[go: up one dir, main page]

WO2025197801A1 - Procédé d'évaluation de la capacité d'inhibition de la formation de caf - Google Patents

Procédé d'évaluation de la capacité d'inhibition de la formation de caf

Info

Publication number
WO2025197801A1
WO2025197801A1 PCT/JP2025/009965 JP2025009965W WO2025197801A1 WO 2025197801 A1 WO2025197801 A1 WO 2025197801A1 JP 2025009965 W JP2025009965 W JP 2025009965W WO 2025197801 A1 WO2025197801 A1 WO 2025197801A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cell
cell structure
cancer
evaluating
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/JP2025/009965
Other languages
English (en)
Japanese (ja)
Inventor
吏惟 森村
イサナ 近藤
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Toppan Holdings Inc
Original Assignee
Toppan Holdings Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Toppan Holdings Inc filed Critical Toppan Holdings Inc
Publication of WO2025197801A1 publication Critical patent/WO2025197801A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • the present invention relates to a method for more reliably evaluating the inhibitory effect on CAF formation in cancer tissues in an in vitro system without using an animal model.
  • the reliability of the evaluation obtained becomes an issue. That is, it is important that the evaluation in the evaluation system reflects the efficacy obtained when the drug is actually administered to a living organism.
  • An evaluation system that has a high probability of matching the evaluation in the evaluation system with the effect obtained when the drug is administered to a living organism is a highly reliable evaluation system.
  • Patent Document 1 discloses a method for producing three-dimensional cellular tissue, including the steps of obtaining a mixture in which cells are suspended in a solution containing at least a cationic buffer solution, an extracellular matrix component, and a strong electrolyte polymer; collecting the cells from the obtained mixture and forming a cell aggregate on a substrate; and culturing the cells to obtain three-dimensional cellular tissue.
  • the three-dimensional cellular tissue can be used, for example, in biological tissue models and solid cancer models, and can be used in various assays such as drug screening.
  • the stroma In the cancer microenvironment (in other words, the environment surrounding cancer cells in the body), the stroma has a significant impact on cancer cells. It has been reported that, particularly in highly malignant cancers, abnormally activated specialized fibroblasts appear in large numbers, and these cells are called “cancer associated fibroblasts (CAFs).” It has also been reported that these CAFs produce various growth factors that promote cancer cell proliferation, and promote angiogenesis, cancer cell proliferation, and invasion (Non-Patent Document 1). Therefore, it is thought that a cancer microenvironment that more closely resembles that found in the body can be created by creating a three-dimensional cellular tissue in which cancer cells and stroma coexist.
  • CAFs cancer associated fibroblasts
  • CAFs have a strong influence on cancer growth and sensitivity to anticancer drugs. Furthermore, in genetically modified T cell therapy (CAR-T cell therapy) using chimeric antigen receptors (CARs) and genetically modified T cell (TCR-T) therapy (TCR-T cell therapy) that introduces T cell receptors, it is essential for CAR-T cells and TCR-T cells to be able to attack cancer cells in order to achieve an anticancer effect, and CAFs function as a physical barrier, such as collagen fibers. Therefore, inhibiting the transformation of fibroblasts in cancer tissue into CAFs is important for achieving greater anticancer effects in anticancer treatment, and there is a need for the development of better agents that inhibit CAF formation.
  • the present invention aims to provide an in vitro method for evaluating the ability to inhibit CAF formation, which allows for more reliable evaluation without using an animal model.
  • the inventors conducted extensive research into various cell culture methods and discovered that by using a cell structure (also known as a three-dimensional cell tissue) containing stromal cells and cancer cells and whose surface has been converted into CAFs, and by using the effect on the anti-cancer effect of immune cells as an indicator, the ability of a compound to inhibit CAF formation can be evaluated, leading to the completion of the present invention.
  • a cell structure also known as a three-dimensional cell tissue
  • a method for evaluating CAF formation inhibitory ability comprising: a CAF formation step of culturing a cell structure containing fibroblasts and cancer cells in a medium containing ascorbic acid or a derivative thereof, TGF- ⁇ , and a compound to be evaluated; a culture step of adding immune cells to the cell structure after the CAF formation step and further culturing the cell structure; and an evaluation step of evaluating the CAF formation inhibitory ability of the compound to be evaluated using the number of live cancer cells in the cell structure after the culture step as an index.
  • a method for evaluating the ability to inhibit CAF formation comprising: [2] The method for evaluating the ability to inhibit CAF formation according to [1], wherein the cell structure is in contact with the culture medium on both the top and bottom surfaces, the cell structure includes a cancer cell layer containing the cancer cells, and the cancer cell layer is located within a range from one-quarter to three-quarters of the height from the bottom surface of the cell structure in the thickness direction. [3] The method for evaluating the ability to suppress CAF formation according to [1] or [2] above, wherein the immune cells are one or more types selected from the group consisting of leukocytes and lymphocytes.
  • the cell structure is (a) mixing cells and extracellular matrix components in a cationic buffer to obtain a mixture; (b) seeding the mixture obtained in step (a) into a cell culture vessel; (c) after the step (b), obtaining a cell structure that is a three-dimensional structure in which cells are stacked in multiple layers in the cell culture vessel; The method for evaluating the CAF formation inhibitory ability of any of [1] to [3] above, wherein the composition is produced by the method. [5] The method for evaluating the ability to suppress formation of CAFs according to any one of [1] to [4] above, wherein the immune cells include CAR-T cells or TCR-T cells.
  • [6] The method for evaluating the ability to suppress CAF formation according to any one of [1] to [5] above, wherein the immune cells include regulatory T cells.
  • [7] The method for evaluating the ability to inhibit CAF formation according to any one of [1] to [6] above, wherein the cell structure comprises multiple cell layers, and the cancer cells are present only in a specific cell layer within the cell structure.
  • [8] The method for evaluating the ability to inhibit CAF formation according to any one of [1] to [7] above, wherein the thickness of the cell structure is 5 ⁇ m or more.
  • [9] The method for evaluating the ability to inhibit CAF formation according to any one of [1] to [8] above, wherein the thickness of the cell structure is 500 ⁇ m or less.
  • the cell structure further comprises one or more cells selected from the group consisting of vascular endothelial cells and lymphatic endothelial cells.
  • the extracellular matrix component is selected from the group consisting of collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, fibrin, proteoglycan, and combinations thereof.
  • the cationic buffer solution further contains a strong electrolyte polymer,
  • the method for evaluating the ability to inhibit CAF formation according to any one of [4] to [11] above, wherein the strong electrolyte polymer is selected from the group consisting of glycosaminoglycan, dextran sulfate, rhamnan sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropanesulfonic acid, polyacrylic acid, and combinations thereof.
  • a method for evaluating CAF formation inhibitory ability comprising: The method comprises: a CAF formation step of culturing a cell structure containing fibroblasts and cancer cells in a medium containing ascorbic acid or a derivative thereof, TGF- ⁇ , and a compound to be evaluated; a culture step of adding immune cells to the cell structure after the CAF formation step and further culturing the cell structure; and an evaluation step of evaluating the ability of the compound to be evaluated to inhibit CAF formation using the number of live cancer cells in the cell structure after the culture step as an index,
  • the cell structure is in contact with the medium on both the top and bottom surfaces, the cell structure comprises a cancer cell layer containing the cancer cells, the cancer cell layer is located within a range from one-quarter to three-quarters of the height from the bottom surface of the cell structure in the thickness direction, The ratio of the ascorbic
  • a compound selection method comprising: culturing a cell structure containing fibroblasts and cancer cells in a medium containing ascorbic acid or an ascorbic acid derivative, TGF- ⁇ , and a compound to be evaluated; after culturing the cell structure, adding immune cells to the cell structure and further culturing the cell structure; and counting the number of viable cancer cells in the cell structure after adding the immune cells and culturing the cell structure.
  • the method for evaluating the ability to inhibit CAF formation evaluates the presence or absence and strength of the ability of a compound to inhibit CAF formation using a cell structure that is in a state that is closest to the in vivo state, specifically, a cell structure in which stromal cells, including epithelial cells, and cancer cells coexist and have been converted into CAFs. Therefore, despite being an in vitro evaluation system, highly reliable evaluations can be obtained.
  • FIG. 1 shows the results of measuring the relative cancer cell survival rate (%) for each number of cells seeded in a cell structure converted into CAFs by AscP and TGF- ⁇ 1 (in the figure, "CAF") and a control cell structure not converted into CAFs (in the figure, “CNT”) in Reference Example 1.
  • FIG. 1 shows stained images of sections of a cell structure converted into CAFs by AscP and TGF- ⁇ 1 (in the figure, "CAF") and a control cell structure that was not converted into CAFs (in the figure, "CNT”) in Reference Example 1.
  • Example 1 these are fluorescence images of a cell structure cultured with fluorescently labeled TCR-T cells, taken immediately after the addition of the TCR-T cells ("0 h” in the figure) and 24 hours after the addition and culture ("24 h” in the figure).
  • FIG. 1 shows a blotting image obtained by Western blotting, using an anti- ⁇ -SMA antibody, an anti-collagen antibody, and an anti-GAPDH antibody, on proteins extracted from a cell structure cultured in a medium supplemented with a combination of AscP, TGF- ⁇ 1, and TSA in Example 1.
  • 1 shows images of the results of microscopic observation of cell structures after 7 days of culture in media containing various concentrations of ascorbic acid and TGF- ⁇ in Experimental Example 2.
  • 10 shows images of hematoxylin-eosin stained cell structures obtained by microscopic observation after 4 days and 7 days of culture in each medium in Experimental Example 2.
  • 1 is a graph showing the thickness of cell structures after 4 days and 7 days of culture in each medium in Experimental Example 2.
  • 10 shows images of the results of microscopic observation of cell structures stained with Sirius Red-saturated picric acid after 4 days and 7 days of culture in each medium in Experimental Example 2.
  • 1 is a graph showing the PSR ratio after 4 days and 7 days of culture in media containing various concentrations of ascorbic acid and TGF- ⁇ in Experimental Example 2.
  • 10 shows images of the results of microscopic observation of cell structures stained with Sirius Red and saturated picric acid after 4 days and 7 days of culture in each medium in Experimental Example 3.
  • a “cell structure” is a three-dimensional structure in which multiple cell layers are stacked.
  • a “cell layer” is a layer composed of a group of cells and stroma that exist in a direction perpendicular to the thickness direction and in which the cell nuclei do not overlap in the thickness direction when observed at a magnification at which cell nuclei can be recognized in a slice image of a cross section of the cell structure in the thickness direction, that is, a magnification at which the entire thickness of the stained slice is visible.
  • “layered” means that two or more different cell layers are stacked in the thickness direction.
  • the “thickness of the cell structure” is the length of the cell structure in the direction of its own weight.
  • the direction of gravity is the direction in which gravity is applied, and is also referred to as the thickness direction.
  • a method for evaluating the ability to inhibit CAF formation according to one embodiment of the present invention involves culturing a cell structure simulating a cancer microenvironment in a medium containing immune cells, a CAF-forming agent, and a target compound (hereinafter sometimes referred to as the "target compound") for evaluation of its ability to inhibit CAF formation, and evaluating the presence or strength of the compound's ability to inhibit CAF formation using as an indicator the cytotoxicity of the immune cells against cancer cells in the cell structure.
  • this evaluation method is a method for evaluating the ability to inhibit CAF formation, and includes a CAF formation step in which a cell structure containing fibroblasts and cancer cells is cultured in a medium containing ascorbic acid or a derivative thereof, TGF- ⁇ , and the target compound; a culture step in which immune cells are added to the cell structure after the CAF formation step and further cultured; and an evaluation step in which the target compound's ability to inhibit CAF formation is evaluated using as an indicator the number of viable cancer cells in the cell structure after the culture step.
  • cancer cells exist surrounded by the interstitium that supports them. Therefore, in order for immune cells to attack cancer cells, the immune cells must infiltrate the interstitium and reach the cancer cells.
  • the extracellular matrix such as collagen fibers
  • the strength of the cytotoxicity of immune cells against cancer cells within the cell structure can be used as an index of the degree of CAF formation in the cell structure.
  • the evaluation method according to this embodiment utilizes this cytotoxicity by immune cells as an index of CAF formation.
  • CAF transformation refers to a state in which ⁇ -SMA ( ⁇ -smooth muscle actin) is expressed or extracellular matrix such as fibronectin and collagen is excessively expressed. Whether fibroblasts have transformed into CAFs can be determined by immunostaining or other methods when there is an excess of extracellular matrix compared to the surrounding fibroblasts.
  • ⁇ -SMA smooth muscle actin
  • Markers such as ⁇ -SMA/ACTA2, COL1, FN1, FAP, MFAP5, COL11A1, TN-C, PDPN, ITGA11, NG2, PDGFR ⁇ / ⁇ , VIM, FSP-1/S100A4, POSTN, EPCAM, CALD1, SMTN, PTPRC, PECAM1, CD70, Vimentin, GPR77, CD10, CD36, CD74, CD146, CAV1, Saa3, P4H, ASPN, OGN, ZEB1, MCT4/SLC16A4, SPARC, MMPs, FOXF1, CAV1, and PTRF can be used to confirm whether fibroblasts have become CAFs by immunostaining.
  • the expression levels of the above-mentioned markers are confirmed and compared with the expression levels of the same markers in cells that have not become CAFs, and if the expression levels of the markers are increased or suppressed, the fibroblasts are determined to have become CAFs.
  • markers whose expression levels increase upon CAF formation include ⁇ -SMA/ACTA2, COL1, FN1, FAP, MFAP5, COL11A1, TN-C, PDPN, ITGA11, NG2, PDGFR ⁇ / ⁇ , VIM, FSP-1/S100A4, POSTN, CD70, Vimentin, GPR77, CD10, CD74, CD146, P4H, ASPN, OGN, ZEB1, MCT4/SLC16A4, SPARC, MMPs, FOXF1, and CAV1.
  • Markers whose expression levels are suppressed upon conversion to CAF include, for example, EPCAM, CALD1, SMTN, PTPRC, PECAM1, Saa3, CD36, CAV1, and PTRF.
  • the cell structure used in the evaluation method according to this embodiment is a cell structure that mimics a cancer microenvironment. Specifically, it is a cell structure that includes fibroblasts and cancer cells. Fibroblasts are the main cells that make up the stroma of cancer tissue and correspond to precursor cells of CAFs.
  • the fibroblasts contained in the cell structure used in this embodiment may be cells derived from any tissue. Examples of such fibroblasts include skin fibroblasts, lung fibroblasts, and cardiac fibroblasts. Furthermore, the fibroblasts contained in the cell structure used in this embodiment may be of one type, or two or more types.
  • the proportion of fibroblasts contained in the cell structure of this embodiment is preferably 10.0% or more, more preferably 20.0% or more, even more preferably 30.0% or more, and even more preferably 40.0% or more, relative to the total number of cells constituting the cell structure.
  • the proportion of fibroblasts contained in the cell structure of this embodiment is preferably 99.9% or less, more preferably 99.0% or less, and even more preferably 90.0% or less.
  • the cell structure used in this embodiment may contain cells other than fibroblasts that make up the interstitium (also referred to as interstitial cells).
  • interstitial cells include endothelial cells, neurons, mast cells, epithelial cells, cardiomyocytes, hepatocytes, pancreatic islet cells, tissue stem cells, and smooth muscle cells.
  • the interstitial cells other than fibroblasts contained in the cell structure used in this embodiment may be of one type, or of two or more types.
  • the type of interstitial cells contained in the cell structure used in this embodiment is not particularly limited, and can be selected appropriately taking into consideration the origin and type of cancer cells to be contained, the type of immune cells to be used for evaluation, the type of CAF formation inhibitor to be used for evaluation, or the in vivo environment in which the desired CAF formation inhibitory activity is exerted.
  • the cell structure used in this embodiment preferably has a vascular network structure.
  • the cell structure used in this embodiment preferably has a three-dimensional vascular network structure, such as lymphatic vessels and/or blood vessels, constructed in at least a portion of its interior, thereby constructing tissue that is closer to that found in a living organism.
  • the vascular network structure may be formed only inside the cell structure, or may be formed so that at least a portion of the vascular network structure is exposed on the surface or bottom of the cell structure.
  • the term "vascular network structure” refers to a network structure, such as a blood vessel network or lymphatic vessel network in biological tissue.
  • a vascular network structure can be formed by including endothelial cells, which form blood vessels, as interstitial cells.
  • the endothelial cells contained in the cell structure used in this embodiment may be vascular endothelial cells or lymphatic endothelial cells.
  • the cell structure may also contain both vascular endothelial cells and lymphatic endothelial cells.
  • the endothelial cells contained in the cell structure may be cells derived from the same biological species as the fibroblasts, or may be cells derived from a different biological species.
  • the number of endothelial cells in the cell structure is preferably sufficient to form a vascular network structure, and can be determined appropriately taking into account the size of the cell structure, the type of endothelial cells and cells other than endothelial cells, etc.
  • a cell structure having a vascular network structure can be prepared by setting the abundance ratio (cell number ratio) of endothelial cells to the total cells that make up the cell structure used in this embodiment to 0.1% or more.
  • the number of endothelial cells in the cell structure used in this embodiment is preferably 0.1% or more of the number of fibroblasts contained in the cell structure, and more preferably 0.1 to 5.0%.
  • the total number of vascular endothelial cells and lymphatic endothelial cells is preferably 0.1% or more of the number of fibroblasts, and more preferably 0.1 to 5.0%.
  • the cell structure used in this embodiment further includes cancer cells.
  • the cancer cells contained in the cell structure used in this embodiment may be one type or two or more types. Cancer cells are cells that are derived from somatic cells and have acquired the ability to proliferate indefinitely.
  • the cancer cells contained in the cell structure used in this embodiment may be established cultured cell lines, or cancer cells collected from a cancer patient. Cancer cells collected from a cancer patient may be cells that have been cultured and grown in advance. Specific examples include primary cancer cells collected from a cancer patient, artificially cultured cancer cells, iPS cancer stem cells, cancer stem cells, and established cancer cell lines prepared in advance for use in cancer treatment research and anticancer drug development. Cancer cells may also be derived from humans or from animals other than humans. When the cell structure used in this embodiment includes cancer cells collected from a cancer patient, it may also contain cells other than cancer cells collected from the cancer patient. Examples of cells other than cancer cells include one or more types of cells contained in solid tissue removed after surgery.
  • Cancers from which the cancer cells contained in the cell structure used in this embodiment are derived include, for example, breast cancer (e.g., invasive ductal carcinoma, ductal carcinoma in situ, and inflammatory breast cancer, etc.), prostate cancer (e.g., hormone-dependent prostate cancer and hormone-independent prostate cancer, etc.), pancreatic cancer (e.g., pancreatic ductal carcinoma, etc.), gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, and adenosquamous carcinoma, etc.), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, and malignant mesothelioma, etc.), colon cancer (e.g., gastrointestinal stromal tumor, etc.), rectal cancer ( gastrointestinal stromal tumors, etc.), colon cancer (e.g., familial colorectal cancer, hereditary non-polyposis colorectal cancer, and gastrointestinal stromal tumor
  • the proportion of cancer cells contained in the cell structure used in this embodiment is preferably 0.01% or more, more preferably 0.05% or more, and even more preferably 0.1% or more, relative to the total number of cells constituting the cell structure.
  • the number of cancer cells in the cell structure is not particularly limited, but in order to more closely resemble the cancer microenvironment in a living body, it is preferable that the ratio of the number of endothelial cells to the number of cancer cells in the cell structure ([number of endothelial cells]/[number of cancer cells]) be greater than 0 (greater than 0) and 50 or less.
  • the cell structure used in this embodiment may be one in which cancer cells are scattered throughout the structure, or one in which cancer cells are present only in a specific cell layer.
  • the cell layer containing these cancer cells (also referred to as the cancer cell layer) is preferably located outside the surface of the cell structure, and more preferably away from the surface.
  • the cancer cell layer is preferably located within a range from the bottom surface (lower surface) of the structure to half the height in the thickness direction, and more preferably within a range from the bottom surface (lower surface) of the structure to one-quarter the height in the thickness direction.
  • the cancer cell layer is preferably located within a range from one-quarter to three-quarters the height in the thickness direction from the bottom surface (lower surface) of the structure. If the distance from the surface of the cell structure to the cancer cell layer is sufficient, the degree of CAF formation on the surface of the cell structure, in other words, the difference in the ease of infiltration of immune cells, can be reflected by the survival rate of cancer cells within the cell structure.
  • the cell structure used in this embodiment may contain cells other than cancer cells and stromal cells.
  • examples of other cells include immune cells, nerve cells, liver cells, pancreatic cells, cardiac muscle cells, smooth muscle cells, bone cells, alveolar epithelial cells, and spleen cells.
  • the cells, including fibroblasts and cancer cells, that make up the cell structure used in this embodiment are not particularly limited and may be cells collected from an animal, cultured cells collected from an animal, cells obtained by subjecting cells collected from an animal to various treatments, or cultured cell lines.
  • the site of collection is not particularly limited and they may be somatic cells derived from bone, muscle, internal organs, nerves, brain, bone, skin, or blood, or may be germ cells or embryonic stem cells (ES cells).
  • the biological species from which the cells that make up the cell structure of this embodiment are derived is not particularly limited. For example, cells derived from animals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, and rats can be used.
  • Cultured cells collected from animals may be primary cultured cells or subcultured cells. Examples of cells that have been subjected to various treatments include induced pluripotent stem cells (iPS cells) and cells after differentiation induction. Furthermore, the cell structure of this embodiment may be composed solely of cells derived from the same biological species, or may be composed of cells derived from multiple biological species.
  • iPS cells induced pluripotent stem cells
  • the cell structure of this embodiment may be composed solely of cells derived from the same biological species, or may be composed of cells derived from multiple biological species.
  • the size and shape of the cell structure used in this embodiment are not particularly limited. Because this allows for the formation of a vascular network structure more similar to the blood vessels formed in tissues in vivo and enables more accurate evaluation, the thickness of the cell structure is preferably 5 ⁇ m or more, more preferably 10 ⁇ m or more, even more preferably 50 ⁇ m or more, and even more preferably 100 ⁇ m or more. The thickness of the cell structure is also preferably 500 ⁇ m or less, more preferably 400 ⁇ m or less, and even more preferably 300 ⁇ m or less.
  • the number of cell layers in the cell structure used in this embodiment is preferably approximately 2 to 60 layers, more preferably approximately 5 to 60 layers, and even more preferably approximately 10 to 60 layers.
  • the number of cell layers that make up a cell structure is measured by dividing the total number of cells that make up the three-dimensional structure by the number of cells per layer (the number of cells required to make up one layer).
  • the number of cells per layer can be determined by culturing cells confluently in advance in a planar manner in the cell culture vessel that will be used to make the cell structure. Specifically, the number of cell layers in a cell structure formed in a certain cell culture vessel can be calculated by counting the total number of cells that make up the cell structure and dividing this by the number of cells per layer in the cell culture vessel.
  • the cell structures used in this embodiment are constructed in a cell culture vessel.
  • the cell culture vessel There are no particular limitations on the cell culture vessel, as long as it is possible to construct a cell structure and to culture the constructed cell structure.
  • Specific examples of the cell culture vessel include dishes, cell culture inserts (e.g., Transwell (registered trademark) inserts, Netwell (registered trademark) inserts, Falcon (registered trademark) cell culture inserts, Millicell (registered trademark) cell culture inserts, etc.), tubes, flasks, bottles, and plates.
  • dish or various cell culture inserts are preferred, as this allows for more appropriate evaluation using the cell structures.
  • the cell structure used in this embodiment may be a structure formed from multiple cell layers containing stromal cells, including fibroblasts, and cancer cells, and the method for constructing the cell structure is not particularly limited. For example, it may be constructed by sequentially stacking layers one by one, or it may be constructed by simultaneously stacking two or more cell layers, or it may be constructed by appropriately combining both construction methods to construct multiple cell layers. Furthermore, the cell structure used in this embodiment may be a multi-layer structure in which each cell layer is composed of a different cell type, or the cell type constituting each cell layer may be the same for all layers of the structure.
  • it may be constructed by forming layers for each cell type and sequentially stacking the cell layers for each cell type, or it may be constructed by previously preparing a cell mixture containing multiple types of cells and simultaneously constructing a multi-layered cell structure from the previously prepared cell mixture containing multiple types of cells.
  • An example of a method for constructing cells layer by layer and layering them sequentially is the method described in Japanese Patent No. 4919464, which involves alternately repeating the steps of forming a cell layer and contacting the formed cell layer with a solution containing ECM (extracellular matrix) components to continuously layer cell layers.
  • ECM extracellular matrix
  • a cell mixture is prepared in advance by mixing all of the cells that make up the cell structure, and each cell layer is formed using this cell mixture. This allows for the construction of a cell structure in which a vascular network structure is formed throughout the structure and cancer cells are scattered throughout the structure.
  • a cell structure can be constructed in which a vascular network structure is formed only in the layer formed from endothelial cells and cancer cells are present only in specific cell layers.
  • An example of a method for constructing two or more cell layers at once is the method described in Japanese Patent No. 5850419.
  • the entire surface of a cell is first coated with a polymer containing an arginine-glycine-aspartic acid (RGD) sequence to which integrins bind, and a polymer that interacts with the RGD sequence-containing polymer.
  • the coated cells, coated with this adhesive film are then placed in a cell culture vessel and the coated cells are allowed to accumulate by centrifugation or other methods, thereby constructing a cell structure formed from multiple cell layers.
  • a cell mixture is prepared in advance by mixing all the cells that make up the cell structure, and an adhesive component is added to this cell mixture to prepare the coated cells.
  • coated cells coating endothelial cells, coated cells coating fibroblasts, and coated cells coating a group of cells collected from a cancer patient can be separately prepared, and after forming multiple layers composed of coated fibroblast cells, a single layer formed from coated endothelial cells can be layered on top of that, and multiple layers formed from coated fibroblast cells can be layered on top of that, and a single layer formed from coated cells containing cancer cells can be layered on top of that.
  • the cell structure used in this embodiment can also be constructed by a method comprising the following steps (a) to (c). (a) mixing cells and extracellular matrix components in a cationic buffer to obtain a mixture; (b) seeding the mixture obtained in step (a) into a cell culture vessel; and (c) obtaining, after step (b), a cell structure in which cells are stacked in multiple layers in the cell culture vessel.
  • step (a) cells are mixed with a buffer solution containing a cationic substance (also referred to as a cationic buffer) and extracellular matrix components, and cell aggregates are formed from this cell mixture, thereby obtaining a three-dimensional cell tissue with few large internal voids. Furthermore, the obtained three-dimensional cell tissue is relatively stable, allowing it to be cultured for at least several days, and the tissue is less likely to collapse even when the culture medium is changed. Furthermore, in this embodiment, step (b) may include allowing the cell mixture seeded in the cell culture vessel to settle within the cell culture vessel. The cell mixture may be sedimented actively by centrifugation or the like, or it may be allowed to settle naturally.
  • a buffer solution containing a cationic substance also referred to as a cationic buffer
  • step (b) may include allowing the cell mixture seeded in the cell culture vessel to settle within the cell culture vessel. The cell mixture may be sedimented actively by centrifugation or the like, or it may be allowed to settle naturally.
  • step (a) it is preferable to further mix the cells with a strong electrolyte polymer.
  • a strong electrolyte polymer By mixing the cells with a cationic substance, a strong electrolyte polymer, and an extracellular matrix component, a thick, three-dimensional cell tissue with few voids can be obtained even when the cells are allowed to settle naturally, without the need for a process such as centrifugation to actively aggregate the cells in step (b).
  • the cationic buffer examples include Tris-HCl buffer, Tris-maleate buffer, Bis-Tris buffer, and HEPES.
  • concentration and pH of the cationic substance (e.g., Tris in Tris-HCl buffer) in the cationic buffer are not particularly limited, as long as they do not adversely affect cell growth and the construction of cell structures.
  • concentration of the cationic substance in the cationic buffer can be 10 to 100 mM, preferably 40 to 70 mM, and more preferably 50 mM.
  • the pH of the cationic buffer can be 6.0 to 8.0, preferably 6.8 to 7.8, and more preferably 7.2 to 7.6.
  • the strong electrolyte polymer examples include, but are not limited to, glycosaminoglycans such as heparin, chondroitin sulfate (e.g., chondroitin 4-sulfate and chondroitin 6-sulfate), heparan sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid; dextran sulfate, rhamnan sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropanesulfonic acid, and polyacrylic acid, or derivatives thereof.
  • glycosaminoglycans such as heparin, chondroitin sulfate (e.g., chondroitin 4-sulfate and chondroitin 6-sulfate), heparan sulfate, dermatan sulfate, keratan sulf
  • the mixture prepared in step (a) may contain only one type of strong electrolyte polymer, or a combination of two or more types.
  • the strong electrolyte polymer is preferably a glycosaminoglycan. It is more preferable to use at least one of heparin, dextran sulfate, chondroitin sulfate, and dermatan sulfate. It is even more preferable that the strong electrolyte polymer used in this embodiment is heparin.
  • the amount of the strong electrolyte polymer to be mixed with the cationic buffer solution is not particularly limited as long as it does not adversely affect cell growth and the construction of cell structures.
  • the concentration of the strong electrolyte polymer in the cationic buffer solution can be greater than 0 mg/mL (higher than 0 mg/mL) and less than 1.0 mg/mL, preferably 0.025 to 0.1 mg/mL, and more preferably 0.05 to 0.1 mg/mL.
  • the mixture can be prepared without adding the strong electrolyte polymer, and then cell structures can be constructed.
  • extracellular matrix components examples include collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, fibrin, proteoglycans, or modified or variant forms thereof.
  • proteoglycans include chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, keratan sulfate proteoglycans, and dermatan sulfate proteoglycans.
  • the mixture prepared in step (a) may contain only one type of extracellular matrix component, or two or more types in combination. In constructing the cell structure used in this embodiment, collagen, laminin, and fibronectin are preferably used, and collagen is more preferred.
  • the amount of extracellular matrix component to be mixed with the cationic buffer solution is not particularly limited, as long as it does not adversely affect cell growth and cell structure formation.
  • the concentration of the extracellular matrix component in the cationic buffer can be greater than 0 mg/mL (greater than 0 mg/mL) and less than 1.0 mg/mL, preferably 0.025 to 0.1 mg/mL, and more preferably 0.05 to 0.1 mg/mL.
  • the blending ratio of the strong electrolyte polymer to the extracellular matrix component mixed with the cationic buffer solution is 1:2 to 2:1.
  • the blending ratio of the strong electrolyte polymer to the extracellular matrix component is preferably 1:1.5 to 1.5:1, and more preferably 1:1.
  • step (c) By repeating steps (a) to (c), specifically by seeding the mixture prepared in step (a) on the cell structure obtained in step (c) as step (b), and then repeating step (c), a cell structure of sufficient thickness can be constructed.
  • the cell composition of the mixture newly seeded on the cell structure obtained in step (c) may be the same as or different from the cell composition that constitutes the cell structure already constructed.
  • step (a) a mixture containing only fibroblasts as cells is prepared, and steps (b) and (c) are performed to obtain a cell structure formed from 10 fibroblast layers in a cell culture vessel.
  • step (a) a mixture containing only vascular endothelial cells as cells is prepared, and steps (b) and (c) are performed to layer one vascular endothelial cell layer on the fibroblast layer in the cell culture vessel.
  • step (a) a mixture containing only fibroblasts as cells is prepared, and steps (b) and (c) are performed to layer 10 fibroblast layers on the vascular endothelial cell layer in the cell culture vessel.
  • step (a) a mixture containing cancer cells collected from a cancer patient is prepared, and steps (b) and (c) are performed to layer one cancer cell layer on the fibroblast layer in the cell culture vessel.
  • steps (b) and (c) are performed to layer one cancer cell layer on the fibroblast layer in the cell culture vessel.
  • This allows the construction of a cell structure in which layers are layered in order by cell type: 10 fibroblast layers - 1 vascular endothelial cell layer - 10 fibroblast layers - 1 cancer cell layer.
  • the thickness of the cell layer laminated in step (c) can be adjusted. The greater the number of cells seeded in step (b), the greater the number of cell layers laminated in step (c).
  • step (a) a mixture of fibroblasts for 20 fibroblast layers and vascular endothelial cells for one vascular endothelial cell layer is prepared, and steps (b) and (c) are performed.
  • a similarly prepared mixture containing cancer cells collected from a cancer patient is then laminated on the resulting multilayer structure, thereby constructing a cell structure having a thickness of 21 layers and in which cancer cell layers are laminated on a structure with a vascular network structure scattered throughout the structure.
  • step (a) a mixture is prepared by mixing fibroblasts equivalent to 20 fibroblast layers, vascular endothelial cells equivalent to one vascular endothelial cell layer, and cells derived from a cancer patient equivalent to one cancer cell layer, and steps (b) and (c) are then performed to construct a cell structure having a thickness of 22 layers, in which both cancer cells and a vascular network structure are independently dispersed within the structure.
  • the resulting cell structure may be cultured after step (c) and before step (b).
  • Culture conditions such as the composition of the culture medium used for culture, culture temperature, culture time, and atmospheric composition during culture, should be set to conditions suitable for culturing the cells that make up the cell structure. Examples of culture media include D-MEM, E-MEM, MEM ⁇ , RPMI-1640, and Ham's F-12.
  • step (a) After step (a), (a'-1) the liquid portion is removed from the resulting mixture to obtain cell aggregates, and (a'-2) the cell aggregates are suspended in a solution, and then step (b) can be carried out. While the desired tissue can be obtained by carrying out steps (a) to (c) above, a more homogeneous tissue can be obtained by carrying out (a'-1) and (a'-2) after step (a) and then carrying out step (b).
  • step (b) the following steps (b'-1) and (b'-2) may be performed instead of step (b).
  • a more homogeneous tissue can also be obtained by performing steps (b'-1) and (b'-2).
  • step (b'-2) may also include allowing the cell mixture seeded in the cell culture vessel to settle within the cell culture vessel.
  • the cell mixture may be sedimented actively by centrifugation or the like, or may be allowed to settle naturally.
  • the term "cellular viscous body" refers to a gel-like cell aggregate as described in Non-Patent Document 4.
  • step (b'-1) A step of seeding the mixture obtained in step (a) into a cell culture vessel, and then removing the liquid component from the mixture to obtain a viscous cell mass.
  • step (b'-2) A step of suspending the viscous cell mass in a solvent in a cell culture vessel.
  • the solvent for preparing the cell suspension is not particularly limited as long as it is non-toxic to cells and does not impair their proliferation or function, and water, buffer solutions, cell culture media, etc. can be used.
  • buffer solutions include phosphate-buffered saline (PBS), HEPES, and Hanks' buffer solution.
  • culture media include D-MEM, E-MEM, MEM ⁇ , RPMI-1640, and Ham's F-12.
  • step (c) A step of forming a layer of cells on a substrate.
  • liquid components may be removed from the seeded mixture.
  • the method for removing liquid components in steps (c) and (c') is not particularly limited, as long as it does not adversely affect cell growth or the construction of cell structures, and can be any method known to those skilled in the art for removing liquid components from a suspension of liquid and solid components. Examples of such methods include aspiration, centrifugation, magnetic separation, and filtration. For example, when a cell culture insert is used as the cell culture vessel, the cell culture insert seeded with the mixture can be centrifuged at 10°C and 400 x g for 1 minute, causing the cell mixture to settle, and the liquid components can then be removed by aspiration.
  • the CAF formation step involves culturing the cell structure in a medium containing ascorbic acid or a derivative thereof (i.e., an ascorbic acid derivative), TGF- ⁇ , and a compound to be evaluated. After the CAF formation step, immune cells are added to the cell structure and further cultured.
  • ascorbic acid or a derivative thereof and TGF- ⁇ are CAF-forming agents that induce the formation of CAFs in fibroblasts in the cell structure.
  • the ascorbic acid or a derivative thereof and TGF- ⁇ used in the evaluation method according to this embodiment may be chemically synthesized products, or may be purified products produced by microorganisms or the like.
  • Immune cells are cells involved in immunity. Specific examples include lymphocytes, macrophages, and dendritic cells. Lymphocytes include T cells, B cells, NK cells, and plasma cells.
  • the immune cells used in the evaluation method according to this embodiment may be of one type, or two or more types.
  • the immune cells used in this embodiment are not particularly limited as long as they are immune cells, but cells that are actually present in the vicinity of the cancer microenvironment and are involved in the mechanism of attacking cancer cells through an immune response are preferred.
  • PBMCs peripheral blood mononuclear cells in plasma
  • PBMCs include lymphocytes and monocytes.
  • Monocytes include macrophages.
  • Lymphocytes include NK cells, B cells, and T cells.
  • PBMCs may be isolated and purified from blood, but buffy coats prepared from blood can also be used as is.
  • Buffy coats contain PBMCs along with other components. Buffy coats can be prepared from blood by standard methods such as centrifugation.
  • Immune cells may have multiple types, such as ABO blood types, that contain the same components but have slightly different properties.
  • any one type of immune cell may be used, or multiple types of immune cells may be used in combination, as needed.
  • the immune cells used in the evaluation method according to this embodiment may be immune cells collected from a living body, a cultured cell line, or cells artificially altered or modified ex vivo.
  • immune cells collected from a cancer patient it is preferable to use immune cells isolated from the peripheral blood or tumor site of the cancer patient, particularly PBMCs.
  • artificially altered or modified immune cells are preferably immune cells whose immune function has been artificially altered to enhance anti-cancer activity. Examples of such immune cells with altered immune function include modified T cells used in CAR-T cell therapy (CAR-T cells) and modified T cells used in TCR-T cell therapy (TCR-T cells).
  • the cell structure is first cultured in a culture medium containing a mixture of ascorbic acid or a derivative thereof, TGF- ⁇ , and a target compound.
  • the culture medium can be the same as the one used to produce the cell structure.
  • Ascorbic acid, TGF- ⁇ , and the target compound may be added simultaneously or separately to the medium in which the cell structure is cultured.
  • Ascorbic acid is an organic compound with a lactone structure, known as vitamin C, a water-soluble vitamin. Ascorbic acid is an optically active compound and exists in both L and D forms, with the L form being preferred. It is the L form that is known as vitamin C. Ascorbic acid may be an ascorbate salt or an ascorbic acid derivative. Examples of ascorbate salts include sodium ascorbate, potassium ascorbate, and calcium ascorbate. Examples of ascorbic acid derivatives include magnesium ascorbyl phosphate, sodium ascorbyl phosphate, and ascorbic acid 2-glucoside.
  • the amounts of ascorbic acid or its derivatives, TGF- ⁇ , and target compound to be mixed into the culture medium can each be determined experimentally, taking into consideration conditions such as the type and number of cells that make up the cell structure, the type and amount of cancer cells contained therein, the type of culture medium, the culture temperature, or the culture time.
  • the amount of immune cells to be mixed into the culture medium can also be determined experimentally, taking into consideration the type and number of cells that make up the cell structure, the type and amount of cancer cells contained therein, the type of culture medium, the culture temperature, or the culture time.
  • the concentration of ascorbic acid in the culture medium is not particularly limited, as long as the thickness of the three-dimensional cell tissue can be made to exceed 50 ⁇ m for at least three days after culture in a culture medium containing ascorbic acid and TGF- ⁇ . It may be 0.02 mM or more and 0.7 mM or less, 0.02 mM or more and 0.5 mM or less, 0.02 mM or more and 0.3 mM or less, 0.02 mM or more and 0.1 mM or less, or 0.05 mM or more and 0.7 mM or less relative to the total volume of the culture medium.
  • It may be 0.05 mM or less, 0.05 mM to 0.5 mM, 0.05 mM to 0.3 mM, 0.05 mM to 0.1 mM, 0.07 mM to 1 mM, 0.07 mM to 0.7 mM, 0.07 mM to 0.5 mM, 0.07 mM to 0.3 mM, or 0.07 mM to 0.1 mM.
  • TGF- ⁇ (transforming growth factor- ⁇ ) is a cytokine belonging to the TGF- ⁇ family, and three isoforms exist in mammals (specifically, TGF- ⁇ 1, TGF- ⁇ 2, and TGF- ⁇ 3).
  • TGF- ⁇ plays an important role in cell proliferation, cell death, cell differentiation, immune regulation, cell motility, and other processes.
  • TGF- ⁇ may be any of TGF- ⁇ 1, TGF- ⁇ 2, and TGF- ⁇ 3.
  • the concentration of TGF- ⁇ in the medium is not particularly limited, as long as it allows the thickness of the three-dimensional cell tissue to exceed 50 ⁇ m for at least three days after culture in a medium containing ascorbic acid and TGF- ⁇ , and may be 0.05 ng/mL to 15 ng/mL, 0.05 ng/mL to 12 ng/mL, 0.05 ng/mL to 10 ng/mL, or 0.05 ng/mL to 7 ng/mL.
  • it may be 0.05 ng/mL or more and 5 ng/mL or less, 0.07 ng/mL or more and 15 ng/mL or less, 0.07 ng/mL or more and 12 ng/mL or less, 0.07 ng/mL or more and 10 ng/mL or less, 0.07 ng/mL or more and 7 ng/mL or less, 0.07 ng/mL or more and 5 ng/mL or less, or 0.1 ng/mL or more and 15 ng/mL or less.
  • the culture time for culturing the cell structure in a medium containing ascorbic acid or a derivative thereof, TGF- ⁇ , and a target compound may be long enough to convert the fibroblasts in the cell structure into CAFs, and may be, for example, 24 to 96 hours, preferably 48 to 96 hours, and more preferably 48 to 72 hours. Furthermore, hydrodynamic stress such as reflux can be applied as needed, as long as it does not significantly change the culture environment.
  • immune cells are added to the cell structure and cultured. Addition of immune cells to the cell structure is preferably carried out by replacing the culture medium with new culture medium containing immune cells.
  • the culture time after adding immune cells is not particularly limited, and can be, for example, 24 to 96 hours, preferably 48 to 96 hours, and more preferably 48 to 72 hours.
  • hydrodynamic stress such as reflux can be applied as needed, as long as it does not significantly change the culture environment.
  • CAFs include the application of TGF- ⁇ family ligands, lipid mediator lysophosphotidic acid, inflammatory cytokines (IL-1, IL-6, TNF), contact with cancer cells (Notch signaling), and physiological stress.
  • Administering ascorbic acid or a derivative thereof and TGF- ⁇ is more preferable as a method for forming CAFs, as this allows the thickness of the cell structure to be maintained for a long period of time.
  • ⁇ Evaluation process> The ability of a target compound to inhibit CAF formation is evaluated using as an index the number of viable cancer cells in the cell structure after the step of adding immune cells to the cell structure and culturing it.
  • a target compound has the ability to inhibit CAF formation
  • the amount of CAFs in the cell structure is lower than when cultured in a medium not containing the target compound, making it easier for immune cells to infiltrate, and therefore the number of surviving cancer cells is lower.
  • the higher the ability of a target compound to inhibit CAF formation the lower the number of surviving cancer cells.
  • the number of surviving cancer cells when cultured in a medium containing the target compound is similar to that when cultured in a medium not containing the target compound.
  • the target compound is evaluated as having the ability to inhibit CAF formation; if the number of surviving cancer cells is the same or higher, the target compound is evaluated as not having the ability to inhibit CAF formation.
  • the compound with a lower number of surviving cancer cells is evaluated as having a higher ability to inhibit CAF formation.
  • the number of viable cancer cells in a cell structure can be evaluated using a signal correlated with the number of viable cancer cells or the amount of viable cancer cells present. It is sufficient to be able to measure the number of viable cancer cells at the time of evaluation; it is not necessary to measure them while they are still alive.
  • cancer cells can be labeled to distinguish them from other cells, and the signal from the label can be used as an indicator for examination. For example, by fluorescently labeling cancer cells and then assessing their viability, it is possible to directly count the viable cancer cells in the cell structure. Image analysis techniques can also be used in this case. Cell viability can be assessed using known cell viability assessment methods such as trypan blue staining and PI (propidium iodide) staining.
  • Fluorescent labeling of cancer cells can be performed using known techniques, such as immunostaining, which uses an antibody against a substance specifically expressed on the cell surface of cancer cells as a primary antibody and a fluorescently labeled secondary antibody that specifically binds to the primary antibody.
  • Cell viability assessment and viable cell count measurement can be performed in the cell structure, or after the cell structure has been disrupted to the single-cell level. For example, after labeling cancer cells and dead cells and destroying the three-dimensional structure of the cell structure, it is possible to directly count only the cancer cells that were alive at the time of evaluation using FACS (fluorescence activated cell sorting) using the label as an indicator.
  • FACS fluorescence activated cell sorting
  • the number of viable cancer cells in the cell structure can also be measured over time.
  • Cancer cells in the cell structure can be labeled after the cell structure is constructed, or they can be labeled in advance before the cell structure is constructed.
  • the cancer cells can be labeled in advance before the cell structure is constructed.
  • Other cells derived from the cancer patient may also be similarly labeled along with the cancer cells.
  • the number of viable cancer cells can also be assessed by measuring the fluorescence intensity of the lysate obtained by lysing the cell structure using a microplate reader, etc.
  • the evaluation method according to this embodiment uses a cell structure with a stroma similar to the structure of the tissue surrounding cancer cells in an actual living organism, and performs evaluation in vitro in an environment closer to that of in vivo, thereby enabling a highly reliable evaluation of the target compound's ability to inhibit CAF formation.
  • Compounds evaluated as having CAF formation inhibitory ability using the evaluation method according to this embodiment are expected to exhibit sufficient CAF formation inhibitory ability when actually administered to cancer patients, and to achieve even greater therapeutic effects when combined with cancer immunotherapy or anticancer drug treatment.
  • the evaluation method according to this embodiment can be used as an unprecedented in vitro drug efficacy evaluation tool in screening candidate CAF formation inhibitor compounds and drug repositioning screening in drug discovery settings, and in selecting and determining anticancer drug treatments (single agents and combination agents) in clinical settings (anticancer drug sensitivity testing).
  • Immunotherapeutic agents are drugs that achieve anti-cancer effects by improving immune function, such as by activating the immune function or motility of immune cells.
  • immunotherapeutic agents include drugs used in biological response modifier therapy (hereinafter abbreviated as "BRM agents"), cytokine-based agents formed from cytokines secreted by immune cells and involved in migration and infiltration, cancer immune checkpoint inhibitors, cancer vaccines, and cancer viruses, which have attracted attention in recent years.
  • BRM agents include krestin, lentinan, and OK-432.
  • cytokine-based agents include interleukins such as IL-8 and IL-2; interferons such as IFN- ⁇ , IFN- ⁇ , and IFN- ⁇ ; and chemokines such as CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11, CXCL16/CXCR6, and CX3CL1/CX3CR1.
  • the compound selection method includes culturing a cell structure containing fibroblasts and cancer cells in a medium containing ascorbic acid or an ascorbic acid derivative, TGF- ⁇ , and a target compound; after culturing the cell structure, adding immune cells to the cell structure and further culturing the cell structure; and counting the number of viable cancer cells in the cell structure after adding the immune cells and culturing the cell structure.
  • the compound selection method of this embodiment involves culturing a cell structure containing fibroblasts and cancer cells in a medium containing ascorbic acid or an ascorbic acid derivative, TGF- ⁇ , and a target compound, and, after culturing the cell structure, adding immune cells to the cell structure and further culturing it.
  • the cell structure is the same as the cell structure described in the method for evaluating the ability to inhibit CAF formation.
  • ascorbic acid or an ascorbic acid derivative and TGF- ⁇ are CAF-forming agents that cause fibroblasts in the cell structure to become CAFs. Therefore, if the target compound does not have the ability to inhibit CAF formation, the cell structure, specifically the fibroblasts in the cell structure, will become CAFs. If the target compound has the ability to inhibit CAF formation, the cell structure, specifically the fibroblasts in the cell structure, will be inhibited from becoming CAFs.
  • the number of viable cancer cells in the cell structure after adding immune cells and culturing is counted. If the target compound has the ability to inhibit CAF formation, when cultured in a medium containing the target compound, the amount of CAFs in the cell structure is lower than when cultured in a medium without the target compound, making it easier for immune cells to infiltrate, and therefore fewer cancer cells survive. The higher the target compound's ability to inhibit CAF formation, the fewer the number of surviving cancer cells. If the target compound does not have the ability to inhibit CAF formation, the number of surviving cancer cells when cultured in a medium containing the target compound will be similar to when cultured in a medium without the target compound.
  • the target compound is selected as a compound capable of inhibiting CAF formation. If the number of surviving cancer cells is the same or higher compared to when cultured under the same conditions except for the absence of the target compound, the target compound is evaluated as not having the ability to inhibit CAF formation.
  • the one with the lower number of surviving cancer cells is selected as the compound with the greater ability to inhibit CAF formation.
  • kits for evaluating CAF formation inhibitors which is a kit containing reagents and the like used in the evaluation method and selection method according to this embodiment.
  • a kit can be formed from a cell structure containing at least fibroblasts and cancer cells and a cell culture vessel for accommodating the cell structure.
  • the kit can also include, instead of the cell structure, cells that constitute the cell structure and a culture vessel used in producing the cell structure.
  • the kit may further comprise other substances used in the evaluation and selection methods.
  • other substances include ascorbic acid or its derivatives, TGF- ⁇ , cell structure culture media, labeling substances for labeling cancer cells, reagents for determining cell viability, and substances used in constructing cell structures (e.g., cationic buffer solutions, strong electrolyte polymers, extracellular matrix components, etc.).
  • DMEM fetal bovine serum
  • general-purpose medium 10% FBS (fetal bovine serum) and 1% P/S
  • general-purpose medium 10% FBS (fetal bovine serum)
  • AIM-V medium containing 1% HEPES, 55 mM 2-ME, and 1% P/S
  • T cell medium was used as the culture medium.
  • the culture vessel used was a "Transwell Culture Insert” (model number: 3470, manufactured by Corning).
  • ⁇ Cell> Normal human dermal fibroblasts (NHDF) (manufactured by Lonza, product number: CC-2509) and human umbilical vein endothelial cells (HUVEC) (manufactured by Lonza, product number: C2517A) were used.
  • Human colon adenocarcinoma-derived HCT15/ ⁇ 2m cells genetically modified to constitutively express green fluorescent protein (GFP) (provided by Sapporo Medical University) were used as cancer cells.
  • GFP green fluorescent protein
  • a stromal cell layer was formed. Specifically, NHDFs (1 x 10 cells) and HUVECs (1.5 x 10 cells) were suspended in Tris-HCl buffer (0.1 mg/mL heparin, 0.1 mg/mL collagen, 50 mM Tris, pH 7.4) containing heparin and collagen to prepare a cell suspension. This cell suspension was centrifuged at 1,000 x g for 2 minutes at room temperature, the supernatant was removed, and the cell suspension was resuspended in 300 ⁇ L of general-purpose medium.
  • Tris-HCl buffer 0.1 mg/mL heparin, 0.1 mg/mL collagen, 50 mM Tris, pH 7.4
  • a cancer cell layer was formed on the formed stromal layer. Specifically, first, the medium outside the culture vessel was removed, and 1 mL of new general-purpose medium was added. Next, the medium inside the culture vessel insert was removed, and a cancer cell suspension (1 x 10 cancer cells suspended in 100 ⁇ L of general-purpose medium) was added to the culture vessel insert, and the culture vessel insert was left to stand in a CO2 incubator (37°C, 5% CO2 ) for 2 hours.
  • a CO2 incubator 37°C, 5% CO2
  • a stromal cell layer was then formed on the cancer cell layer.
  • NHDFs (1 x 10 cells) and HUVECs (1.5 x 10 cells) were first suspended in a Tris-HCl buffer solution containing heparin and collagen (0.1 mg/mL heparin, 0.1 mg/mL collagen, 50 mM Tris, pH 7.4).
  • the resulting cell suspension was centrifuged at 1,000 x g for 2 minutes at room temperature. The supernatant was removed and the cell suspension was resuspended in 200 ⁇ L of general-purpose medium.
  • the resulting cell suspension was then added to the culture vessel insert containing the cancer cell layer, centrifuged at 400 x g for 2 minutes at room temperature, and then allowed to stand for 2 hours in a CO2 incubator (37°C, 5% CO2 ). This resulted in the formation of a cell structure in which the cancer cell layer was sandwiched between stromal cell layers.
  • ⁇ CAF conversion> The general-purpose medium outside the culture vessel insert in which the cell constructs were formed was removed, and 2.3 mL of general-purpose medium containing 100 ⁇ M AscP and 1 ng/mL TGF- ⁇ 1 was added. The culture vessel was then cultured in a CO2 incubator (37°C, 5% CO2 ) for 3 days (CAF-formed cell constructs). As a control, 2.3 mL of general-purpose medium not containing AscP and TGF- ⁇ 1 was added, and the cells were cultured in the same manner to prepare a control (CTL) cell construct.
  • CTL control
  • TCR-T ⁇ Addition of TCR-T>
  • the medium outside the culture vessel was removed, 1 mL of T cell medium was added, and then the medium inside the culture vessel insert was removed.
  • a TCR-T cell suspension (a suspension of TCR-T cells in 300 ⁇ L of T cell medium) was added to the culture vessel insert, and the cells were cultured for 3 to 7 days in a CO2 incubator (37°C, 5% CO2 ).
  • TCR-T ⁇ Fluorescently labeled TCR-T>
  • the TCR-T added to the cell construct was fluorescently labeled in advance, and the state of infiltration of TCR-T into cancer cells in the cell construct was evaluated.
  • the TCR-T was fluorescently labeled with an anti-CD8 antibody (CD8_Alexa647, 557708, manufactured by BD) labeled with a fluorescent substance (Alexa647).
  • TCR-T cells (2 x 10 cells) were suspended in 50 ⁇ L of 1% BSA-containing PBS, and 2 ⁇ L of fluorescently labeled antibody solution was added and mixed. The mixture was then left to stand at 4°C for at least 30 minutes.
  • T cell medium After washing with 500 ⁇ L of 1% BSA-containing PBS, the cells were suspended in T cell medium to achieve an appropriate cell number. The resulting fluorescently labeled TCR-T cell suspension was added to a culture vessel insert and cultured for 3 to 7 days in a CO2 incubator (37°C, 5% CO2 ).
  • GFP expressed in cancer cells was photographed using a microscope system ("OperettaCLS," manufactured by PerkinElmer) so that the entire cell structure in the culture vessel insert was captured within the imaging range.
  • the obtained images were analyzed to calculate the percentage of live cancer cells (cells in which GFP was detected) occupying the insert (the number of cells in a confluent state is set to 100%) as the cancer cell survival rate (%).
  • the cancer cell survival rate (%) of the control cell structure to which TCR-T was not added was set to 100%, and the cancer cell survival rate (%) of the cell structure to which TCR-T was added was calculated as the relative cancer cell survival rate (%).
  • ⁇ Section evaluation> The cell structures subjected to viable cell count analysis were washed twice with PBS and then fixed by adding 300 ⁇ L of 10% formalin buffer and leaving it to stand for at least 15 minutes. The fixed cell structures were then washed three times with PBS. The fixed cell structures were then embedded in paraffin using a low-toxicity solvent ("G-Nox,” manufactured by Genostaff) and a paraffin embedding device ("CT-Pro20,” manufactured by Genostaff), and 6 mm-thick sections were prepared.
  • G-Nox low-toxicity solvent
  • CT-Pro20 paraffin embedding device
  • paraffin sections were immersed in citrate buffer (pH 6) and heated in a microwave oven to activate the antigen, and then stained with anti- ⁇ -SMA (alpha smooth muscle actin) antibody (mouse monoclonal antibody, M0851, manufactured by Dako) (antibody concentration: 0.2 ⁇ g/mL).
  • anti- ⁇ -SMA alpha smooth muscle actin
  • the paraffin sections were then immersed in proteinase K solution (5 ⁇ g/mL) for enzymatic treatment to activate the antigen, and then stained with anti-fibronectin antibody (mouse monoclonal antibody, ab6328, manufactured by abcam) (antibody concentration: 0.4 ⁇ g/mL).
  • the stained paraffin sections were placed on glass slides and observed using an optical microscope (MX51, Olympus), and images were obtained.
  • Figure 1 shows the results of measuring the relative cancer cell survival rate (%) for each number of cells seeded for cell structures converted into CAFs using AscP and TGF- ⁇ 1, and for control cell structures that had not been converted into CAFs.
  • the relative cancer cell survival rate for cell structures converted into CAFs (referred to as “CAF” in the figure) was clearly higher than for control cell structures (referred to as "CNT” in the figure). This is thought to be because CAF conversion made it difficult for TCR-T to infiltrate into the cell structures, resulting in a decrease in the amount of TCR-T that reached the cancer cells.
  • FIG 2 shows stained images of paraffin sections of each cell structure.
  • "HE” indicates a hematoxylin-eosin stained image.
  • the cell structure converted into CAF (“CAF” in Figure 2) had increased amounts of ⁇ -SMA, fibronectin, and collagen compared to the control cell structure ("CNT" in Figure 2).
  • Example 1 Several drugs (low molecular weight compounds) were examined for their ability to inhibit CAF formation. Specifically, the relative cancer cell survival rate (%) of CAF-formed cell structures and control cell structures was calculated in the same manner as in Reference Example 1, except that drugs were added to the cell structures simultaneously with AscP and TGF- ⁇ 1 at various concentrations. For drug-free controls, DMSO was added in an amount equal to that of the drug. For comparison, the relative cancer cell survival rate (%) was also calculated for cell structures cultured in the same manner with the addition of only the drug, without the addition of TCR-T cells.
  • HDAC histone deacetylase
  • TSA histone deacetylase
  • JQ-1 BET family inhibitor JQ-1
  • Figure 4(A) shows the general behavior of relative cancer cell survival rate (%) when a drug without CAF formation inhibitor was added
  • Figure 4(B) shows the results of measuring the relative cancer cell survival rate (%) of cell structures transformed into CAFs with the addition of TSA
  • Figure 4(C) shows the results of measuring the relative cancer cell survival rate (%) of cell structures transformed into CAFs with the addition of JQ-1.
  • drug only indicates the relative cancer cell survival rate (%) of cell structures to which only the drug was added
  • drug + immune cells indicates the relative cancer cell survival rate (%) of cell structures to which TCR-T cells were added after the drug was added.
  • the relative cancer cell survival rate (%) is lower when TCR-T cells are added, but the relative cancer cell survival rate (%) does not change even when the drug concentration is changed. This is because the drug itself does not have the effect of promoting the cytotoxicity of TCR-T cells.
  • the relative cancer cell survival rate (%) of the TSA-added cell constructs on days 7 and 11 of culture was measured using drug combination effect analysis software (Synergy Finder) to determine whether TCR-T cells and TSA had a synergistic effect on cancer cell cytotoxicity.
  • the resulting synergy score was 21.651 from the results on day 7 of culture, and 28.214 from the results on day 11 of culture, indicating that there was a synergistic effect between TCR-T cells and TSA.
  • FIG. 5 shows fluorescent images (GFP/CD8-Alexa647) taken immediately after the addition of the TCR-T cells ("0 h” in the figure) and 24 hours after the addition and culture ("24 h” in the figure).
  • the accumulation of fluorescently labeled TCR-T cells around the cancer cells was suppressed and not observed in the cell constructs without TSA ("DMSO" in the figure).
  • TSA in the cell constructs with TSA added
  • TSA and JQ-1 promoted the cytotoxicity of TCR-T cells against cancer cells, and accumulation of TCR-T cells in cancer cells was also observed. Therefore, TSA and JQ-1 were assessed to have the ability to suppress CAF formation.
  • a cell structure consisting of NHDF and HUVEC was formed in the same manner as in Reference Example 1, except that a cancer cell layer was not formed.
  • the cell structure was cultured for three days in a general-purpose medium containing AscP, TGF- ⁇ 1, and TSA in the combinations shown in Figure 6.
  • the cell structures were lysed in SDS lysis buffer, heated at 100°C for 5 minutes, and then stirred in a vortex mixer for 30 seconds. The supernatant was then centrifuged at 16,500 x g for 10 minutes and collected. The protein content of the supernatant was quantified using a protein quantification kit (Protein Assay BCA Kit, Fujifilm Wako Pure Chemical Corporation). 2 ⁇ g of supernatant protein was mixed with 6x SDS-PAGE Sample Buffer (final concentration 1x, Cosmo Bio Co., Ltd.) and RIPA Buffer (adjusted to 10 ⁇ L, Fujifilm Wako Pure Chemical Corporation) to prepare the electrophoresis sample.
  • a protein quantification kit Protein Assay BCA Kit, Fujifilm Wako Pure Chemical Corporation
  • the prepared electrophoresis samples and molecular weight markers were electrophoresed (SDS-PAGE). Proteins were transferred from the gel after SDS-PAGE to a PVDF membrane. After transfer, the membrane was blocked with 4% BSA/TBS-T (1% Tween-20, 1x TBS Buffer) and then reacted with primary antibody solution overnight at 4°C.
  • the primary antibodies used were anti-COL1A1 (E8F4L) antibody (72026, CST), anti- ⁇ -SMA (D4K9N) antibody (19245, CST), and anti-GAPDH monoclonal antibody (016-25523, Fujifilm Wako Pure Chemical Industries). The membrane was then washed with TBS-T and reacted with secondary antibody solution for 1 hour with shaking.
  • the secondary antibodies used were Amersham ECL-specific HRP-conjugated rabbit IgG full-length antibody (NA934V, Cytiva) and Amersham ECL-specific HRP-conjugated mouse IgG full-length antibody (NA931, Cytiva). After washing again with TBS-T, the bands detected by each antibody were detected using the detection reagent "SuperSignal® West Femto Maximum Sensitivity Substrate” (34095, Thermo Fisher Scientific) and the image analyzer "Amersham Imager 600" (GE Healthcare).
  • 1.0 x 106 cells of human neonatal dermal fibroblasts NHDF (product number "CC-2509”, Lonza) and 1.5 x 104 cells of human umbilical vein endothelial cells HUVEC (product number "C2517A”, Lonza) were suspended in 50 mM Tris-HCl buffer solution (pH 7.4) containing 0.1 mg/mL heparin (product number "H3149-100KU”, Sigma) and 0.1 mg/mL collagen (product number "ASC-1-100-100", Sigma).
  • the cell suspension was then centrifuged at 1,000 x g (gravitational acceleration) for 2 minutes at room temperature, the supernatant removed, and the cells resuspended in 300 ⁇ L of general-purpose medium.
  • the resulting cell suspension was then seeded into a 24-well cell culture insert (product number "3470", Corning) pre-coated with 0.1 mg/mL fibronectin, with 1 mL of general-purpose medium added to the outside.
  • the cell culture insert was then centrifuged at 400 ⁇ g for 2 minutes at room temperature and then placed in a CO2 incubator (37°C, 5% CO2 ) for 2 hours. 1 mL of general-purpose medium was then added to the outside of the cell culture insert, and the insert was cultured in a CO2 incubator (37°C, 5% CO2 ) for 24 hours (the start of this culture was defined as the start of the culture).
  • ⁇ Dissemination of cancer cells>> The medium outside the cell culture insert was removed, and 1 mL of general-purpose medium was added. Subsequently, the medium inside the culture insert was removed, and 100 ⁇ L of a suspension of 1 ⁇ 10 3 human colon adenocarcinoma cells HCT15/ ⁇ 2m was suspended in 100 ⁇ L of general-purpose medium. The resulting suspension was seeded into the cell culture insert, and the insert was left to stand in a CO 2 incubator (37°C, 5% CO 2 ) for 2 hours.
  • ⁇ Layer of interstitial cells 1.0 ⁇ 10 NHDF cells and 1.5 ⁇ 10 HUVEC cells were suspended in 50 mM Tris-HCl buffer solution (pH 7.4) containing 0.1 mg/mL heparin (product number "H3149-100KU”, Sigma) and 0.1 mg/mL collagen (product number "ASC-1-100-100", Sigma).
  • the resulting cell suspension was then centrifuged at 1,000 x g for 2 minutes at room temperature, the supernatant was removed, and the cells were resuspended in 200 ⁇ L of general-purpose medium.
  • the 200 ⁇ L of general-purpose medium containing the suspended cells was then layered onto the cell culture insert on which the cancer cells had been seeded.
  • the cell culture insert obtained above was left to stand in a CO 2 incubator (37° C., 5% CO 2 ) for 2 hours.
  • the medium inside and outside the culture insert was then removed, and 2.3 mL of general-purpose medium with an ascorbic acid concentration of 0 mM, 0.05 mM, or 0.1 mM and a TGF- ⁇ concentration of 0 ng/mL, 0.1 ng/mL, 1 ng/mL, 5 ng/mL, or 10 ng/mL was added, followed by culturing for 3 days in a CO2 incubator (37°C, 5% CO2 ).
  • Figure 7 shows images of cell structures observed under a microscope after 7 days of culture in media containing various concentrations of ascorbic acid and TGF- ⁇ . As shown in Figure 7, when cells were cultured in media containing ascorbic acid, cell structures were formed. In contrast, when cells were cultured in media containing only TGF- ⁇ without ascorbic acid, the cell tissue shrank and detached from the cell culture insert, and no cell structures were formed.
  • ⁇ Measuring the thickness of cell structures>> The cell construct was then removed from the cell culture insert and embedded in paraffin. 6 mm thin sections were prepared along a line passing through the center of gravity of the cell construct as viewed from the top of the cell culture insert. The thin sections were then stained with hematoxylin and eosin (HE). The HE-stained thin sections were photographed under an optical microscope (MX51, Olympus Corporation), and the maximum thickness of the cell construct was measured using ImageJ.
  • HE hematoxylin and eosin
  • Figure 8 shows images of hematoxylin-eosin stained cell structures obtained by microscopy after 4 and 7 days of culture in each medium.
  • Figure 9 is a graph showing the thickness of the cell structures after 4 and 7 days of culture in each medium.
  • "General Medium” indicates cell structures cultured in a general medium that does not contain ascorbic acid or TGF- ⁇
  • “Ascorbic Acid-Added Medium” indicates cell structures cultured in a general medium containing 0.1 mM ascorbic acid
  • “Ascorbic Acid/TGF- ⁇ -Added Medium” indicates cell structures cultured in a general medium containing 0.1 mM ascorbic acid and 10 ng/L TGF- ⁇ .
  • general-purpose medium indicates cell constructs cultured in a general-purpose medium containing no ascorbic acid or TGF- ⁇
  • "ascorbic acid added” indicates cell constructs cultured in a general-purpose medium containing 0.1 mM ascorbic acid
  • "ascorbic acid/TGF- ⁇ added” indicates cell constructs cultured in a general-purpose medium containing 0.1 mM ascorbic acid and 1 ng/L TGF- ⁇ .
  • Example 3 (Production of cell structure 3) As NHDF, 2.0 ⁇ 10 6 cells of human neonatal dermal fibroblast NHDF were seeded in a square dish (model number "166508", Nunc) containing 90 mL of general-purpose medium or general-purpose medium containing 0.1 mM ascorbic acid, and cultured for 24 hours in a CO 2 incubator (37°C, 5% CO 2 ). Except for this, the preparation of stromal cells, seeding of cancer cells, and layering of stromal cells were carried out in the same manner as in Experimental Example 1.
  • the medium inside and outside the cell culture insert was removed, and 2.3 mL of general-purpose medium or general-purpose medium containing 0.1 mM ascorbic acid was added, followed by culturing for 3 days in a CO2 incubator (37°C, 5% CO2 ). Then, the medium inside and outside the culture insert was removed, and 2.3 mL of general-purpose medium or general-purpose medium containing 0.1 mM ascorbic acid was added, followed by culturing for 3 days in a CO2 incubator (37°C, 5% CO2 ).
  • the evaluation method according to this embodiment is a method that can provide a more reliable evaluation of the ability to inhibit CAF formation in cancer tissues without using an animal model. Therefore, this evaluation method can be used in drug discovery for the development of new CAF formation inhibitors and drug repositioning screening, etc.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé d'évaluation d'une capacité d'inhibition de la formation de CAF qui comprend : une étape de formation de CAF pour la culture d'une structure cellulaire contenant des fibroblastes et des cellules cancéreuses dans un milieu de culture contenant de l'acide ascorbique ou un dérivé de celui-ci, du TGF-β et un composé cible d'évaluation ; une étape de culture pour ajouter des cellules immunitaires à la structure cellulaire après l'étape de formation de CAF et cultiver par après la structure cellulaire ; et une étape d'évaluation pour évaluer une capacité d'inhibition de formation CAF du composé cible d'évaluation en utilisant, en tant qu'indicateur, le nombre de cellules vivantes hors des cellules cancéreuses dans la structure cellulaire après l'étape de culture.
PCT/JP2025/009965 2024-03-21 2025-03-14 Procédé d'évaluation de la capacité d'inhibition de la formation de caf Pending WO2025197801A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2024-045761 2024-03-21
JP2024045761 2024-03-21

Publications (1)

Publication Number Publication Date
WO2025197801A1 true WO2025197801A1 (fr) 2025-09-25

Family

ID=97139662

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2025/009965 Pending WO2025197801A1 (fr) 2024-03-21 2025-03-14 Procédé d'évaluation de la capacité d'inhibition de la formation de caf

Country Status (1)

Country Link
WO (1) WO2025197801A1 (fr)

Similar Documents

Publication Publication Date Title
JP7452609B2 (ja) 抗がん効果の評価方法、及びがん免疫療法の奏効性予測方法
JP7331979B2 (ja) 抗がん剤の評価方法
JP7589282B2 (ja) 初代培養方法
JP2022105712A (ja) 被検化合物の脈管新生阻害活性の評価方法
JP2024087068A (ja) 細胞構造体及びその使用
Zhang et al. Adjustable extracellular matrix rigidity tumor model for studying stiffness dependent pancreatic ductal adenocarcinomas progression and tumor immunosuppression
JP2022022300A (ja) ガン化の可能性評価方法
JP7251595B2 (ja) 薬剤耐性を有する細胞を作製する方法、及び抗ガン剤をスクリーニングするための方法
US20240218418A1 (en) Method for evaluating migration ability of migratory cells
WO2025197801A1 (fr) Procédé d'évaluation de la capacité d'inhibition de la formation de caf
US20240426809A1 (en) Evaluation method of anticancer drug and selection method of anticancer drug
US20250120372A1 (en) Humanized mouse model of fibrosis

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25773430

Country of ref document: EP

Kind code of ref document: A1