JP2022165800A - Medium composition for feline breast tumor organoid, and method for producing feline breast tumor organoid - Google Patents

Abstract

To provide a medium composition optimal for culture of feline breast tumor organoids.SOLUTION: A composition comprises at least one component selected from the group consisting of GF2, FGF7, FGF10 and TGF-α.SELECTED DRAWING: Figure 5

Description

The present invention relates to a medium composition for culturing organoids prepared from feline mammary gland tumors and a method for producing feline mammary gland tumor organoids using the medium composition.

Three-dimensional organoid culture method (Non-Patent Document 1: Sato et al., Nature, 2009) mixes epithelial cells isolated from various organs with Matrigel to enhance stemness of Wnt, Noggin, R-spondin, etc. It was developed as a method that reproduces a three-dimensional epithelial tissue structure on a culture dish by culturing in a special medium containing factors. In recent years, an organoid culture method using surgical specimens from patients such as human colon cancer and pancreatic cancer has been established, and structural similarities with tissues immediately after excision and correlations with gene mutations have been shown (Non-Patent Document 2: Wetering et al., Cell, 2015 and Non-Patent Document 3: Boj et al., Cell, 2015), it is also expected to be a useful tool for personalized medicine.

In addition, a technology was developed to non-invasively culture bladder cancer organoids using urine samples from dogs with bladder cancer. It has been clarified that it can be applied to tumor re-formation ability tests in deficient mice and anticancer drug sensitivity tests of individual patient animals (Non-Patent Document 4: Elbadawy and Usui et al., Cancer Sci. 2019).

By the way, mammary gland tumors account for about 17% of cat tumors, the third most common, and are known to be malignant tumors with high malignancy and low survival rate. Since mammary gland tumors have a high recurrence rate, long-term treatment poses a problem of physical burden on patients and economic burden on owners. Currently, surgical therapy is the first-choice treatment for mammary gland tumors in cats, and chemotherapy is used for highly malignant cases, cases unsuitable for surgery, and palliative care. anticancer drug therapy after surgical resection is recommended.

Anticancer agents are often empirically selected by veterinarians, and there are no established treatment protocols. On the other hand, since there are no commercially available cultured cells for feline mammary tumors, research leading to new treatment methods has stalled, and the establishment of effective anticancer drug treatments is an urgent issue. It's becoming

Sato et al., Nature, 459(7244):262-5, 2009 Wetering et al., Cell, 161(4):933-45, 2015 Boj et al., Cell, 160(1-2):324-38, 2015 Elbadawy and Usui et al., Cancer Sci. 110(9):2806-2821, 2019

The organoid culture method, which is currently attracting attention, uses a gel and a culture solution that stimulates stem cells and promotes proliferation, thereby maintaining the diversity and stemness of cells and improving the in vivo microenvironment. It is a reproducible method. In recent years, attention has been paid to the use of cancer organoids obtained by applying this method for basic cancer and clinical research, but no organoid culture method has been established for feline mammary gland tumors.

Therefore, the present invention prepares a feline mammary gland tumor organoid using mammary gland tumor tissue surgically removed from a cat suffering from mammary gland tumor, and provides an optimal medium composition for culturing the feline mammary gland tumor organoid, and the feline mammary gland tumor organoid medium. , to provide a method for producing feline mammary tumor organoids.

As a result of intensive studies conducted by the present inventors in order to achieve the above-described object, the presence of specific components in culturing feline mammary gland tumor organoids found that the proliferation rate was significantly improved, and completed the present invention. reached. The present invention includes the following.

[1] A composition for feline mammary tumor organoid medium, comprising at least one component selected from the group consisting of FGF2, FGF7, FGF10 and TGF-α.
[2] The feline mammary gland tumor organoid medium composition of [1], further comprising at least one component selected from the group consisting of Wnt agonists, BMP inhibitors, EGF and TGFβ inhibitors.
[3] The composition for feline mammary gland tumor organoid medium according to [1], wherein the component is FGF10.
[4] A feline mammary gland tumor organoid medium containing at least one component selected from the group consisting of FGF2, FGF7, FGF10 and TGF-α.
[5] The feline mammary tumor organoid medium according to [4], further comprising at least one component selected from the group consisting of Wnt agonists, BMP inhibitors, EGF and TGFβ inhibitors.
[6] The feline mammary gland tumor organoid medium according to [4], wherein the component is FGF10.
[7] A method for producing feline mammary gland tumor organoids, comprising culturing feline mammary gland tumor organoids in the feline mammary gland tumor organoid medium according to any one of [4] to [6].
[8] The method for producing feline mammary gland tumor organoids according to [7], further comprising the step of producing the feline mammary gland tumor organoids from mammary tissue collected from a cat suffering from mammary gland cancer.
[9] preparing feline mammary tumor organoids from mammary glands collected from cats suffering from mammary adenocarcinoma;
a step of culturing the feline mammary gland tumor organoids in the feline mammary gland tumor organoid medium according to any one of [4] to [6]; A method of treating a cat with mammary adenocarcinoma, comprising the step of measuring susceptibility.
[10] The method of treatment according to [9], further comprising the step of administering a drug having high drug susceptibility as a result of measuring the drug susceptibility to the mammary adenocarcinoma-affected cat.

The feline mammary gland tumor organoid culture medium composition according to the present invention can significantly improve the cell growth rate of feline mammary gland tumor organoids by containing specific components. Therefore, by using the feline mammary gland tumor organoid medium composition, it is possible to efficiently produce feline mammary gland tumor organoids that can be used for basic research on feline mammary gland tumors and for treatment of cats suffering from mammary gland tumors.

In addition, since the method for producing feline mammary gland tumor organoids according to the present invention uses a medium containing specific components, feline mammary gland tumor organoids can be propagated with excellent cell culture efficiency. Therefore, by using the method for producing feline mammary gland tumor organoids, it is possible to efficiently produce feline mammary gland tumor organoids that can be used for basic research on feline mammary gland tumors and for treatment of cats suffering from mammary gland tumors.

FIG. 10 is a photograph of three types of feline mammary gland tumor organoids produced in Examples. FIG. It is a photograph of HE staining of feline mammary gland tumor organoids and tumor tissues. FIG. 11 is a photograph of immunohistochemical staining showing the expression pattern of hormone receptors (HER2, ER and PR) for feline mammary tumor organoids and tumor tissues. 1 is a photograph of feline mammary gland tumor organoids produced in Examples, cultured in the presence of specific medium components. FIG. 2 is a characteristic diagram showing the results of measuring cell proliferation efficiency when feline mammary tumor organoids prepared in Examples were cultured in the presence of specific medium components. FIG. 2 is a characteristic diagram showing the results of an anticancer drug sensitivity test on feline mammary gland tumor organoids produced in Examples.

The present invention will be described in detail below.
The feline mammary gland tumor organoid medium composition according to the present invention comprises at least one component selected from the group consisting of FGF2, FGF7, FGF10 and TGF-α. Each of these FGF2, FGF7, FGF10 and TGF-α can significantly improve the growth efficiency of feline mammary tumor organoids. That is, at least one component selected from the group consisting of FGF2, FGF7, FGF10 and TGF-α is used in a medium for culturing feline mammary tumor organoids. The feline mammary gland tumor organoid is not particularly limited, and can be obtained, for example, by a method referring to Sato et al., Nature, 2009 or Sato T et al., Gastroenterology. 2011 Nov;141(5):1762-72. be able to. The feline mammary gland tumor organoid medium composition according to the present invention is used as a medium for culturing feline mammary gland tumor organoids obtained by the method.

The feline mammary gland tumor organoid culture medium composition according to the present invention may further contain a part or all of the medium components for culturing feline mammary gland tumor organoids described below. When the feline mammary gland tumor organoid medium composition according to the present invention contains part of the medium components for culturing feline mammary gland tumor organoids described later, it can be used together with the rest of the medium components as a feline mammary gland tumor organoid medium. . Moreover, when the feline mammary gland tumor organoid medium composition according to the present invention contains all of the medium components for culturing feline mammary gland tumor organoids described later, it can be used as it is as a medium for feline mammary gland tumor organoids.

In the feline mammary gland tumor organoid medium composition according to the present invention, the concentration of at least one component selected from the group consisting of FGF2, FGF7, FGF10 and TGF-α is not particularly limited, and the dilution when used as a medium It can be appropriately defined according to the magnification. In addition, when the feline mammary gland tumor organoid medium composition further contains some or all of the medium components for culturing feline mammary gland tumor organoids, the concentration of these medium components is not particularly limited, and the concentration of these medium components is not particularly limited. It can be appropriately defined according to the dilution ratio.

FGF2 is a basic fibroblast growth factor known as non-glycosylated pepperin-binding growth factor. FGF2 is not particularly limited, and any animal-derived FGF2 can be used. For example, commercially available FGF2 derived from various animals such as human FGF2, mouse FGF2, or rat FGF2 can be used, or feline FGF2 prepared as a recombinant by isolating the FGF2 gene in cats can be used. good.

The FGF2 concentration contained in the medium is not particularly limited, but can be, for example, 2 ng/mL to 500 ng/mL, preferably 5 ng/mL to 500 ng/mL, and 5 ng/mL to 400 ng/mL. more preferably 5 ng/mL to 300 ng/mL, more preferably 5 ng/mL to 200 ng/mL, more preferably 5 ng/mL to 100 ng/mL, more preferably 5 ng/mL More preferably ~50 ng/mL. More specifically, the concentration of FGF2 contained in the medium can be 10 ng/mL.

FGF7 is a fibroblast growth factor, also called keratinocyte growth factor (KGF). FGF7 is not particularly limited, and any animal-derived FGF7 can be used. For example, commercially available FGF7 derived from various animals such as human FGF7, mouse FGF7, or rat FGF7 can be used, or feline FGF7 prepared as a recombinant by isolating the FGF7 gene in cats can be used. good.

The FGF7 concentration contained in the medium is not particularly limited, but can be, for example, 0.4 ng/mL to 100 ng/mL, preferably 1 ng/mL to 100 ng/mL, and 1 ng/mL to 80 ng/mL. More preferably, 1 ng / mL to 60 ng / mL, more preferably 1 ng / mL to 40 ng / mL, more preferably 1 ng / mL to 20 ng / mL, 1 ng /mL to 10 ng/mL is more preferable. More specifically, the concentration of FGF7 contained in the medium can be 5 ng/mL.

FGF10 is a fibroblast growth factor known as heparin binding growth factor. FGF10 is not particularly limited, and any animal-derived FGF10 can be used. For example, commercially available FGF10 derived from various animals such as human FGF10, mouse FGF10 or rat FGF10 can be used, or feline FGF10 prepared as a recombinant by isolating the FGF10 gene in cats can be used. good.

The FGF10 concentration contained in the medium is not particularly limited, but can be, for example, 4 ng/mL to 1000 ng/mL, preferably 10 ng/mL to 1000 ng/mL, and 10 ng/mL to 800 ng/mL. more preferably 10 ng/mL to 600 ng/mL, more preferably 10 ng/mL to 400 ng/mL, more preferably 10 ng/mL to 200 ng/mL, more preferably 10 ng/mL More preferably ~100 ng/mL. More specifically, the concentration of FGF10 contained in the medium can be 20 ng/mL.

TGF-α is a transforming growth factor-α (or transforming growth factor-α) known as a cytokine produced by monocytes, keratinocytes (keratinocytes) and various tumor cells. TGF-α is not particularly limited, and any animal-derived TGF-α can be used. For example, commercially available TGF-α derived from various animals such as human TGF-α, mouse TGF-α or rat TGF-α can be used. You may use feline TGF-alpha produced as.

The TGF-α concentration contained in the medium is not particularly limited, but can be, for example, 4 ng/mL to 1000 ng/mL, preferably 10 ng/mL to 1000 ng/mL, and 10 ng/mL to 800 ng/mL. More preferably, 10 ng / mL to 600 ng / mL, more preferably 10 ng / mL to 400 ng / mL, more preferably 10 ng / mL to 200 ng / mL, 10 ng /mL to 100 ng/mL is more preferable. More specifically, the concentration of TGF-α contained in the medium can be 20 ng/mL.

The medium components used with the feline mammary gland tumor organoid medium composition according to the present invention include those contained in a medium for culturing ordinary three-dimensional organoids. Specifically, the medium for culturing ordinary three-dimensional organoids is not particularly limited, but a serum-free basal cell culture medium can be used. The serum-free basal medium for cell culture includes, for example, a synthetic medium adjusted to about pH 7.0 to 7.6 with a carbonate-based buffer. More specifically, Dulbecco's Modified Eagle Medium/Ham's F-12 mixed medium (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12; DMEM/F12) supplemented with glutamine, insulin, penicillin or streptomycin, and transferrin. . Also included is RPMI 1640 medium (Roswell Park Memorial Institute 1640 medium) supplemented with glutamine, insulin, penicillin or streptomycin, and transferrin. Also included are Advanced-DMEM/F12 supplemented with glutamine and penicillin or streptomycin, and Advanced RPMI medium supplemented with glutamine and penicillin or streptomycin.

The serum-free basal cell culture medium may also be supplemented with further purified natural, semi-synthetic or synthetic growth factors. Growth factors include, for example, B-27 Supplement (manufactured by Thermo Fisher SCIENTIFIC), N-acetyl-L-cysteine (manufactured by Sigma), N-2 Supplement (manufactured by Thermo Fisher SCIENTIFIC) and the like.

In addition to these basal media, the organoid medium should contain a Wnt agonist that activates Wnt signaling, a BMP inhibitor that inhibits BMP signaling, an epidermal growth factor (EGF), and a TGFβ inhibitor. can be done. It is produced by culturing stem cells embedded in a scaffold support such as an extracellular matrix using these organoid-producing media. Scaffold supports used in fabricating three-dimensional organoids include polymers capable of absorbing and retaining water such as collagen and agarose, and sponge-like membranes such as porous polystyrene. More specifically, Matrigel containing collagen (manufactured by Corning Life Sciences) can be used as the scaffold support.

Wnt agonists include, for example, Wnt, Wnt-3a, Noggin, GSK inhibitors, R-spondins such as R-spondin1, R-spondin2, R-spondin3 and R-spondin4.

BMP inhibitors include Noggin, Chordin, a chordin-like protein containing a chordin domain, Follistatin, a follistatin-related protein containing a follistatin domain, DAN, a DAN-like protein containing a DAN cysteine-knot domain. , sclerostin/SOST, decorin and α-2 macroglobulin.

EGF is a 6045 Da protein consisting of 53 amino acid residues and three intramolecular disulfide bonds, and binds as a ligand to the epidermal growth factor receptor (EGFR) present on the cell surface. The concentration of EGF contained in the medium is not particularly limited, but can be, for example, 2 ng/mL to 500 ng/mL, preferably 5 ng/mL to 500 ng/mL, and 10 ng/mL to 400 ng/mL. more preferably 20 ng/mL to 300 ng/mL, more preferably 30 ng/mL to 200 ng/mL, and more preferably 40 ng/mL to 100 ng/mL. More specifically, the concentration of EGF can be 50 ng/mL.

TGFβ (transforming growth factor β) inhibitors include, for example, A83-01 (3-(6-methylpyridin-2-yl)-1-phenylthiocarbamoyl-4-quinolin-4-ylpyrazole), ALK5 Inhibitor I (3-(pyridin-2-yl)-4-(4-quinonyl)-1H-pyrazole), LDN193189 (4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a ]pyrimidin-3-yl)quinoline), SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-pyridin-2-yl-1H-imidazol-2-yl]benzamide) , SB-505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride hydrate), SD-208 ((2-(5-chloro-2-fluorophenyl)pteridin-4-yl)pyridin-4-yl-amine), SB-525334 (6-[2-(1,1-dimethylethyl)-5-( 6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline), LY-364947 (4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline), LY2157299 (4 -[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-quinoline-6-carboxylic acid amide), TGF- β RI Kinase Inhibitor II 616452 (2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine), TGF-β RI Kinase Inhibitor III 616453 (2- (5-benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, HCl), TGF-β RI Kinase Inhibitor IX 616463 (4-( (4-((2,6-dimethylpyridin-3-yl)oxy)pyridin-2-yl)amino)benzenesulfonamide), TGF-β RI Kinase Inhibitor VII 616458 (1-2-((6,7- Dimethoxy-4-quinolyl)oxy)-(4,5-dimethylphenyl)-1-ethanone), naphthyridine (6-(2-tert-butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl)-quinoxaline), AP12009 (TGF-β2 antisense compound “Trabedersen”), Belagenpumatucel -L (TGF-β2 antisense gene-modified allogeneic tumor cell vaccine), CAT-152 (Glaucoma-lerdelimumab (anti-TGF-β-2 monoclonal antibody)), CAT-192 (Metelimumab (human IgG4 monoclonal antibody)), GC-1008 (anti-TGF-β monoclonal antibody), and the like.

The concentration of the TGF-β inhibitor contained in the medium varies depending on its type and is not particularly limited. For example, when A83-01 is used as a TGF-β inhibitor, it can be 0.1 μM to 5 μM, preferably 0.2 μM to 3 μM, more preferably 0.3 μM to 1 μM, More preferably, it is 0.4 μM to 0.8 μM. More specifically, the concentration of A83-01 can be 0.5 μM.

Culture conditions and the like for feline mammary gland tumor organoids are not particularly limited. See, for example, Sato et al., Nature, 2009 and Sato T et al., Gastroenterology. be able to. When culturing feline mammary gland tumor organoids with reference to these, the culture temperature can be 30 to 40°C, most preferably about 37°C.

By using the feline mammary gland tumor organoid culture medium composition according to the present invention, feline mammary gland tumor organoids can be cultured more efficiently than by conventional culture methods. In other words, by using the feline mammary gland tumor organoid culture medium composition according to the present invention, the growth rate of feline mammary gland tumor organoids can be increased as compared to conventional culture methods. Thus, by applying the present invention, the time required for subculturing feline mammary gland tumor organoids can be shortened, and they can be obtained more efficiently than when using a normal culture medium.

The cultured feline mammary gland tumor organoids are not particularly limited and can be used for various bioassays. For example, a bioassay confirming the efficacy of an anticancer drug can be performed using cultured feline mammary tumor organoids. In particular, by applying the present invention, an organoid is prepared from a mammary gland tumor collected from a cat suffering from mammary adenocarcinoma, and the effect of an anticancer agent (drug sensitivity ) can be verified and an anticancer agent effective for the treatment of the cat can be appropriately selected. This allows for rapid determination of the optimal treatment for cats with mammary adenocarcinoma.

In addition, by applying the present invention, feline mammary gland tumor organoids that can be used for the development of novel therapeutic agents (anticancer agents) for feline mammary gland cancer can be rapidly supplied.

EXAMPLES The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[Example 1]
[Preparation of feline mammary gland tumor organoids]
Surgical excision of the tumor site was performed from cats with mammary tumors (3 cases). FMT20001, FMT20002 and FMT20003, respectively. Thereafter, feline mammary gland tumor organoids were produced according to the following method. Specifically, first, the collected tissue was transferred to a 10 cm dish containing 2 ml of PBS to prevent the tissue from drying. Then, using surgical scissors, tissue pieces about 1 cm square were cut out in the dish. The obtained tissue piece was transferred to another 10 cm dish and washed 3 times with 2 ml of PBS to remove adhered blood components. After that, the whole dish was transferred to ice, and the piece of tissue was cut with ophthalmic scissors until it became viscous. The tissue piece was transferred to a 15 ml tube containing 500 μl of Liberase TH 1.25 mg/ml (sigma) and 450 μl of Advanced DMEM medium (gibco), and pipetted 10 times using a 1 ml Pipetman. Then, the tube was inserted into a constant temperature bath heated to 37° C. and shaken for 15 minutes. After 15 minutes, the tube was once taken out, pipetting was performed 10 times using a 1 ml Pipetman, and the mixture was further shaken for 15 minutes. The tube was taken out and pipetting was repeated 10 times to confirm that the cell turbidity was in a translucent state, and it was passed through a 100 µm cell strainer. When tissue pieces were visible even after shaking for 30 minutes, centrifugation was performed at 600×g for 3 minutes, the supernatant was removed, and 1 ml of TrypLE (gibco) was added and placed in a constant temperature bath for 5 minutes. The tube was taken out and passed through a cell strainer after pipetting as before. The filtered cell solution was centrifuged at 600 xg for 3 minutes. After removing the supernatant, 8 ml of PBS was added and pipetting was performed 10 times using a 1 ml Pipetman. I did this work a total of 3 times. The supernatant was removed, 40 μl of Matrigel (Corning) was added per well of a 24-well plate according to the amount of cell sedimentation, and the mixture was gently mixed several times using a 200 μl Pipetman. Matrigel containing cell components was seeded in 40 μl aliquots and placed in an incubator at 37° C. for 30 minutes. After that, 500 μl/well of medium warmed to 37° C. was added and culture was started.

Three types of feline mammary gland tumor organoids were produced by the above method (Fig. 1). As shown in Fig. 1, it was observed that the three types of feline mammary tumor organoids prepared in this example each had a different morphology.

[Evaluation of homology between feline mammary gland tumor organoids and excised tissues]
The feline mammary gland tumor organoids prepared as described above were subjected to HE staining according to a standard method. Tumor tissues FMT20001 and FMT20002 excised as described above were also subjected to HE staining according to a standard method. FIG. 2 shows the HE-stained image of the feline mammary gland tumor organoid and the HE-stained images of the original tumor tissues FMT20001 and FMT20002, respectively. As shown in FIG. 2, it was found that the feline mammary gland tumor organoids and the original tumor tissue are very similar in pathological structure.

[Comparison of hormone receptor expression between feline mammary gland tumor organoid and tumor tissue]
The feline mammary gland tumor organoid prepared as described above and the original tumor tissue FMT20001 were evaluated for similarity in hormone receptor expression according to the following method. In this example, expression of HER2 (human epidermal receptor 2: epidermal growth factor receptor 2), ER (estrogen receptor) and PR (progesterone receptor) was evaluated as hormone receptors. .

First, cryosections were prepared from organoids and excised tissues. The slide glass was washed with PBS for 5 minutes with shaking. After removing from PBS, the water was removed, 50 μl of 1.5% normal goat serum (NGS) was added to each section, and the section was allowed to stand at room temperature for 30 minutes in a wet box. After removing the NGS, the primary antibody was diluted 1:100 with PBS, 50 μl was added to each section in the same manner as described above, and allowed to stand at 4° C. overnight. On the next day, the sections were washed three times with PBS for 5 minutes each, and fluorescent secondary antibodies and Hexst were each diluted with PBS, 50 μl of which was placed on each section, and placed under light shielding for 60 minutes. After that, the cells were washed three times with PBS for 5 minutes while shielding from light, dried, sealed with a cover glass, dried, and then observed using a confocal laser microscope (Zeiss).

FIG. 3 shows the results of immunochemical staining experiments for HER2, ER, and PR for feline mammary gland tumor organoids and the original tumor tissue FMT20001. As shown in FIG. 3, it was found that the feline mammary gland tumor organoids and the original tumor tissue FMT20001 have similar expression patterns of HER2 and ER (PR expression was observed in both organoids and tumor tissue). Nakata).

[Examination of optimal supplements for feline mammary gland tumor organoid culture]
In culturing feline mammary gland tumor organoids prepared as described above, a search was made for medium components that would further promote cell growth and organoid formation. Specifically, a culture solution was prepared by adding various medium components to the medium composition shown in Table 1, and the control was set to 100%, and the cell proliferation rate due to the addition of the culture components was comparatively analyzed.

Table 2 shows the medium components examined in this example.

Feline mammary gland tumor organoids (derived from tumor tissue FMT20001) were cultured for 7 days using a culture solution to which each of the medium components shown in Table 2 was added. FIG. 4 shows the results of imaging the organoids after culture, and FIG. 5 shows the results of measuring the cell proliferation rate. In addition, the cell proliferation rate was calculated as follows. That is, a culture solution was prepared by adding various medium components shown in Table 2 to the medium composition shown in Table 1. Fluorescence intensity was measured with a plate reader (TECAN), and the cell growth rates of other culture medium components were compared with the control group with only the medium composition shown in Table 1 as 100%.

As shown in FIGS. 4 and 5, FGF2, FGF7, FGF10, and TGF-α are active in feline mammary tumor organoids, other than the known media components used to produce three-dimensional organoids, such as the Wnt agonists Wnt-3A and EGF. had a proliferative effect on In particular, FGF7 was found to have a superior growth-enhancing effect compared to Wnt-3A and EGF.

[Anticancer drug sensitivity test using feline mammary gland tumor organoids]
It was clarified that the feline mammary gland tumor organoids prepared in this example show a high degree of similarity to the original feline mammary gland tumor tissue both structurally and in the expression pattern of hormone receptors. Therefore, we evaluated the responsiveness of anticancer drugs by comparing the cell viability of organoids when using carboplatin and doxorubicin, which are anticancer drugs commonly used for feline mammary gland tumors. rice field.

First, the medium was completely removed with an aspirator, and 500 µl/well of PBS was added. After PBS was aspirated, 500 μl/1 well of EDTA/PBS was added and allowed to stand on ice for 30 minutes. Then, the adhering Matrigel was peeled off using a 1 ml Pipetman, and the plate was allowed to stand again on ice for 60 minutes. All the peeled Matrigel was transferred together with EDTA/PBS to a 15 ml tube using a 1 ml Pipetman, and centrifuged at 600 xg for 3 minutes. The supernatant was removed with an aspirator, 1 ml of PBS was added, pipetting was performed 10 times using a 1 ml Pipetman, and centrifugation was performed in the same manner. The supernatant was removed, 1 ml of TrypLE (gibco) warmed to 37°C was added, pipetting was performed 10 times, and the mixture was allowed to stand in a constant temperature bath at 37°C for 5 minutes. The tube was taken out and pipetting was performed again 10 times, and a 70 μl cell strainer was attached to a 50 ml tube containing 100 μl FBS to filter the cell turbidity. 10 µl was taken out from the prepared cell solution and used to calculate the number of cells. A necessary amount of solution for seeding cells in a 96-well plate was taken from the prepared cell solution, transferred to a 1.5 ml tube, and centrifuged at 600 xg for 3 minutes. The supernatant was removed, and 10 μl of Matrigel was added to each well of a 96-well plate and gently mixed several times using a 200 μl Pipetman. Matrigel containing cell components was seeded in 10 μl portions and placed in an incubator at 37° C. for 30 minutes. After that, 100 μl/well of a medium warmed to 37° C. was added and cultured. Carboplatin and doxorubicin were added the next day. After the containing medium was aspirated, 4 concentrations of anticancer drug solutions diluted with the medium were prepared and added at 100 µl/well. In the control group, DMSO was used as an alternative to anticancer drugs. Measurement of cell viability was performed after 3 days. 10 µl/well of PrestoBlue reagent (invitrogen) was added and incubated at 37°C. After 3 to 4 hours, the fluorescence intensity was measured with a plate reader (TECAN) under the condition of Gain 45, and the sensitivity to each anticancer drug was examined.

FIG. 6 shows the sensitivity of the three feline mammary tumor organoids (derived from FMT20001, FMT20002 and FMT20003) produced above to carboplatin and doxorubicin. As shown in Figure 6, the three feline mammary tumor organoids used were found to exhibit different sensitivities to carboplatin and doxorubicin, respectively. In particular, the sensitivity to carboplatin was shown to be extremely high for FMT20001-derived and FMT20003-derived of the three feline mammary tumor organoids used. These results suggest that treatment with carboplatin is desirable for FMT20001 and FMT20003 cats with mammary adenocarcinoma.

Claims (10)

A composition for feline mammary tumor organoid medium, comprising at least one component selected from the group consisting of FGF2, FGF7, FGF10 and TGF-α. 2. The composition for feline mammary gland tumor organoid culture medium according to claim 1, further comprising at least one component selected from the group consisting of Wnt agonists, BMP inhibitors, EGF and TGFβ inhibitors. The composition for feline mammary gland tumor organoid medium according to claim 1, wherein the component is FGF10. A feline mammary tumor organoid medium containing at least one component selected from the group consisting of FGF2, FGF7, FGF10 and TGF-α. 5. The feline mammary tumor organoid medium according to claim 4, further comprising at least one component selected from the group consisting of Wnt agonists, BMP inhibitors, EGF and TGFβ inhibitors. 5. The feline mammary tumor organoid culture medium according to claim 4, wherein said component is FGF10. A method for producing a feline mammary gland tumor organoid, comprising culturing a feline mammary gland tumor organoid in the feline mammary gland tumor organoid medium according to any one of claims 4 to 6. 8. The method for producing feline mammary gland tumor organoids according to claim 7, further comprising the step of producing said feline mammary gland tumor organoids from mammary tissue collected from a cat suffering from mammary gland cancer. producing a feline mammary tumor organoid from mammary glands taken from a cat with mammary adenocarcinoma;
culturing the feline mammary gland tumor organoid in the feline mammary gland tumor organoid medium according to any one of claims 4 to 6;
further culturing the feline mammary gland tumor organoids obtained by the culture in the presence of a drug to measure drug sensitivity;
A method for treating a cat with mammary adenocarcinoma. 10. The treatment method according to claim 9, further comprising the step of administering a drug having high drug sensitivity to the mammary cancer-affected cat as a result of measuring the drug sensitivity.

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