tissue

Abstract

To provide a structure effective for engraftment of tumor tissue.SOLUTION: Provided is a structure including a hydrogel and a tumor tissue covered with the hydrogel. The hydrogel is composed of gelatin crosslinked with a polyether.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a structure in which tumor tissue is covered with a hydrogel, a method for transplanting tumor tissue using said structure, and a scaffold material suitable for said structure. [Background technology]

[0002] Traditionally, anticancer drug discovery involves in vitro screening using cancer cell lines established from patients and in vivo testing using subcutaneously implanted cancer cell lines in rodents before proceeding to clinical trials. However, even drug candidates that demonstrate efficacy in preclinical trials often fail to demonstrate sufficient efficacy when administered to humans. One possible reason for this is that cancer cell lines adapt to an artificial environment during establishment and lose their intrinsic properties through repeated passage in nutrient-rich media. To address this issue, interest has grown in patient-derived xenograft (PDX) models as an experimental model that recapitulates the intrinsic properties of tumors (Non-Patent Document 1). PDX models are generated by implanting a portion of patient-derived tumor tissue into mice and are expected to be a tool for accurately predicting drug efficacy in clinical trials.

[0003] However, because tumor tissue as is does not necessarily have a high engraftment rate in model animals such as rodents, scaffolds that promote tumor tissue engraftment are used. Among these, Matrigel basement membrane matrix (hereinafter simply referred to as "Matrigel") is widely used (Non-Patent Document 2). "Matrigel (registered trademark)" is a product sold by BD Biosciences and other companies, and is a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins, and contains laminin (main component), collagen IV, heparan sulfate proteoglycan, entactin / nitogen, etc.

[0004] However, the effectiveness of Matrigel as a scaffold to support engraftment varies depending on the type of cancer, and research on bladder cancer, for example, has even suggested that it may have a negative effect (Non-Patent Document 3). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Eugenia R. Zenella et al., Nature Reviews Clinical Oncology 2022 Nov.; 19(11): 719-732. doi: 10.1038 / s41571-022-00682-6 [Non-patent document 2] John W. Cassidy et al., Cancer Research 2015 Aug 1; 75(15): 2963-2968. doi: 10.1158 / 0008-5472.CAN-15-0727 [Non-patent document 3] Yuki Kita et al., Blader Cancer, vol. 6, no. 2, pp. 131-141, 2020. doi: 10.3233 / BLC-200281 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, an object of the present invention is to provide a structure that is effective for tumor tissue engraftment. Another object of the present invention is to provide a method for transplanting tumor tissue using the structure and a scaffold material suitable for the structure. [Means for solving the problem]

[0007] As a result of intensive research to achieve the above-mentioned object, the inventors discovered that a hydrogel composed of gelatin cross-linked with polyether is effective as a scaffold to aid in the engraftment of tumor tissue, and thus completed the present invention.

[0008] Therefore, the structure of the present invention is a structure comprising a hydrogel and tumor tissue covered with the hydrogel, wherein the hydrogel is composed of gelatin cross-linked with polyether.

[0009] In a preferred embodiment of the structure of the present invention, the hydrogel forms a chemical bond with a protein in the tumor tissue.

[0010] Another preferred embodiment of the structure of the present invention is a polymer having a polyalkylene glycol skeleton.

[0011] In another preferred embodiment of the structure of the present invention, the polyether comprises a di-, tri-, tetra- or octa-branched polyethylene glycol.

[0012] In another preferable embodiment of the structure of the present invention, the polyether has a total of one or more electrophilic functional groups on the side chain or at the end.

[0013] In another preferred embodiment of the structure of the present invention, the electrophilic functional group is selected from the group consisting of an N-hydroxysuccinimidyl group, a maleimide group, and a sulfosuccinimidyl group.

[0014] In another preferred embodiment of the structure of the present invention, the tumor tissue includes bladder cancer tissue.

[0015] Furthermore, the scaffold material of the present invention is a scaffold material for use in transplanting tumor tissue, and is a scaffold material containing a hydrogel composed of gelatin cross-linked with polyether.

[0016] Furthermore, the method for transplanting tumor tissue of the present invention is a method for transplanting tumor tissue, which comprises using the above-described structure of the present invention to transplant tumor tissue covered with the hydrogel into a subject.

[0017] A preferred embodiment of the method for transplanting a tumor tissue of the present invention includes promoting angiogenesis around the transplanted tumor tissue.

[0018] In another preferable embodiment of the tumor tissue transplantation method of the present invention, the subject is a non-human animal. [Effects of the Invention]

[0019] The structure of the present invention can provide a structure that is effective for tumor tissue engraftment. Furthermore, the method for transplanting tumor tissue of the present invention can provide a method for transplanting tumor tissue using the structure. Furthermore, the scaffold material of the present invention can provide a scaffold material suitable for the structure. [Brief explanation of the drawings]

[0020] [Figure 1] This figure shows the change in graft volume obtained from the experiment conducted in the Examples. In the figure, the circles represent the volume of the graft in Group 1 (tumor tissue only), the squares represent the volume of the graft in Group 2 (Matrigel), and the triangles represent the volume of the graft in Group 3 (GP hydrogel). [Figure 2] Representative images obtained by microscopic observation in the histopathological examination performed in the examples are shown. Panels A to D on the left show the overall image, and panels A to D on the right show enlarged images. In the figure, A is a microscopic image of TG2 bladder cancer tissue before cryopreservation. B is a microscopic image of a tissue fragment obtained on the final day (day 46) of the experiment for Group 1 (tumor tissue only). C is a microscopic image of a tissue fragment obtained on the final day (day 46) of the experiment for Group 2 (Matrigel). D is a microscopic image of a tissue fragment obtained on the final day (day 46) of the experiment for Group 3 (GP hydrogel). [Figure 3]These are representative images obtained by microscopic observation in the histopathological examination performed in the examples, focusing particularly on blood vessels. In the figure, A is a microscopic image of TG2 bladder cancer tissue before cryopreservation. B is a microscopic image of a tissue fragment obtained on the final day (day 46) of the experiment for Group 1 (tumor tissue only). C is a microscopic image of a tissue fragment obtained on the final day (day 46) of the experiment for Group 2 (Matrigel). D is a microscopic image of a tissue fragment obtained on the final day (day 46) of the experiment for Group 3 (GP hydrogel). The lower images in A, B, C, and D show enlarged views of the red squares in the upper images. In each image, the arrow indicates a blood vessel with maintained morphology, and the triangle indicates a blood vessel with thrombus formation. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention relates to a structure in which tumor tissue is covered with a hydrogel, a method for transplanting tumor tissue using the structure, and a scaffold material suitable for the structure.

[0022] The structure of the present invention is a structure comprising a hydrogel and tumor tissue covered with the hydrogel.

[0023] In the present invention, a hydrogel refers to a gel containing water as a dispersion medium. The amount of water contained in the hydrogel is, for example, 82 to 98.5% by mass, preferably 88 to 98% by mass, and more preferably 91 to 94% by mass. The dispersion medium is not limited to water, and a water-soluble organic solvent may be used in combination. Examples of water-soluble organic solvents include alcohols such as ethanol. Furthermore, various aqueous buffer solutions or saline solutions capable of adjusting the pH are preferably used as the dispersion medium, and phosphate-buffered saline is more preferably used.

[0024] A gel is generally a dispersion of polymers that has high viscosity and lost fluidity, and is a state in which the storage modulus G' and loss modulus G'' satisfy the relationship G' ≥ G'', and is typically a substance with a three-dimensional network structure. The storage modulus G' and loss modulus G'' can be obtained by dynamic viscoelasticity testing using a rheometer, and one example of measurement conditions is a temperature of 25°C, a shear amplitude of 1%, and a vibration frequency of 1.0 Hz.

[0025] In the present invention, the hydrogel is composed of gelatin cross-linked with polyether. Here, cross-linking refers to chemical cross-linking. Covering tumor tissue with such a hydrogel can suppress necrosis of the transplanted tumor tissue and promote angiogenesis around the transplanted tumor tissue. Therefore, such a hydrogel is effective as a scaffold for patient-derived xenograft models.

[0026] The amount of polyether-crosslinked gelatin contained in the hydrogel is, for example, 1 to 6 mass %, preferably 1 to 4 mass %, and more preferably 1 to 3 mass %.

[0027] Gelatin is a widely used material in tissue engineering and cell therapy due to its availability, biocompatibility, and good cell adhesion. However, gelatin itself is highly soluble in water at 37°C and does not function as a support for retaining materials at the desired site. Therefore, in this invention, gelatin cross-linked with polyether is used.

[0028] The polyether used for crosslinking gelatin is preferably a polymer having a structure in which alkylene glycols such as ethylene glycol and propylene glycol are polymerized. Examples include linear polyethylene glycol, linear polypropylene glycol, linear polybutylene glycol, and polyethers in which alkylene glycols are polymerized to some or all of the hydroxyl groups of a polyhydric alcohol (e.g., glycerin, diglycerin, pentaerythritol, sorbitol, etc.) (e.g., polyethylene glycol chain, polypropylene glycol chain, polybutylene glycol chain, etc.). The polyether used for crosslinking gelatin is more preferably a polyether having multiple polyalkylene glycol chains such as polyethylene glycol chains, polypropylene glycol chains, polybutylene glycol chains, etc. (so-called branched polyalkylene glycol).

[0029] In the present invention, the polyether used for cross-linking gelatin is more preferably a polymer having a polyalkylene glycol skeleton, and even more preferably a polymer having a polyethylene glycol skeleton. That is, in the present invention, the hydrogel is more preferably composed of gelatin cross-linked with a polymer having a polyalkylene glycol skeleton, and even more preferably composed of gelatin cross-linked with a polymer having a polyethylene glycol skeleton.

[0030] In this specification, the term "polyalkylene glycol backbone" refers to a structure in which -RO- structural units (R is an alkylene group) are repeated, and is also referred to as a "polyalkylene glycol chain." The term "polyethylene glycol backbone" refers to a structure in which -CH2CHO- structural units are repeated, and is also referred to as a "polyethylene glycol chain."

[0031] In this specification, the term "polyalkylene glycol" refers to both linear polyalkylene glycol and branched polyalkylene glycol. The term "polyethylene glycol" also refers to both linear polyethylene glycol and branched polyethylene glycol. This also applies to "polypropylene glycol" and the like.

[0032] Furthermore, a hydrogel composed of gelatin cross-linked with polyethylene glycol (PEG) is also called a "GP hydrogel."

[0033] Polyethylene glycol (PEG) is preferably a polymer having multiple polyethylene glycol backbones in the molecule, for example, a polymer having two polyethylene glycol backbones is also called a bi-branched PEG, a polymer having three polyethylene glycol backbones is also called a tri-branched PEG, a polymer having four polyethylene glycol backbones is also called a tetra-branched PEG, a polymer having eight polyethylene glycol backbones is also called an octa-branched PEG, etc. A polymer having multiple polyethylene glycol backbones in the molecule is also called a "multi-arm PEG."

[0034] The polyether used for cross-linking gelatin preferably comprises a di-, tri-, tetra- or octa-branched polyethylene glycol.

[0035] The polyether used for crosslinking gelatin preferably has an electrophilic functional group so that it can be easily chemically bonded to the amino acid residues of gelatin. The polyether used for crosslinking gelatin can have a total of one or more electrophilic functional groups on the side chain or end. The polyether can have a hydroxyl group derived from the raw material, and by utilizing this hydroxyl group, the electrophilic functional group can be easily introduced. The electrophilic functional group is preferably introduced to the end of a structure in which alkylene glycol, such as a polyethylene glycol backbone, is polymerized via a linker moiety as necessary.

[0036] Examples of electrophilic functional groups include maleimide, N-hydroxysuccinimidyl (NHS), succinimidyl carbonate, sulfosuccinimidyl, phthalimidyl, imidazolyl, acryloyl, nitrophenyl, and -CO2PhNO2 (where Ph represents o-, m-, or p-phenylene). Those skilled in the art can appropriately use known electrophilic functional groups. Preferably, the electrophilic functional group is selected from the group consisting of N-hydroxysuccinimidyl, maleimide, and sulfosuccinimidyl. When the polyether used for crosslinking gelatin has multiple electrophilic functional groups, the electrophilic functional groups may be the same or different, but it is preferable that they are the same.

[0037] The polyether used for crosslinking gelatin preferably has an electrophilic functional group, more preferably has an electrophilic functional group selected from the group consisting of an N-hydroxysuccinimidyl group, a maleimide group, and a sulfosuccinimidyl group, and particularly preferably has an N-hydroxysuccinimidyl (NHS) group from the viewpoint of binding to lysine residues in gelatin.

[0038] The polyether used for crosslinking gelatin preferably has a weight average molecular weight of 1×10 3 ~1×10 5 More preferably, it is in the range of 0.5×10 4 ~5×10 4 and more preferably in the range of 1×10 4 ~2×10 4 The range is.

[0039] In the present invention, the weight average molecular weight of the polyether is a weight average molecular weight calculated as polyethylene glycol by gel permeation chromatography (GPC).

[0040] Non-limiting examples of preferred PEGs having an electrophilic functional group at their termini include compounds represented by the following formula (I), which have four polyethylene glycol backbones and an N-hydroxysuccinimidyl (NHS) group at the termini of each polyethylene glycol backbone via a linker moiety: [ka]

[0041] In the above formula (I), R 21 ~R 24 are each the same or different (preferably the same), and are a C1-C7 alkylene group, a C2-C7 alkenylene group, -NH-R 25 -, -CO-R 25 -, -R 26 -OR 27 -, -R 26 -NH-R 27 -, -R 26 -CO2-R 27 -, -R 26 -CO2-NH-R 27 -, -R 26 -CO-R 27 - or -R 26 -CO-NH-R 27 - where R 25 represents a C1-C7 alkylene group. 26 represents a C1-C3 alkylene group, and R 27 represents a C1-C5 alkylene group. 21 ~R 24 is a linker moiety connecting the N-hydroxysuccinimidyl (NHS) group and the polyethylene glycol backbone.

[0042] In the above formula (I), n 21 ~n 24 may be the same or different. 21 ~n 24 The closer the values ​​of n are, the better, and it is particularly preferable that they are the same. 21 ~n 24is, for example, an integer value of 5 to 600, preferably 25 to 250, more preferably 50 to 120, and even more preferably 110 to 120.

[0043] As used herein, the term "C1-C7 alkylene group" refers to an alkylene group having from 1 to 7 carbon atoms, which may have a branch, and refers to a linear C1-C7 alkylene group or a C2-C7 alkylene group having one or more branches (having from 2 to 7 carbon atoms, including the branches). Examples of C1-C7 alkylene groups include -CH2-, -(CH2)2-, -(CH2)3-, -CH(CH3)-, -(CH2)3-, -(CH(CH3))2-, -(CH2)2-CH(CH3)-, -(CH2)3-CH(CH3)-, -(CH2)2-CH(C2H5)-, -(CH2)6-, -(CH2)2-C(C2H5)2-, and -(CH2)3C(CH3)2CH2-. The term "C1-C3 alkylene group" refers to an alkylene group having 1 to 3 carbon atoms, which may be branched, and the term "C1-C5 alkylene group" refers to an alkylene group having 1 to 5 carbon atoms, which may be branched.

[0044] As used herein, the term "C2-C7 alkenylene group" refers to a linear or branched alkenylene group having 2 to 7 carbon atoms and having one or more double bonds in the chain, and examples thereof include divalent groups having one or more double bonds formed by removing hydrogen atoms from adjacent carbon atoms of the alkylene group.

[0045] In this specification, alkylene groups and alkenylene groups may have one or more optional substituents. Examples of the substituents include, but are not limited to, alkoxy groups, halogen atoms (which may be fluorine, chlorine, bromine, or iodine atoms), amino groups, mono- or di-substituted amino groups, substituted silyl groups, acyl groups, and aryl groups. When an alkylene group or alkenylene group has two or more substituents, the substituents may be the same or different. Furthermore, when the substituent is a substituent containing a hydrocarbon group (e.g., an alkoxy group, an acyl group, an aryl group, etc.), the hydrocarbon group may also have one or more optional substituents.

[0046] In addition, in this specification, the expression "may have a substituent" does not specify the type, position, or number of the substituent.

[0047] For crosslinking of gelatin, the polyether is preferably used in an amount of 0.25 to 5 parts by mass, more preferably 2 to 3 parts by mass, per 100 parts by mass of gelatin.

[0048] The hydrogel preferably has an electrophilic functional group. The hydrogel having such a functional group can bind to and adhere to proteins in tumor tissue, allowing it to stably exist around the tumor tissue. Here, the electrophilic functional group is preferably a functional group derived from the polyether used for crosslinking gelatin, and is an unreacted functional group that was not used for crosslinking gelatin. The electrophilic functional group is as described in the description of the polyether used for crosslinking gelatin.

[0049] In the structure of the present invention, the hydrogel preferably forms a chemical bond with a protein in the tumor tissue.

[0050] The hydrogel can be produced by mixing the components constituting the hydrogel, namely gelatin, water, and polyether, which causes crosslinking of the gelatin and ultimately gelation.

[0051] A preferred method for producing a hydrogel involves mixing a solution X containing gelatin and water with a solution Y containing polyether and water. By adjusting the concentrations of gelatin and polyether in the solutions and the pH of the solutions, the time from mixing of solution X and solution Y until gelation (i.e., the time until the storage modulus G' = loss modulus G'') can be appropriately adjusted. In this specification, gelation is also referred to as solidification.

[0052] The concentration of gelatin in solution X is preferably in the range of 20 to 120 g / L, more preferably in the range of 20 to 80 g / L, and even more preferably in the range of 20 to 60 g / L.

[0053] The concentration of the polyether in the solution Y is preferably in the range of 10 to 240 g / L, more preferably in the range of 20 to 160 g / L, and even more preferably in the range of 60 to 120 g / L.

[0054] The pH of solution X and solution Y is preferably in the range of 6.5 to 11.5, more preferably in the range of 7.0 to 10.5, and even more preferably in the range of 7.0 to 8.5.

[0055] Solution X and Solution Y contain water, and may contain a water-soluble organic solvent in combination. To prepare Solution X, it is preferable to use an aqueous buffer solution capable of adjusting the pH, and it is more preferable to use phosphate buffered saline.

[0056] It is preferable to mix Solution X and Solution Y so that the amount of polyether is preferably in the range of 0.25 to 5 parts by mass, more preferably 2 to 3 parts by mass, per 100 parts by mass of gelatin.

[0057] The temperatures of solution X and solution Y when they are mixed may be such that gelatin and polyether are dissolved in each solution and solution X and solution Y have fluidity. Although the temperatures of solution X and solution Y when they are mixed may be different from each other, it is preferable to mix solution X and solution Y at the same temperature.

[0058] In the present invention, in order to produce a structure, it is preferable to cover the tumor tissue with a mixture of the components that make up the hydrogel, i.e., gelatin, water, and polyether, before gelation, preferably a mixture in a state where the viscosity is increasing and the fluidity is decreasing.

[0059] The structure of the present invention includes tumor tissue covered with a hydrogel. Tumor tissue is a cell population containing autonomously growing cells, and includes both benign and malignant tumor tissue. However, considering the intended use of the structure of the present invention, it is more suitable to use malignant tumor tissue as the tumor tissue, and more suitable to use malignant tumor tissue derived from humans. Examples of malignant tumors include lung cancer, breast cancer, gastric cancer, colon cancer, uterine cancer, ovarian cancer, head and neck cancer, bladder cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, and angiosarcoma. In one embodiment of the structure of the present invention, the tumor tissue includes bladder cancer tissue. Note that, although the examples demonstrate the effectiveness of the structure of the present invention against bladder cancer tissue, the tumor tissue is not limited to this.

[0060] The structure of the present invention has a three-dimensional structure due to the hydrogel, and a desired structure can be obtained by scraping off excess hydrogel, etc. Examples of the shape of the structure of the present invention include a sphere, granule, cube, rectangular parallelepiped, etc.

[0061] The structure of the present invention has a volume of 1 to 125 mm 3 (specifically, equivalent to 1 mm square to 5 mm square) is preferable, and 3In the structure of the present invention, the amount of tumor tissue is preferably in the range of 25 to 87% by volume of the structure (specifically, equivalent to a hydrogel coating with a thickness of 0.05 to 1 mm), and more preferably in the range of 40 to 77% by volume (specifically, equivalent to a hydrogel coating with a thickness of 0.5 to 0.1 mm).

[0062] The structure of the present invention can be produced by covering tumor tissue with a mixture of the components that make up the hydrogel, i.e., gelatin, water, and polyether, before gelation, preferably a mixture in a state where the viscosity has increased and the fluidity is decreasing, during the hydrogel production process.

[0063] The method for producing a structure of the present invention preferably includes the following steps. Step 1: A mixture is prepared by mixing a solution X containing gelatin and water with a solution Y containing a polyether and water. Step 2: A portion of the mixture prepared in step 1 is placed on a support. Step 3: Place the tumor tissue on the mixture placed on the support in step 2. Step 4: The remainder of the mixture prepared in step 1 is placed on top of the tumor tissue placed on the mixture in step 3, and the tumor tissue is covered with the mixture. Step 5: The structure obtained in step 4 is left standing on the support until it solidifies. Here, steps 2 to 4 are carried out before the mixture prepared in step 1 gels (ie, solidifies).

[0064] In step 1, the "solution X containing gelatin and water" and the "solution Y containing polyether and water" are as described in the explanation of the method for producing the hydrogel.

[0065] The mixture prepared in step 1 is adjusted so that the time from mixing Solution X and Solution Y until gelation (i.e., the time until storage modulus G' = loss modulus G'') is preferably 0.5 to 5 minutes, taking into account ease of pipetting, and more preferably 0.5 to 3 minutes, taking into account damage to tumor tissue.

[0066] In step 2, the support is not particularly limited, but is preferably a hydrophobic film, for example, a plastic film such as Parafilm.

[0067] In step 2, the method for placing the mixture prepared in step 1 on the support is not particularly limited, and examples thereof include coating and dropping.

[0068] In step 3, the means for placing the tumor tissue on the mixture is not particularly limited, and examples thereof include placement with tweezers.

[0069] In step 4, the means for placing the mixture prepared in step 1 on the tumor tissue is not particularly limited, and examples include painting and dropping.

[0070] In step 4, the mixture prepared in step 1 is also placed on the surface of the mixture placed on the support in step 2 that is not covered with tumor tissue.

[0071] In step 5, the structure is preferably left to stand at a temperature of 15°C to 30°C.

[0072] The method for transplanting tumor tissue of the present invention comprises transplanting tumor tissue covered with a hydrogel using the above-described structure of the present invention into a subject (particularly a non-human animal).

[0073] In the present invention, non-human animals include, for example, non-human mammals (e.g., rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, sheep, goats, pigs, horses, cows, monkeys, etc.), and among these, rodents such as mice, rats, and guinea pigs are preferred, with mice and rats being particularly preferred.

[0074] In the present invention, the method for transplanting tumor tissue into a non-human animal is not particularly limited, and examples include a method in which a transplantation needle is inserted subcutaneously and the structure of the present invention filled in the transplantation needle is delivered subcutaneously to the non-human animal.

[0075] The method for transplanting tumor tissue of the present invention preferably includes promoting angiogenesis around the transplanted tumor tissue.

[0076] The tumor tissue transplantation method of the present invention can improve the growth and survival of transplanted tumor tissue by using the structure of the present invention for transplantation, and is therefore suitable for use in patient-derived xenograft models.

[0077] The scaffold material of the present invention is a scaffold material for use in transplanting tumor tissue, and includes a hydrogel composed of gelatin cross-linked with polyether. Here, the "hydrogel composed of gelatin cross-linked with polyether" is as described in the explanation of the "hydrogel" constituting the structure of the present invention. [Example]

[0078] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples in any way.

[0079] Materials and Methods 1. Tumor Tissue In this example, the tumor tissue used was highly malignant bladder cancer tissue maintained subcutaneously as TG1 (first passage, Trans Generation 1) in immunodeficient mice at HOIST Co., Ltd. The experiments performed in this example were approved by the ethics committee of HOIST Co., Ltd.

[0080] 2.Animals CB-17 / IcrHsd-Prkdcscid mice (Japan SLC, Inc.) were obtained at 6 weeks of age and maintained under specific pathogen-free conditions. These immunodeficient mice were used in experiments at 7 weeks of age. All experiments and procedures related to the care and treatment of mice used in this example were conducted in accordance with the Animal Care and Experiment Committee of Japan SLC, Inc.

[0081] 3. Preparation of Frozen Stocks of Patient-derived Bladder Cancer Tissue Bladder cancer tissue (TG1 bladder cancer tissue) was transported from HOIST Co., Ltd. to Japan SLC Co., Ltd. at 4°C. TG1 bladder cancer tissue was minced on ice with a razor into cubes with sides of approximately 2 mm per sample block and then subcutaneously implanted into immunodeficient mice. The implanted tissue was allowed to grow in the mice until its volume increased approximately 10-fold, after which the tissue was excised from the mice to obtain TG2 bladder cancer tissue. A portion of the obtained TG2 bladder cancer tissue was used for histopathological examination. The remaining tissue was again minced into cubes with sides of approximately 2 mm per sample block and frozen using CELLBANKER® (Xenogen Pharma Co., Ltd.) according to the manufacturer's instructions.

[0082] 4. Preparation of a construct with GP hydrogel covering tissue fragments for transplantation A gelatin solution was prepared by dissolving APAT high-grade gelatin (Nippi Corporation) in D-PBS(-) (FUJIFILM Wako Pure Chemical Industries, Ltd.) to a concentration of 80 g / L. The gelatin solution was stored in a refrigerator until use. The gelatin solution, which had lost fluidity due to refrigeration, was warmed in a 37°C thermostatic water bath for 30 minutes immediately before use and then returned to room temperature (approximately 25°C), whereby it regained fluidity. The gelatin solution thus treated was diluted 2-fold with a HEPES solution prepared by dissolving 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) (Dojindo Laboratories, Inc.) in water for injection (Hikari Pharmaceutical Co., Ltd.) to a concentration of 0.2 mol / L (pH 8.2). Separately, 4-arm PEG-SC (PEG-NHS, molecular weight 10,000) manufactured by SINOPEG, a 4-branched PEG with N-hydroxysuccinimidyl groups at its termini, was dissolved in water for injection (Hikari Pharmaceutical Co., Ltd.) to a concentration of 100 g / L. A 1:1 mixture of the resulting 40 g / L gelatin solution and 100 g / L PEG-NHS solution was prepared. 20 μL of the unsolidified mixture was placed on parafilm, and a tissue fragment for transplantation was placed on top of the unsolidified mixture. Another 20 μL of the unsolidified mixture was then dropped onto the tissue fragment for transplantation. The mixture was incubated at room temperature (approximately 25°C) for 5 minutes, yielding a construct in which the tissue fragment for transplantation was covered with GP hydrogel. Excess gel was scraped off with a razor and used for subcutaneous implantation in mice.

[0083] 5. Preparation of Matrigel-soaked Tissue Fragments for Implantation A sufficient amount of liquid BD Matrigel basement membrane matrix (BD Biosciences) was placed in a plastic dish, and the tissue fragments were placed therein, ensuring that the tissue fragments were completely submerged in the liquid. The treated tissue fragments were then used for subcutaneous implantation in mice. The Matrigel containing the tissue fragments was kept on ice until transplantation.

[0084] 6. Subcutaneous Implantation of Patient-Derived Bladder Cancer Tissue and Evaluation of Changes in Implanted Tissue Size The TG2 bladder cancer tissue (approximately 2 mm cubes per sample block) frozen as described in "3. Preparation of frozen stocks of patient-derived bladder cancer tissue" above was thawed in a 37°C water bath on the day of transplantation. The thawed TG2 bladder cancer tissue (hereinafter also referred to as tissue fragments) was immersed in phosphate-buffered saline (PBS, Thermo Fisher Scientific) and gently rinsed. Excess PBS was wiped off from the rinsed tissue fragments, and these were used as tissue fragments for transplantation. The tissue fragments for transplantation were then divided into three groups and subjected to different treatments. Each group contained four samples. The first group was a group in which the transplant tissue fragment itself was transplanted subcutaneously into mice as a sample, without using a scaffold such as GP hydrogel or Matrigel. The second group was a group in which the samples (tissue fragments for transplantation soaked in Matrigel) prepared according to the above "5. Preparation of tissue fragments for transplantation soaked in Matrigel" were subcutaneously transplanted into mice. The third group was a group in which a sample (a structure in which a tissue fragment for transplantation was covered with GP hydrogel) prepared according to the above "4. Preparation of a structure in which a tissue fragment for transplantation was covered with GP hydrogel" was subcutaneously implanted into mice. Samples from Group 1 (tumor tissue itself), Group 2 (tissue fragments for transplantation soaked in Matrigel), and Group 3 (constructs of tissue fragments for transplantation covered with GP hydrogel) were each subcutaneously delivered to 7-week-old immunodeficient mice (described above in "2. Animals") using a transplant needle (inner diameter 3.5 mm, length 85 mm, product number KN-391-35, Natsume Seisakusho Co., Ltd.). After delivery, the mice were not sutured. After transplantation, the major and minor diameters of the tissue fragments were measured using a digital caliper (Mitutoyo Corporation). Based on the measured length, the volume of the transplanted tissue fragments (hereinafter also referred to as grafts) was calculated using the following formula 1. Graft volume (mm 3 ) = major axis (mm) × minor axis (mm) × minor axis (mm) × 0.5 (Equation 1) However, the volume of the graft in Group 3 includes the volume of the hydrogel. In Group 2, the surface of the graft was coated with a very thin layer of Matrigel, but the increase in volume due to this coating was negligible.

[0085] 7. Histopathological Examination On the final day (day 46) of the experiment conducted in "6. Subcutaneous implantation of patient-derived bladder cancer tissue and evaluation of changes in size of implanted tissue fragments," mice were euthanized, and subcutaneous tumor tissue was excised for histopathological evaluation. The excised tissue was fixed in 10% neutral buffered formalin, and hematoxylin-eosin stained specimens were prepared and examined histopathologically.

[0086] (result) 8. Size change of transplanted tissue fragments The changes in the volume of the grafts obtained from the experiment performed in "6. Evaluation of subcutaneous transplantation of patient-derived bladder cancer tissue and changes in the size of the transplanted tissue fragments" above are shown in Figure 1. On day 3 after implantation, the volume of the tissue fragments in Group 3 (GP hydrogel) was approximately 2.5 times larger than that in Groups 1 (tumor tissue only) and 2 (Matrigel). In Groups 1 (tumor tissue only) and 2 (Matrigel), the size of the tissue fragments remained almost unchanged until day 17 after implantation, then rapidly increased. In Group 3 (GP hydrogel), the size of the tissue fragments decreased slightly until day 21 after implantation, but then rapidly increased. By the final day of the experiment (day 46), the size of the tissue fragments in Group 3 (GP hydrogel) had increased to approximately six times its initial volume. Group 1 (tumor tissue only) saw an approximately five-fold increase in the initial volume of the tissue fragments, while Group 2 (Matrigel) saw an approximately three-fold increase in the initial volume of the tissue fragments. Therefore, Group 3 (GP hydrogel) was found to most effectively promote the growth of implanted tissue fragments.

[0087] 9. Histopathological characteristics of transplanted tissue fragments Representative images obtained by microscopic observation during the examination performed in "7. Histopathological Examination" above are shown in Figures 2 and 3. In Group 1 (tumor tissue only), a large necrotic area was observed in the center of the tumor tissue compared to the TG2 bladder cancer tissue before cryopreservation. Furthermore, increased degeneration and necrosis were observed around the tumor tissue in Group 1 (tumor tissue only). In contrast, in Groups 2 (Matrigel) and 3 (GP hydrogel), degeneration and necrosis of the tumor tissue were suppressed, and the morphology was almost unchanged from the TG2 bladder cancer tissue before cryopreservation. Furthermore, slightly more blood vessels with thrombi were observed in the periphery of the tumor tissue in Groups 1 (tumor tissue only) and 2 (Matrigel) compared to Group 3 (GP hydrogel). Furthermore, an increased number of degenerated cancer cells was observed around the tumor tissue in Groups 1 (tumor tissue only) and 2 (Matrigel) compared to Group 3 (GP hydrogel) and the TG2 bladder cancer tissue before cryopreservation. In Group 2 (Matrigel) and Group 3 (GP hydrogel), a small amount of scaffold material was observed at the edge of the implanted tissue fragments, but the amount was small enough not to affect the tumor volume. In Group 3 (GP hydrogel), a small amount of macrophage infiltration was observed around the GP hydrogel.

[0088] (Consideration) In this example, we investigated the effectiveness of GP hydrogel in establishing PDX models from cryopreserved tumor tissue. As shown in Figure 1, the size of tumor tissue implanted subcutaneously in immunodeficient mice was larger in Group 3 (GP hydrogel) than in Group 1 (tumor tissue only) or Group 2 (Matrigel). Furthermore, histopathological examination showed that cryopreserved tumor tissue, even when using scaffolds such as Group 2 (Matrigel) and Group 3 (GP hydrogel), had a larger necrotic area in the center of the implanted tumor tissue compared to the TG2 bladder cancer tissue before cryopreservation. This increase in necrotic area is thought to be due to the cryopreservation and thawing processes. On the other hand, both GP hydrogel and Matrigel scaffolds were shown to ameliorate peripheral degeneration and necrosis of tumor tissue. This demonstrates that the scaffolds actually support tumor tissue engraftment. Furthermore, as shown in Figure 3, in Group 3 (GP hydrogel), blood vessels were formed around the transplanted tumor tissue, maintaining a morphology comparable to that of the TG2 bladder cancer tissue before cryopreservation. In contrast, in Groups 1 (tumor tissue only) and 2 (Matrigel), blood vessels with thrombi were observed around the transplanted tumor tissue. These results suggest that Group 3 (GP hydrogel) has better blood flow and nutrient supply around the transplanted tumor tissue compared with Groups 1 (tumor tissue only) and 2 (Matrigel). This may explain why the size of the transplanted tumor tissue in Group 3 (GP hydrogel) was larger than that in Groups 1 (tumor tissue only) and 2 (Matrigel). It is also possible that the good cell adhesive properties of gelatin in the GP hydrogel promoted the adhesion of transplanted cancer cells and tumor tissue engraftment. Another possibility is that cancer stem cells (CSCs) were activated by the GP hydrogel, promoting the growth of the transplanted tumor tissue. The optimal stiffness for CSCs is known to be a Young's modulus of 4 to 25 kPa, and the Young's modulus of the GP hydrogel used in this study was approximately 8 kPa. The initial size of the transplanted tumor tissue in Group 3 (GP hydrogel) was approximately 2.5 times larger than that of Group 1 (tumor tissue only) and Group 2 (Matrigel).The size of the implanted tumor tissue in Group 3 (GP hydrogel) decreased slightly for three weeks after implantation and then began to increase. It is believed that the GP hydrogel remained at the implantation site and degraded during the first three weeks. Indeed, in Group 3 (GP hydrogel), scaffold debris and phagocytosing macrophages were observed around the implanted tumor tissue. The increase in size of the implanted tumor tissue in Group 1 (tumor tissue only) was greater than in Group 2 (Matrigel). While this result seems inconsistent with the fact that Matrigel is often used as a good scaffold for PDX models, it is consistent with a report in Non-Patent Document 3, which showed no benefit of Matrigel in a bladder cancer PDX model.

[0089] (Conclusion) The use of GP hydrogel promoted the growth of transplanted tumor tissue compared to the use of no scaffold or Matrigel. Furthermore, the use of GP hydrogel suppressed necrosis of transplanted tumor tissue compared to the use of no scaffold, and promoted the formation of blood vessels around the transplanted tumor tissue with maintained morphology compared to the use of Matrigel. These findings suggest that GP hydrogel is an effective scaffold for patient-derived xenograft models.

Claims

1. A structure comprising a hydrogel and tumor tissue covered with said hydrogel, said hydrogel being composed of gelatin cross-linked with a polyether.

2. The structure of claim 1 , wherein the hydrogel forms chemical bonds with proteins in the tumor tissue.

3. The structure according to claim 1 , wherein the polyether is a polymer having a polyalkylene glycol backbone.

4. 10. The structure of claim 1, wherein the polyether comprises a di-, tri-, tetra-, or octa-arm polyethylene glycol.

5. The structure according to claim 1 , wherein the polyether has a total of one or more electrophilic functional groups on a side chain or at an end.

6. 6. The structure of claim 5, wherein the electrophilic functional group is selected from the group consisting of an N-hydroxysuccinimidyl group, a maleimide group, and a sulfosuccinimidyl group.

7. The structure of claim 1 , wherein the tumor tissue comprises bladder cancer tissue.

8. A scaffold material for use in tumor tissue transplantation, comprising a hydrogel composed of gelatin cross-linked with a polyether.

9. A method for transplanting tumor tissue, comprising transplanting tumor tissue covered with the hydrogel using the structure according to any one of claims 1 to 7 into a subject.

10. The transplantation method of claim 9, further comprising promoting angiogenesis around the transplanted tumor tissue.

11. The transplantation method according to claim 9 , wherein the subject is a non-human animal.

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