Esophageal cancer organoid culture medium, culture method and application thereof

Abstract

The present invention relates to a culture medium for culturing esophageal cancer organoids, comprising an MST1 / 2 kinase inhibitor, at least one cell culture additive selected from N2 and B27, hepatocyte growth factor, SB202190, Y27632, A83-01, epidermal growth factor, gastrin, keratinocyte growth factor, GlutaMAX, and nicotinamide. The present invention also relates to a culture method for esophageal cancer organoids and its use. By using the esophageal cancer organoid culture medium of the present invention, effective and rapid expansion of esophageal cancer organoids can be achieved. The expanded organoids retain the patient's pathological characteristics, improve the culture success rate and expansion rate of esophageal cancer organoids, and provide a research basis for personalized treatment of patients.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a culture medium for culturing esophageal cancer organoids, a method for culturing esophageal cancer organoids using the culture medium, and an application of the culture medium in evaluating and screening drug efficacy. Background Art

[0002] Esophageal cancer is a common digestive tract tumor with high morbidity and mortality in my country. Most patients are diagnosed in the mid-to-late stage, with limited treatment options. Targeted therapies for esophageal cancer have received significant attention in recent years, but effective targeted drugs are still unavailable. This is due to the lack of robust cell models for pathogenesis and drug research. Therefore, new models are needed to develop novel therapeutics for esophageal cancer. However, traditional esophageal cancer cell lines no longer meet this need, and a growing number of studies are using primary cells derived from patient tissue.

[0003] Traditional clinical drug sensitivity testing mostly uses two-dimensional cell culture. However, these cells only simulate tissue physiological conditions to a limited extent and lack authentic in vivo tissue structure, which can easily lead to low differentiation levels and loss of cellular physiological function. This, in turn, makes the experimental results difficult to predict actual clinical outcomes. Organoids, a three-dimensional (3D) cell culture, are primarily derived from human embryonic stem cells, induced pluripotent stem cells, and adult stem cells with the ability to differentiate. Endogenous tissue stem cells exist in various tissues and organs and play a vital role in maintaining the functional morphology of each organ. Under certain inductive conditions in vitro, these stem cells can self-organize into miniature structures measuring only a few millimeters in diameter. Tumor organoids are miniature 3D tumor cell models cultured in the laboratory using primary tumors taken from patients. Tumor organoids closely mimic the characteristics of the original tumor tissue, preserving the heterogeneity between individual tumors. They can be used for functional testing, such as high-throughput drug screening and personalized precision medicine.

[0004] Currently, esophageal cancer organoid culture methods often rely on expensive protein factors such as R-spondin-1, WNT3A, and Noggin, resulting in high costs. Furthermore, the technology is complex and technically challenging, limiting its large-scale commercial application. Therefore, a low-cost, simple, and highly successful organoid culture method and culture medium are needed. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a culture medium and a culture method for rapidly expanding esophageal cancer organoids in vitro.

[0006] One aspect of the present invention is to provide a culture medium for esophageal cancer organoids, comprising an MST1 / 2 kinase inhibitor, at least one cell culture additive selected from N2 and B27, hepatocyte growth factor, SB202190, Y27632, A83-01, epidermal growth factor, gastrin, keratinocyte growth factor, GlutaMAX, and nicotinamide. The MST1 / 2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof.

[0007]

[0008] in,

[0009] R1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and aryl (e.g., phenyl and naphthyl, etc.) optionally substituted by 1-2 independently R6, aryl C1-C6 alkyl (e.g., benzyl, etc.) and heteroaryl (e.g., thienyl, etc.);

[0010] R2 and R3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;

[0011] R4 and R5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylaminoC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, and C3-C6 heterocyclylC1-C6 alkyl (the heterocyclyl is selected from, for example, piperidinyl, tetrahydropyranyl, etc.);

[0012] R6 is selected from halogen (preferably fluorine and chlorine, more preferably fluorine), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy), and C1-C6 haloalkyl (preferably trifluoromethyl).

[0013] In a preferred embodiment, the MST1 / 2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt or solvate thereof,

[0014]

[0015] in,

[0016] R1 is selected from C1-C6 alkyl, phenyl optionally substituted by 1-2 independently R6, thienyl optionally substituted by 1-2 independently R6, and benzyl optionally substituted by 1-2 independently R6, more preferably phenyl optionally substituted by 1-2 independently R6;

[0017] R5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R5 is more preferably hydrogen;

[0018] R6 are each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, and R6 is more preferably fluorine, methyl or trifluoromethyl.

[0019] Preferably, the MST1 / 2 inhibitor is at least one selected from the following compounds or pharmaceutically acceptable salts or solvates thereof.

[0020]

[0021]

[0022]

[0023]

[0024]

[0025] Most preferably, the MST1 / 2 kinase inhibitor of the present invention is Compound 1.

[0026] In an embodiment of the present invention, the content of each component in the culture medium of the present invention satisfies any one, multiple or all of the following:

[0027] (1) The concentration of MST1 / 2 kinase inhibitor is preferably 2.5-10 μM;

[0028] (2) The volume ratio of B27 or N2 cell culture supplement to culture medium is 1:25 to 1:100;

[0029] (3) The concentration of hepatocyte growth factor is preferably 5 to 40 ng / mL;

[0030] (4) The concentration of SB202190 is preferably 200-1000 nM;

[0031] (5) The concentration of Y27632 is preferably 2.5 to 10 μM;

[0032] (6) The concentration of A83-01 is preferably 200 to 1000 nM;

[0033] (7) The concentration of epidermal growth factor is preferably 1 to 40 ng / mL;

[0034] (8) The concentration of gastrin is preferably 5 to 20 ng / mL;

[0035] (9) The concentration of keratinocyte growth factor is preferably 2 to 40 ng / mL;

[0036] (10) The volume ratio of GlutaMAX to culture medium is preferably 1:50 to 1:200;

[0037] (11) The concentration of nicotinamide is preferably 1 to 10 mM.

[0038] In an embodiment of the present invention, the culture medium further contains an initial culture medium selected from DMEM / F12, DMEM, F12 or RPMI-1640; and one or more antibiotics selected from streptomycin / penicillin, amphotericin B and primocin.

[0039] In a preferred embodiment, when the antibiotic is selected from streptomycin / penicillin, the concentration of streptomycin is in the range of 25 to 400 μg / mL, and the concentration of penicillin is in the range of 25 to 400 U / mL. When the antibiotic is selected from amphotericin B, the concentration range is 0.25 to 4 μg / mL. When the antibiotic is selected from Primocin, the concentration range is 25 to 400 μg / mL.

[0040] The present invention also provides a method for culturing esophageal cancer organoids. In the method for culturing esophageal cancer organoids of the present invention, the esophageal cancer organoids are cultured using the esophageal cancer organoid culture medium of the present invention.

[0041] The esophageal cancer organoid culture method of the present invention comprises the following steps.

[0042] 1. Isolate samples from esophageal cancer solid tumor tissue to obtain esophageal cancer primary cells. The process includes the following steps:

[0043] (1) Esophageal cancer tissue samples were isolated and digested with basal culture medium and tissue digestion solution in a 1:1 ratio (approximately 10 mL of tissue digestion solution was added per 1 g of tumor tissue) in a constant temperature shaker for digestion at a temperature of 4–37°C, a shaker speed of 200–300 rpm, and a digestion time of 3–6 h.

[0044] (2) After digestion, centrifuge and discard the supernatant. The centrifugation speed is 1200-1600 rpm and the centrifugation time is 2-6 minutes.

[0045] The basal culture medium includes an initial culture medium selected from DMEM / F12, DMEM, F12, or RPMI-1640; and one or more antibiotics selected from streptomycin / penicillin, amphotericin B, and primocin. The tissue digestion solution includes 1640 culture medium, collagenase II (1-2 mg / mL), collagenase IV (1-2 mg / mL), DNase (50-100 U / mL), hyaluronidase (0.5-1 mg / mL), calcium chloride (1-5 mM), and bovine serum albumin (BSA) (5-10 mg / mL).

[0046] 2. Prepare the esophageal cancer organoid culture medium of the present invention, and culture the esophageal cancer primary cells obtained in the above steps.

[0047] The primary esophageal cancer cells obtained in step 1 were resuspended in the esophageal cancer organoid culture medium of the present invention and counted, and the cell density was diluted to 5-10×10 5 / mL, take out the diluted cell suspension and add it to an equal volume of Matrigel matrix gel to mix well, then inoculate the mixture into a multi-well plate, put the inoculated multi-well plate into the incubator for 30-60 minutes, wait for Matrigel to completely solidify, and then add esophageal cancer organoid culture medium for expansion culture.

[0048] The present invention also provides a method for evaluating or screening drugs for treating esophageal cancer, comprising the following steps:

[0049] (1) culturing esophageal cancer organoids using the culturing method of the present invention;

[0050] (2) Select the drug to be tested and dilute it according to the required concentration gradient;

[0051] (3) adding the diluted drug to the organoids cultured in (1);

[0052] (4) Conduct organoid size or organoid viability tests.

[0053] The beneficial effects of the present invention include:

[0054] (1) Improve the success rate of culturing organoids derived from esophageal cancer tissue to over 85%;

[0055] (2) Ensure that the primary cultured esophageal cancer organoids in vitro can maintain the patient's pathological characteristics;

[0056] (3) High amplification efficiency, capable of rapidly culturing esophageal cancer organoids, which can also be continuously passaged;

[0057] (4) The culture cost is controllable, and the culture medium does not need to be added with expensive Wnt agonists, R-spondin family proteins, and Noggin proteins;

[0058] (5) The technology can produce a large number of esophageal cancer organoids, which are suitable for high-throughput screening of candidate compounds and providing high-throughput in vitro drug sensitivity functional testing for patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figures 1A-1K This is a graph showing the effects of different concentrations of factors added to the esophageal cancer organoid culture medium of the present invention on the proliferation of esophageal cancer organoids.

[0060] Figures 2A-2D This is a photograph of esophageal cancer organoids cultured using the esophageal cancer organoid culture medium of the present invention observed under a microscope. Figure 2A Photograph showing organoid culture day 0 obtained from sample OE1; Figure 2B The photo shows the organoids obtained from sample OE1 after 14 days of culture; Figure 2C A photograph showing organoids obtained from sample OE2 after 15 days of culture; Figure 2D show Figure 2C A partial enlarged view of .

[0061] Figure 3 These are the results of pathological and immunohistochemical identification of esophageal cancer organoids obtained by culturing sample OE4 using the esophageal cancer organoid culture medium of the present invention.

[0062] Figure 4A and 4B The results of the comparison of culturing esophageal cancer organoids using the esophageal cancer organoid culture medium of the present invention and the existing culture medium are shown in FIG. Figure 4A A photograph showing the cultured cells after 15 days using the EOM medium of the present invention; Figure 4B The photograph shows the cells after 15 days of culture using the literature medium ROM.

[0063] Figures 5A-5D The results of drug concentration sensitivity testing of esophageal cancer organoids obtained by culturing the esophageal cancer organoid culture medium of the present invention are shown, wherein Figure 5A and 5B These are photos of organoid growth in the untreated group and 7 days after drug treatment, taken under a 4x objective microscope. Figure 5C and 5D The inhibition rate curve of esophageal cancer organoid growth inhibited by different concentrations of test drugs. DETAILED DESCRIPTION

[0064] For a better understanding of the present invention, the present invention will be further described below in conjunction with the embodiments and drawings. The following embodiments are merely illustrative of the present invention and are not intended to limit the present invention.

[0065] [Preparation Example of MST1 / 2 Kinase Inhibitor]

[0066] As used herein, an MST1 / 2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1 / 2 signaling. Generally, an MST1 / 2 kinase inhibitor, for example, binds to and reduces the activity of MST1 / 2 kinases. Due to the structural similarities between MST1 and MST2, an MST1 / 2 kinase inhibitor may also be a compound that binds to and reduces the activity of either MST1 or MST2.

[0067] 1. Preparation of MST1 / 2 kinase inhibitor compound 1

[0068] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzene Sulfonamide 1

[0069]

[0070] Methyl 2-amino-2-(2,6-difluorophenyl)acetate (A2): Add 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) to a round-bottom flask, followed by methanol (30 mL). Thionyl chloride (1.2 mL) was then added dropwise under an ice bath. The reaction was allowed to react at 85°C overnight. After completion of the reaction, the solvent was evaporated under reduced pressure to yield a white solid, which was used directly in the next step.

[0071] Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (A3): To a round-bottom flask, add methyl 2-amino-2-(2,6-difluorophenyl)acetate (2 g), followed by acetone (30 ml) and potassium carbonate (2.2 g). The mixture was then cooled to -10°C in an ice-salt bath. An acetone solution of 2,4-dichloro-5-nitropyrimidine (3.1 g) was then slowly added. The reaction mixture was stirred at room temperature overnight. After completion of the reaction, the mixture was filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by pressurized silica gel column chromatography to yield compound A3. LC / MS: M+H 359.0.

[0072] 2-Chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (A4): To a round-bottom flask, add methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (2.5 g), followed by acetic acid (50 ml) and iron powder (3.9 g). The reaction system was stirred at 60°C for two hours. After completion of the reaction, the solvent was evaporated under reduced pressure, and the resulting product was neutralized with saturated sodium bicarbonate until alkaline. Extraction was performed with ethyl acetate, and the organic phase was washed with water and saturated brine, respectively, and dried over anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to obtain the crude product. The crude product was washed with diethyl ether to obtain compound A4. LC / MS: M+H 297.0.

[0073] 2-Chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (A5): 2-Chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (2 g) and N,N-dimethylacetamide (10 mL) were added to a round-bottom flask. The mixture was cooled to -35°C, and iodomethane (0.9 mL) was added, followed by sodium hydride (615 mg). The reaction system was stirred for two hours. After completion of the reaction, the mixture was quenched with water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine, then dried over anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to obtain the crude product. The crude product was washed with diethyl ether to obtain compound A5. LC / MS: M+H 325.0.

[0074] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzenesulfonamide (1): 2-chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (100 mg), sulfonamide (53 mg), p-toluenesulfonic acid (53 mg), and sec-butanol (5 mL) were added to a round-bottom flask. The reaction system was stirred at 120°C overnight. After completion of the reaction, the mixture was filtered and washed with methanol and diethyl ether to obtain compound 1. LC / MS: M+H 461.1.

[0075] 2. Preparation of other MST1 / 2 inhibitor compounds of the present invention

[0076] Other MST1 / 2 inhibitor compounds of the present invention were synthesized in a similar manner to compound 1, and their structures and mass spectrometry data are shown in the following table.

[0077]

[0078]

[0079]

[0080]

[0081]

[0082] Example 1 Effects of various added factors in esophageal cancer organoid culture medium on the proliferation of esophageal cancer organoids

[0083] (1) Preparation of esophageal cancer organoid culture medium

[0084] First, a basal medium containing an initial culture medium is prepared. The initial culture medium can be selected from DMEM / F12, DMEM, F12, or RPMI-1640, which are commonly used in the art. In this embodiment, the basal medium is formulated as follows: DMEM / F12 medium (purchased from Corning) + 100 μg / mL Primocin (purchased from InvivoGen, 0.2% (v / v), commercially available at a concentration of 50 mg / ml).

[0085] Different types of additives (see Table 1) were added to the basal culture medium to prepare esophageal cancer organoid culture medium containing different additives.

[0086] (2) Isolation and processing of primary esophageal cancer cells

[0087] 1. Sample selection

[0088] Esophageal cancer solid tumor tissue samples (intraoperative) were obtained from patients by professional medical staff of professional medical institutions, and the patients signed informed consent. 3 Commercial tissue preservation solution (manufacturer: Miltenyi Biotec) was used for storage and transportation.

[0089] 2. Material Preparation

[0090] After disinfecting the surfaces of sterile 15mL centrifuge tubes, pipettes, 10mL pipettes, and sterile pipette tips, place them in a clean bench and irradiate with UV light for 30 minutes. Remove the basal culture medium from the 4°C refrigerator 30 minutes in advance, and remove the tissue digestion solution from the -20°C refrigerator 30 minutes in advance.

[0091] Tissue digestion solution formula: 1640 culture medium (Corning, 10-040-CVR), collagenase II (2 mg / mL), collagenase IV (2 mg / mL), DNase (50 U / mL), hyaluronidase (0.75 mg / mL), calcium chloride (3.3 mM), and bovine serum albumin (BSA) (10 mg / mL).

[0092] The above-mentioned collagenase II, collagenase IV, DNA enzyme, and hyaluronidase were all purchased from Sigma; calcium chloride and BSA were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.

[0093] 3. Sample separation

[0094] 3.1 Take the tissue sample in the clean bench and place it in a culture dish. Remove the blood-stained tissue and rinse it twice with basal culture medium. Transfer the tissue to another culture dish and perform mechanical separation with a sterile scalpel. Cut the tissue into 1*2*1mm pieces. 3 size;

[0095] 3.2 Aspirate the cut tissue into a 15 mL centrifuge tube, add 5 mL of basal culture medium, mix well, and centrifuge at 1500 rpm for 3 minutes;

[0096] 3.3 Discard the supernatant and add basal culture medium and tissue digestion solution in a 1:1 ratio (Note: the amount of tissue digestion solution added is approximately 10 mL for 1 g of tumor tissue). Label the sample name and number, seal with sealing film, and digest at 37°C in a shaker (Zhichu Instrument ZQLY-180N) at 300 rpm. Observe the digestion completion every 30 minutes, judging by the absence of visible particulate matter.

[0097] 3.4 After digestion, filter out undigested tissue clumps through a 70 μm filter. Rinse the tissue clumps on the filter with basal culture medium into a centrifuge tube to reduce cell loss, and centrifuge at 1500 rpm for 3 minutes at room temperature.

[0098] 3.5 Discard the supernatant and observe whether there are blood cells. If there are blood cells, add 8 mL of blood cell lysis buffer (purchased from Sigma), mix well, and lyse at 4°C for 20 minutes, inverting once during the process, and centrifuge at 1500 rpm for 4 minutes at room temperature;

[0099] 3.6 Discard the supernatant and add 2 mL of basal culture medium to resuspend the cells for later use.

[0100] 4. Cell Counting and Processing

[0101] 4.1 Observation under microscope: Pipette a small amount of resuspended cells and spread them flatly in a culture dish. Observe the density and morphology of cancer cells under a microscope (CNOPTEC, BDS400).

[0102] 4.2 Live cell counting: Take 12 μL of the resuspended cell suspension and 12 μL of trypan blue dye (manufacturer: Sangon Biotech (Shanghai) Co., Ltd.) and mix thoroughly. Then, take 20 μL and add it to a cell counting plate (manufacturer: Countstar, specification: 50 plates / box). Using a cell counter (Countstar, IC1000), calculate the percentage of live large cells (cell size >10 μm) = number of live cells / total number of cells*100%.

[0103] (3) Culture of esophageal cancer organoids

[0104] The primary esophageal cancer cells obtained in the above steps were resuspended in pre-cooled DMEM / F12 and counted, and the cell density was diluted to 5-10×10 5 / mL, take out 400μL of the diluted cell suspension and add it to an equal volume of Matrigel matrix gel (Corning) and mix gently, then inoculate the mixture into a 96-well plate at 8μL / well. Place the inoculated culture plate in the incubator for 30 minutes, wait for the Matrigel to completely solidify, and then add the culture medium shown in Table 1 that has been restored to room temperature in advance, and expand the culture by replacing the culture medium every five days. After 10 days, the cultured organoids were photographed, and the diameter of the organoids was measured and counted to compare the promoting effect of each factor on the proliferation of esophageal cancer organoids. Among them, as an experimental control, a basal culture medium without any additives was used, and the experimental results are shown in Table 1.

[0105] Table 1 Additives in culture medium and their effects on promoting organoid proliferation

[0106]

[0107]

[0108] Among them, "+" indicates that compared with the basic culture medium, the culture medium with the addition of this additive has a proliferation-promoting effect on at least two esophageal cancer organoids isolated from esophageal cancer tissue; "-" indicates that the culture medium with the addition of this additive has an inhibitory effect on the proliferation of at least one esophageal cancer organoid isolated from esophageal cancer tissue; "○" indicates that the culture medium with the addition of this additive has no obvious effect on the proliferation of at least two esophageal cancer organoids isolated from esophageal cancer tissue.

[0109] Based on the above results, factors such as compound 1, Y27632, SB202190, keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), A83-01, B27, GlutaMAX, gastrin, nicotinamide, and epidermal growth factor (EGF) are planned to be selected for further culture experiments.

[0110] Example 2 Effects of Different Concentrations of Culture Medium Additives on the Proliferation of Esophageal Cancer Organoids

[0111] According to the method of Example 1 (2), primary esophageal cancer cells were obtained from intraoperative tissue samples (numbered OE1 and OE2), and organoid culture was performed using the culture medium formula in Table 2 below.

[0112] Table 2 Culture medium formula (concentration is final concentration)

[0113]

[0114] When using medium from Recipe 1, add 200 μL of B27 prepared in Recipe 1 to each well of a 96-well plate seeded with organoids, achieving final B27 concentrations of 1:25, 1:50, and 1:100, respectively. A control well (BC) was also established using medium from Recipe 1. The final concentrations of other factors added to this series of media were the same as for EOM medium. The following experiments with Recipes 1-11 were performed in the same manner and are not further detailed here.

[0115] When using the culture medium of Formula 2, add 200 μL of prepared HGF to each well of the 96-well plate seeded with organoids based on Formula 2, with final HGF concentrations of 40 ng / mL, 10 ng / mL, and 5 ng / mL, respectively; and set up control wells (BC) using the culture medium of Formula 2.

[0116] When using the culture medium of Formula 3, add 200 μL of the prepared SB202190 cell culture supplement based on Formula 3 to each well of the 96-well plate seeded with organoids. The final concentrations of SB202190 cell culture supplement are 200 nM, 500 nM, and 1000 nM, respectively. Set up control wells (BC) using the culture medium of Formula 3.

[0117] When using the culture medium of Formula 4, 200 μL of the prepared Y27632 was added to each well of the 96-well plate seeded with organoids based on Formula 4. The final concentrations of Y27632 were 2.5 μM, 5 μM, and 10 μM, respectively. Control wells (BC) were set up using the culture medium of Formula 4.

[0118] When using the culture medium of Formula 5, 200 μL of prepared A83-01 was added to each well of a 96-well plate seeded with organoids based on Formula 5. The final concentrations of A83-01 were 200 nM, 500 nM, and 1000 nM, respectively. Control wells (BC) were set up using the culture medium of Formula 5.

[0119] When using the culture medium of Formula 6, add 200 μL of prepared EGF to each well of the 96-well plate seeded with organoids based on Formula 6, with final EGF concentrations of 1 ng / mL, 5 ng / mL, and 40 ng / mL, respectively; and set up control wells (BC) using the culture medium of Formula 6.

[0120] When using the culture medium of Formula 7, 200 μL of prepared gastrin was added to each well of a 96-well plate seeded with organoids based on Formula 7. The final concentrations of gastrin were 5 ng / mL, 10 ng / mL, and 20 ng / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 7.

[0121] When using the culture medium of Formula 8, 200 μL of prepared KGF was added to each well of the 96-well plate seeded with organoids based on Formula 8. The final concentrations of KGF were 2 ng / mL, 10 ng / mL, and 40 ng / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 8.

[0122] When using the medium of Formula 9, add 200 μL of prepared GlutaMAX to each well of a 96-well plate seeded with organoids, with final concentrations of GlutaMAX of 1:200, 1:100, and 1:50, respectively; and set up control wells (BC) using the medium of Formula 9.

[0123] When using the culture medium of Formula 10, 200 μL of compound 1 prepared on the basis of Formula 10 was added to each well of a 96-well plate seeded with organoids, with the final concentrations of compound 1 being 2.5 μM, 5 μM, and 10 μM, respectively; and control wells (BC) were set using the culture medium of Formula 10.

[0124] When using the culture medium of Formula 11, 200 μL of prepared nicotinamide was added to each well of a 96-well plate seeded with organoids, with final concentrations of nicotinamide of 1 mM, 2.5 mM, and 10 mM, respectively; and control wells (BC) were set up using the culture medium of Formula 11.

[0125] After 12 days, the cultured organoids were photographed and their diameters were measured and counted to compare the effects of each factor concentration on the proliferation of esophageal cancer organoids. The data collected from the two samples are summarized and shown in Figures 1A to 1K . Figures 1A to 1K The ratio is the ratio of the organoid diameter obtained after 12 days of culture using each medium to the diameter of the organoid obtained after 12 days of culture using the corresponding BC control well. A ratio greater than 1 indicates that the medium containing the factor or small molecule at the different concentrations promotes proliferation more effectively than the medium in the control well. A ratio less than 1 indicates that the medium containing the factor or small molecule at the different concentrations promotes proliferation less effectively than the medium in the control well.

[0126] according to Figures 1A to 1KAccording to the results, the volume concentration of B27 is preferably 1:25 to 1:100; the content of hepatocyte growth factor HGF is preferably 5 to 40 ng / mL; the content of SB202190 is preferably 200 to 1000 nM; the content of Y27632 is preferably 2.5 to 10 μM; the content of A83-01 is preferably 200 to 1000 nM; the content of epidermal growth factor EGF is preferably 1 to 40 ng / mL; the content of gastrin is preferably 5 to 20 μg / mL; the content of keratinocyte growth factor is preferably 2 to 40 ng / mL; the volume concentration of GlutaMAX is preferably 1:50 to 1:200; the content of MST1 / 2 kinase inhibitor compound 1 is preferably 2.5 to 10 μM; and the content of nicotinamide is preferably 1 to 10 mM.

[0127] Example 3 Esophageal cancer organoid culture and identification

[0128] The primary esophageal cancer cells (OE1, OE2, OE4) obtained according to the method described in Example 1 (2) were resuspended in the esophageal cancer organoid culture medium EOM of the present invention and counted. The cell density was diluted to 5-10×10 5 Cells were plated at a concentration of 400 μL / mL. 400 μL of the diluted cell suspension was added to an equal volume of Matrigel (Corning) and gently mixed. The mixture was then seeded into a 24-well plate at a rate of 40 μL / well. The plate was placed in an incubator for 30 minutes to allow the Matrigel to completely solidify. Then, 500 μL of esophageal cancer organoid culture medium (EOM), previously brought to room temperature, was added to each well. The culture was expanded by replacing the medium every five days.

[0129] On days 0 to 15, the cultured esophageal cancer organoids were observed using a microscope (Invitrogen EVOS M500). Figures 2A-2D The following images show esophageal cancer organoids obtained from samples OE1 (day 0), OE1 (day 14), OE2 (day 15, 10x), and OE2 (day 15, 20x) cultured at a 4x objective. As shown, organoids increase in size over the course of culture; different organoid types can form from the same sample, mimicking tumor heterogeneity in vitro.

[0130] The cultured esophageal cancer organoids were subjected to pathological and immunohistochemical identification, and the corresponding original tissue samples were also subjected to pathological and immunohistochemical identification to compare the consistency of organoid and tissue pathological indicators.

[0131] Figure 3The images below show the results of pathological and immunohistochemical characterization of esophageal cancer organoids obtained from sample OE4 after in vitro culture. These images were taken at a 20x objective. As shown, the organoids' structural morphology resembles that of cancerous tissue; immunohistochemical markers indicate that the cells obtained from this sample are esophageal cancer cells. These results demonstrate that the diagnostic results of esophageal cancer organoids cultured using the culture medium EOM presented in this invention are consistent with those of the pre-cultured esophageal cancer tissue.

[0132] Comparison of Example 4 with existing culture medium culture effects

[0133] (1) Preparation of control culture medium

[0134] The culture medium used in the preparation literature (Yuta Kasagi et al., (Cell Mol Gastroenterol Hepatol 2018; 5: 333–352) was prepared, and its formula was Advanced DMEM / F12 medium (purchased from Invitrogen) + 1:100 Penicillin / Streptomycin (purchased from Corning) + 1 mM L-glutamine (purchased from Corning) + 10 mM HEPES (purchased from Thermo Fisher Scientific) + 1:50 B27 (purchased from Gibco) + 1:100 N2 (purchased from Gibco) + 1 mmol / L N-acetylcysteine ​​(purchased from MCE) + 10 mmol / L nicotinamide (purchased from MCE) + 100 ng / mL R-Spondin 1 (purchased from Sino Biological) + 100 ng / mL wnt3A (purchased from RD) + 25 ng / mL gastrin (purchased from MCE) + 10 ng / mL epidermal growth factor (purchased from Sino Biological) + 100 ng / mL Noggin (purchased from Sino Biologica) + 500 nmol / L SB202190 (purchased from MCE) + 500 nmol / L LA8301 (purchased from MCE) + 10 μmol / L Y27632 (purchased from MCE) + 1 mmol / L calcium chloride (purchased from Shanghai Bioengineering). Hereinafter referred to as ROM medium.

[0135] (2) Esophageal cancer organoid culture

[0136] According to the method of Example 1 (2), primary esophageal cancer cells were obtained from the intraoperative tissue sample OE8, and organoid culture was performed using EOM medium and ROM medium according to the method of Example 3.

[0137] On the 15th day of culture, the cultured esophageal cancer organoids were observed using a microscope (Invitrogen EVOS M500). Figure 4A and 4B These are photos of organoids obtained by culturing in EOM medium and ROM medium for 15 days, taken under a 4x objective lens.

[0138] according to Figure 4A and 4B The results showed that compared with ROM medium, EOM medium can significantly promote the formation and expansion culture of esophageal cancer organoids.

[0139] Example 5: Use of esophageal cancer organoids expanded using the culture medium of the present invention for drug screening

[0140] (1) Esophageal cancer organoid culture

[0141] Esophageal cancer primary cells were isolated from an intraoperative esophageal cancer sample (OE6) according to the method of Example 1 (2), and organoid culture was performed using EOM medium. Drug screening was performed when the diameter of the esophageal cancer organoid exceeded 50 μm.

[0142] (2) Screening drug preparation

[0143] According to the table below, 10 concentration gradients of two drugs (hydroxycamptothecin and sorafenib; both purchased from MCE) were prepared and stored for use.

[0144] Preparation of different concentrations of hydroxycamptothecin and sorafenib drug additive solutions: Hydroxycamptothecin and sorafenib were prepared into 10 stock solutions with different concentrations, with the highest concentration being 20,000 μM. The solutions were then diluted at a 2-fold dilution ratio to obtain stock solutions with different concentrations of 10,000 μM, 5,000 μM, 2,500 μM, 1,250 μM, 625 μM, 312.5 μM, 156.25 μM, 78.13 μM, 39.06 μM and 19.53 μM.

[0145] (3) Dosing

[0146] The prepared drug storage solution was taken out and placed at room temperature. The drug was diluted 1000 times with EOM medium to obtain 20000nM, 10000nM, 5000nM, 2500nM, 1250nM, 625nM, 312.5nM, 156.25nM, 78.13nM and 39.06nM. The organoids obtained by culture according to step (1) were taken out from the incubator, the culture medium in the culture wells was removed, and the culture medium containing the drug was slowly poured into the 96-well transparent culture plate along the well wall at 100μL per well. After the addition of the drug, the surface of the 96-well plate was disinfected and moved to the incubator for further culture. The viability of the organoids was measured after 7 days.

[0147] (4) Organoid viability test

[0148] Take out the CellTiter-Glo luminescent reagent (purchased from Promega) from the 4°C refrigerator, take 10 ml of the reagent into the sample tank, take out the 96-well plate to be tested from the incubator, add 50 μL of CellTiter-Glo luminescent reagent to each well, let it stand for 30 minutes, and observe the status of the cells in the 96-well plate. If most of the cells have been lysed, gently shake to mix, and pipette 100 μL into another white 96-well plate, and use a multi-function microplate reader (Perkin Elmer Envision) for detection.

[0149] (5) Data processing

[0150] According to the formula, drug inhibition rate (%) = 100% - (chemiluminescence value of the culture well on the 7th day of the drug treatment group / chemiluminescence value of the culture well on the 0th day of the drug treatment group) / (chemiluminescence value of the culture well on the 7th day of the drug treatment group) DMSO / Chemiluminescence value of culture wells on day 0 DMSO )*100%, calculate the inhibition rate of different drugs, and show the results in Figures 5A to 5D . Figure 5A and 5B The following are photos of organoid growth in the untreated group taken under a 4x objective microscope (Invitrogen EVOS M500), as well as photos of organoid growth after 7 days of treatment with hydroxycamptothecin and sorafenib. Figure 5C and 5D The inhibition rate curve of esophageal cancer organoid growth inhibited by different concentrations of test drugs.

[0151] Depend on Figure 5A and 5B It can be confirmed that the organoids cultured using the esophageal cancer organoid culture medium of the present invention have good growth status. After treatment with hydroxycamptothecin and sorafenib, the growth inhibition of the organoids has a certain concentration dependence. After treatment with high concentrations of drugs, the growth of the organoids is significantly inhibited, and the cells are observed to be obviously shrunken, smaller, and even lysed under the microscope. Figure 5C and 5D Figure 2 shows the inhibition rate curves of esophageal cancer organoids at different concentrations of two test drugs. The inhibitory effects of hydroxycamptothecin and sorafenib at different concentrations varied somewhat, showing a dose-dependent pattern. This suggests that organoids from the same patient exhibit varying efficacy and sensitivity to different drugs. These results can be used to assess the effectiveness and effective dosage of these drugs in clinical use in esophageal cancer patients.

[0152] Industrial Applicability

[0153] The present invention provides a culture medium and method for culturing esophageal cancer organoids. The cultured organoids can be used for evaluating and screening drug efficacy. Therefore, the present invention is suitable for industrial applications.

[0154] Although the present invention is described in detail herein, the present invention is not limited thereto. Those skilled in the art may make modifications based on the principles of the present invention. Therefore, all modifications made in accordance with the principles of the present invention should be understood to fall within the scope of protection of the present invention.

Claims

1. A culture medium for esophageal cancer organoids, characterized in that: Made with the following ingredients: MST1 / 2 kinase inhibitor; B27 supplement; hepatocyte growth factor; SB202190; Y27632; A83-01; epidermal growth factor; gastrin; keratinocyte growth factor; GlutaMAX; nicotinamide; an initial culture medium selected from DMEM / F12, DMEM, F12, or RPMI-1640; and one or more antibiotics selected from streptomycin / penicillin, amphotericin B, and primocin, Wherein, the MST1 / 2 kinase inhibitor is compound 1 or a pharmaceutically acceptable salt or solvate thereof, in, The concentration of the MST1 / 2 kinase inhibitor is 2.5 to 10 μM; The volume ratio of the B27 additive to the culture medium of the esophageal cancer organoid is 1:25 to 1:100; The concentration of the hepatocyte growth factor is 5 to 40 ng / mL; The concentration of SB202190 is 200-1000 nM; The concentration of Y27632 is 2.5-10 μM; The concentration of A83-01 is 200-1000 nM; The concentration of the epidermal growth factor is 1 to 40 ng / mL; The concentration of gastrin is 5 to 20 ng / mL; The concentration of the keratinocyte growth factor is 2 to 40 ng / mL; The volume ratio of the GlutaMAX to the culture medium of the esophageal cancer organoid is 1:50 to 1:200; The concentration of the nicotinamide is 1-10 mM.

2. A method for culturing esophageal cancer organoids, characterized in that The following steps are involved: (1) Isolate samples from esophageal cancer solid tumor tissue to obtain esophageal cancer primary cells; (2) preparing a culture medium for esophageal cancer organoids according to claim 1, and performing organoid culture on the primary esophageal cancer cells obtained in step (1).

3. A method for screening drugs for treating esophageal cancer, characterized in that: The following steps are involved: (1) culturing esophageal cancer organoids using the culturing method of esophageal cancer organoids according to claim 2; (2) Select the drug to be tested and dilute it according to the required concentration gradient; (3) adding the diluted drug to the organoids cultured in (1); (4) Conduct organoid size or organoid viability testing.

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