WO2023060709A1 - Esophageal cancer organoid culture medium and culture method and use thereof - Google Patents

Abstract

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, a hepatocyte growth factor, SB202190, Y27632, A83-01, an epidermal growth factor, gastrin, a keratinocyte growth factor, GlutaMAX and nicotinamide. The present invention further relates to an esophageal cancer organoid culture method and the use thereof. The effective and rapid expansion of esophageal cancer organoids can be achieved by means of using the esophageal cancer organoid culture medium, and the organoids obtained by means of such expansion maintain the pathological characteristics of the patient, increasing the culture success rate and the expansion rate of esophageal cancer organoids, and providing a research basis for individualized treatment of patients.

Description

Culture medium, culture method and application of esophageal cancer organoids technical field

The invention belongs to the field of biotechnology, and in particular relates to a medium for culturing esophageal cancer organoids, a method for cultivating esophageal cancer organoids using the medium, and its application in drug efficacy evaluation and screening.

Background technique

Esophageal cancer is a common digestive tract tumor with high morbidity and mortality in my country. Most of the patients are clinically diagnosed in the middle and advanced stages, and treatment options are limited. In recent years, targeted therapy for esophageal cancer has attracted attention, but there is no effective targeted drug for the treatment of esophageal cancer. The reason is that there is no good cell model for pathogenesis and drug research, so new models are needed To develop new drugs for the treatment of esophageal cancer. However, the traditional esophageal cancer cell lines have been unable to meet this need, and more and more studies have been carried out using primary cells obtained from patients' own tissues.

Traditional clinical drug sensitivity testing mostly uses two-dimensional cell culture. However, two-dimensional cultured cells can only simulate the physiological conditions of tissues to a limited extent, and lack the real tissue structure in vivo, which will easily lead to low differentiation level and loss of cell physiological functions, which makes it difficult to predict the actual clinical results from the obtained experimental results. Organoids, which belong to three-dimensional (3D) cell cultures, are mainly derived from human embryonic stem cells, induced pluripotent stem cells and adult stem cells with differentiation ability. Endogenous tissue stem cells exist in different tissues and organs, and play an important role in maintaining the functional morphology of various organs. Under certain induction conditions in vitro, these stem cells can self-organize to form a miniature structure with a diameter of only a few millimeters. Tumor organoids are some miniature 3D tumor cell models grown in the laboratory using primary tumors taken from patients. Tumor organoids highly simulate the characteristics of the source tumor tissue, retain the tumor heterogeneity among individuals, and can be used for functional testing, such as high-throughput drug screening and individualized precision therapy.

At present, expensive protein factors such as R-spondin-1, WNT3A, and Noggin are mostly used in esophageal cancer organoid culture methods, resulting in high cost of organoid culture; and this technology is complicated to operate and technically difficult, leading to its large-scale commercialization Application is limited. Therefore, there is a need to develop a low-cost, simple and high success rate organoid culture method and medium.

Contents of the invention

In order to solve the above technical problems, the present invention provides a culture medium and a culture method for rapid expansion of esophageal cancer organoids in vitro.

One aspect of the present invention is to provide a medium for esophageal cancer organoids, said medium comprising MST1/2 kinase inhibitors, 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 Niacinamide. Wherein, the MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof,

in,

R ₁ is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted by _1-2 independent R (such as phenyl and naphthyl, etc.), aryl C1-C6 alkyl (such as benzyl, etc.) and heteroaryl (such as thienyl, etc.);

R ₂ and R ₃ are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;

R ₄ and R ₅ are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxyl, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclyl C1-C6 alkyl (the heterocyclyl is selected from, for example, piperidinyl, tetrahydropyran base, etc.);

_R 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 trifluoro methyl).

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

in,

_R is selected from C1-C6 alkyl, phenyl optionally substituted by 1-2 independently _R6 , thienyl optionally substituted by 1-2 independently _R6 , and optionally substituted by 1 -2 independently _R6 substituted benzyl, _R1 is more preferably optionally 1-2 independently _R6 substituted phenyl;

R ₅ is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R ₅ is more preferably hydrogen;

R ₆ is each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, and R ₆ is more preferably fluorine, methyl or trifluoromethyl.

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

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

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

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

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

(3) The concentration of hepatocyte growth factor is preferably 5-40 ng/mL;

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

(5) The concentration of Y27632 is preferably 2.5-10 μM;

(6) The concentration of A83-01 is preferably 200-1000nM;

(7) The concentration of epidermal growth factor is preferably 1-40 ng/mL;

(8) The concentration of gastrin is preferably 5-20 ng/mL;

(9) The concentration of keratinocyte growth factor is preferably 2-40 ng/mL;

(10) The volume ratio of GlutaMAX to the medium is preferably 1:50 to 1:200;

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

In an embodiment of the present invention, the medium also contains an initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and one selected from streptomycin/penicillin, amphotericin B and Primocin one or more antibiotics.

In a preferred embodiment, when the antibiotic is selected from streptomycin/penicillin, the streptomycin concentration ranges from 25 to 400 μg/mL, and the penicillin concentration ranges from 25 to 400 U/mL; when the antibiotic is selected from amphotericin B, The concentration range is 0.25-4 μg/mL, and when the antibiotic is selected from Primocin, the concentration range is 25-400 μg/mL.

The invention also provides a method for culturing esophagus cancer organoids. In the method for culturing esophageal cancer organoids of the present invention, the esophageal cancer organoid culture medium of the present invention is used to culture esophageal cancer organoids.

The method for culturing esophageal cancer organoids of the present invention includes the following steps.

1. Separating samples from esophageal cancer solid tumor tissues to obtain primary esophageal cancer cells. The process includes the following steps:

(1) Separate esophageal cancer tissue samples, add 1:1 ratio of basal medium and tissue digestion solution (the amount of tissue digestion solution is about 10 mL of tissue digestion solution per 1 g of tumor tissue) and place in a constant temperature shaker for digestion , the digestion temperature is 4-37°C, the shaker speed is 200rpm-300rpm, and the digestion time is 3-6 hours;

(2) After the digestion is completed, discard the supernatant after centrifugation, the centrifugation speed is 1200-1600 rpm, and the centrifugation time is 2-6 minutes.

Wherein, the basal medium formula includes an initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and one or more antibiotics selected from streptomycin/penicillin, amphotericin B and Primocin. The formula of tissue digestion solution includes 1640 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 (1~5mM), bovine serum albumin BSA (5~10mg/mL).

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

Resuspend and count the primary esophageal cancer cells obtained in the above step 1 with initial medium, basal medium or esophageal cancer organoid medium of the present invention, dilute the cell density to 5-10×10 ⁵ cells/mL, Take out the diluted cell suspension and add it to an equal volume of Matrigel and mix well, then inoculate the mixture on a multi-well plate, put the inoculated multi-well plate in the incubator for 30-60 minutes, wait until the Matrigel is completely solidified, and then add the esophageal cancer Organoid culture medium for expansion.

The present invention also provides a method for evaluating or screening drugs for the treatment of esophageal cancer, which includes the following steps:

(1) using the method for culturing esophageal cancer organoids of the present invention to cultivate esophageal cancer organoids;

(2) Select the drug to be detected and dilute it according to the required concentration gradient;

(3) adding the diluted drug to the organoid cultured in (1);

(4) Perform organoid size or organoid viability test.

The beneficial effects of the present invention include:

(1) Improve the success rate of esophageal cancer tissue-derived organoid culture, and the success rate reaches more than 85%;

(2) Ensure that the primary cultured esophageal cancer organoids in vitro can maintain the pathological characteristics of the patient;

(3) The amplification efficiency is high, and esophageal cancer organoids can be rapidly cultured, and the amplified esophageal cancer organoids can also be continuously passaged;

(4) The culture cost is controllable, and the medium does not need to add expensive Wnt agonists, R-spondin family proteins and Noggin proteins;

(5) The number of esophageal cancer organoids obtained by the technique is large, which is suitable for high-throughput screening of candidate compounds and providing patients with high-throughput drug sensitivity functional tests in vitro.

Description of drawings

1A-1K are graphs showing the effects of different concentrations of factors added to the esophageal cancer organoid medium of the present invention on the proliferation of esophageal cancer organoids.

Figures 2A-2D are photographs of esophageal cancer organoids cultured using the esophageal cancer organoid medium of the present invention under a microscope, wherein Figure 2A shows photos of organoids obtained from sample OE1 cultured for 0 days; Figure 2B shows photos obtained from sample OE1 The photos of the organoids obtained from OE1 after 14 days of culture; where Fig. 2C shows the photos of the organoids obtained from sample OE2 after 15 days of culture; Fig. 2D shows the partial enlarged view of Fig. 2C.

Fig. 3 is a graph showing the results of pathological and immunohistochemical identification of the original tissue of esophageal cancer sample OE4 and the esophageal cancer organoid cultured from OE4 using the esophageal cancer organoid medium of the present invention.

Figures 4A and 4B are graphs showing the comparison results of culturing esophageal cancer organoids using the esophageal cancer organoid medium of the present invention and the existing medium, wherein Figure 4A shows a photograph after 15 days of culture with the EOM medium of the present invention ; Figure 4B shows photographs after 15 days of culture with literature medium ROM.

Figures 5A-5D are graphs showing the results of drug concentration sensitivity testing of esophageal cancer organoids obtained by culturing the esophageal cancer organoid medium of the present invention, wherein Figures 5A and 5B are pictures taken under a microscope with a 4x objective lens without drug treatment The photos of organoid growth in the same group, and the photos of organoid growth after 7 days of treatment with hydroxycamptothecin and sorafenib respectively. Inhibition rate curves for esophageal cancer organoid growth.

Detailed ways

In order to better understand the present invention, the present invention will be further described below in conjunction with the embodiments and accompanying drawings. The following examples are only to illustrate the present invention but not to limit it.

[Preparation Example of MST1/2 Kinase Inhibitor]

In the present specification, an MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1/2 signal transduction. In general, MST1/2 kinase inhibitors, for example, bind to MST1/2 kinase and reduce its activity. Due to the similarity in the structures of MST1 and MST2, MST1/2 kinase inhibitors may also be, for example, compounds that bind to MST1 or MST2 and reduce their activity.

1. Preparation of MST1/2 Kinase Inhibitor Compound 1

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

2-Amino-2-(2,6-difluorophenyl)acetic acid methyl ester (A2): In a round bottom flask was added 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) Methanol (30 mL) was then added, followed by the dropwise addition of thionyl chloride (1.2 mL) under ice-cooling. The reaction system was reacted overnight at 85°C. After the reaction, the system was evaporated to dryness under reduced pressure to obtain a white solid, which was directly used in the next step.

Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (A3): Add 2-amino- After adding acetone (30 ml) and potassium carbonate (2.2 g) to 2-(2,6-difluorophenyl) methyl acetate (2 g), the system was cooled to -10 ° C with an ice-salt bath, and then slowly added 2,4-Dichloro-5-nitropyrimidine (3.1 g) in acetone. The reaction was stirred overnight at room temperature. After the reaction was completed, it was filtered, and the solvent was removed from the filtrate under reduced pressure, and the residue was purified by pressurized silica gel column chromatography to obtain compound A3. LC/MS: M+H 359.0.

2-Chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (A4): add 2-((2-chloro- Methyl 5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (2.5 g) was added followed by acetic acid (50 ml) and iron powder (3.9 g). The reaction system was stirred at 60°C for two hours. After the reaction, the system was evaporated to dryness under reduced pressure, and the resultant was neutralized to alkalinity with saturated sodium bicarbonate. Extracted with ethyl acetate, the organic phase was washed with water and saturated brine, and dried over anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to obtain a crude product. The crude product was washed with ether to obtain compound A4. LC/MS: M+H 297.0.

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

4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzene Sulfonamide (1): Add 2-chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridine-6(5H) to a round bottom flask - Ketone (100 mg), sulfonamide (53 mg), p-toluenesulfonic acid (53 mg) and sec-butanol (5 ml). The reaction system was stirred overnight at 120°C. After the reaction, filter and wash with methanol and ether to obtain compound 1. LC/MS: M+H 461.1.

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

Other MST1/2 inhibitor compounds of the present invention were synthesized according to a method similar to compound 1, and their structures and mass spectrometry data are shown in the table below.

Example 1 Effects of Added Factors in Esophageal Cancer Organoid Culture Medium on the Proliferation of Esophageal Cancer Organoids

(1) Preparation of esophageal cancer organoid culture medium

First prepare the basal medium containing the initial medium. The initial medium can be selected from DMEM/F12, DMEM, F12 or RPMI-1640 commonly used in the art. In this embodiment, the formulation of the basal medium is: DMEM/F12 medium (purchased from Corning Company)+100 μg/mL Primocin (purchased from InvivoGen Company, 0.2% (v/v), commercially available product concentration 50mg/ml ).

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

(2) Isolation and processing of primary esophageal cancer cells

1 sample selection

Esophageal cancer solid tumor tissue samples (intraoperative) were obtained from patients by professional medical staff of professional medical institutions, and all patients signed informed consent. Intraoperative samples of 5-10 mm ³ were stored and transported in commercial tissue preservation solution (manufacturer: Miltenyi Biotec).

2 material preparation

After surface disinfection of 15mL sterile centrifuge tubes, pipette guns, 10mL pipettes, sterile pipette tips, etc., put them in the ultra-clean workbench and irradiate them with ultraviolet light for 30 minutes. Take out the basal medium from the 4°C refrigerator 30 minutes in advance, and take out the tissue digestion solution from the -20°C refrigerator 30 minutes in advance.

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

Collagenase II, collagenase IV, DNase, and hyaluronidase mentioned above were all purchased from Sigma; calcium chloride and BSA were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.

3 Sample Separation

3.1 Take the tissue samples in the culture dish in the ultra-clean bench, remove the tissue with blood, wash it twice with the basal medium, transfer the tissue to another culture dish for mechanical separation with a sterile scalpel, and divide the tissue block into 1 *2*1mm ³ size;

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

3.3 Discard the supernatant, add basal medium and tissue digestion solution in a ratio of 1:1 (note: the amount of tissue digestion solution added is about 10 mL of tissue digestion solution for 1 g of tumor tissue), mark the sample name and number, and seal it with a parafilm. Digestion was carried out at 37°C on a 300rpm shaker (ZQLY-180N), during which the digestion was observed every 30 minutes to see if there were no particles visible to the naked eye;

3.4 After the digestion is completed, filter the undigested tissue mass through a 70 μm filter, wash the tissue mass on the filter with the basal medium into the centrifuge tube to reduce cell loss, and centrifuge at room temperature for 3 minutes at 1500 rpm;

3.5 Discard the supernatant and observe whether there are blood cells. If there are blood cells, add 4 mL of blood cell lysate (purchased from Sigma Company), mix well, and lyse at 4°C for 10 minutes.

3.6 Discard the supernatant, add 2mL basal medium to resuspend the cells, and set aside.

4 Cell Counting and Processing

4.1 Observation under a microscope: transfer a small amount of resuspended cells to a culture dish, and observe the density and shape of cancer cells under a microscope (CNOPTEC, BDS400);

4.2 Viable cell counting: Take 12 μL of the resuspended cell suspension and 12 μL of trypan blue staining solution (manufacturer: Sangon Bioengineering (Shanghai) Co., Ltd.) and mix thoroughly, then take 20 μL and add it to a cell counting plate (manufacturer: Countstar , specification: 50 pieces/box), the percentage of living large cells (cell size > 10 μm) calculated by a cell counter (Countstar, IC1000) = number of viable cells/number of total cells * 100%.

(3) Culture of esophageal cancer organoids

Resuspend and count the primary esophageal cancer cells obtained in the above steps with pre-cooled DMEM/F12, dilute the cell density to 5-10× ¹⁰⁵ cells/mL, take out 400 μL of the diluted cell suspension and add an equal volume of Matrigel Matrigel (Corning) was mixed gently, and then the mixture was inoculated in a 96-well plate at 8 μL/well. Put the inoculated culture plate into the incubator for 30 minutes, wait until the Matrigel is completely solidified, then add the culture medium shown in Table 1 that has been returned to room temperature beforehand, and expand the culture by replacing the culture medium every five days. After 10 days, the cultured organoids were photographed, and the diameters of the organoids were measured and counted, and the promotion effects of various factors on the proliferation of esophageal cancer organoids were compared. Among them, as an experimental control, a basal medium without any additives was used, and the experimental results are shown in Table 1.

Table 1 Added ingredients in the medium and their effect on promoting organoid proliferation

serial number	Types of medium additives	supplier	Final concentration	Grading of proliferative degree
1	N2	Gibco	1:50	+
2	R-spondin1	Beijing Yiqiao	20ng/mL	○
3	gastrin	MCE	10ng/mL	+
4	B27	Gibco	1:50	+
5	A8301	MCE	100nM	+
6	SB202190	MCE	200nM	+
7	Fibroblast growth factor/FGF	Beijing Yiqiao	10ng/mL	○
8	hepatocyte growth factor HGF	Beijing Yiqiao	50ng/mL	+
9	Noggin	Beijing Yiqiao	50ng/mL	○
10	Fetal bovine serum/FBS		Excell	5%	+
11	Keratinocyte Growth Factor/KGF	Beijing Yiqiao	25ng/mL	+
12	GlutaMAX	Gibco	1:100	+
13	Nicotinamide	MCE	2.5mM	+
14	epidermal growth factor EGF	Beijing Yiqiao	10ng/mL	+
15	Y27632	MCE	10μM	+
16	ITS Cell Culture Supplement	Gibco	1:100	○

17	Compound 1	Preparation example	5μM	+
18	CHIR99021	MCE	2.5μM	-

Among them, "+" means that compared with the basal medium, the medium added with this additive has the effect of promoting the proliferation of at least two cases of esophageal cancer organoids isolated from esophageal cancer tissue; "-" means that the medium with this additive added The culture medium showed an inhibitory effect on at least one case of esophageal cancer organoids isolated from esophageal cancer tissues; Proliferation was not significantly affected in both cases.

According to the above results, compound 1, Y27632, SB202190, keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), A83-01, B27, GlutaMAX, gastrin, nicotinamide, epidermal growth factor ( EGF) and other factors for further culture experiments.

Example 2 Effects of Different Concentrations of Medium Added Factors on the Proliferation of Esophageal Carcinoma Organoids

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

Table 2 Medium formula (concentration is the final concentration)

When the medium of formula 1 was used, 200 μL of B27 prepared on the basis of formula 1 was added to the 96-well plate inoculated with organoids, and the final concentrations of B27 were 1:25, 1:50, 1 : 100; and use the medium of formula 1 to set up control wells (BC). The final concentration of other added factors in this series of medium is the same as that of EOM medium. The experiments of the following recipes 1-11 were also carried out in the same manner, so no further description was given.

When using the medium of formula 2, add 200 μL of HGF prepared on the basis of formula 2 to the 96-well plate inoculated with organoids, and the final concentrations of HGF are 40 ng/mL, 10 ng/mL, and 5 ng respectively. /mL; and use the medium of formula 2 to set up control wells (BC).

When using the medium of Formula 3, 200 μL of SB202190 cell culture additive prepared on the basis of Formula 3 was added to the 96-well plate inoculated with organoids, and the final concentration of SB202190 cell culture additive was 200 nM, 500 nM, 1000 nM; and set up control wells (BC) using the medium of recipe 3.

When using the medium of formula 4, add 200 μL of Y27632 prepared on the basis of formula 4 to the 96-well plate inoculated with organoids, and the final concentrations of Y27632 are 2.5 μM, 5 μM, and 10 μM respectively; and use The media of formulation 4 set up control wells (BC).

When using the medium of Formula 5, add 200 μL of A83-01 prepared on the basis of Formula 5 to the 96-well plate inoculated with organoids, and the final concentrations of A83-01 are 200 nM, 500 nM, and 1000 nM respectively. ; and set up control wells (BC) using the medium of formula 5.

When using the medium of Formula 6, add 200 μL of EGF prepared on the basis of Formula 6 to the 96-well plate inoculated with organoids, and the final concentrations of EGF are 1 ng/mL, 5 ng/mL, 40 ng/mL, respectively. mL; and set up control wells (BC) using the medium of recipe 6.

When using the medium of Formula 7, add 200 μL of prepared gastrin per well on the basis of Formula 7 to the 96-well plate inoculated with organoids, and the final concentrations of gastrin are 5 ng/mL and 10 ng respectively. /mL, 20ng/mL; and set up control wells (BC) using the medium of formula 7.

When using the medium of Formula 8, add 200 μL of KGF prepared on the basis of Formula 8 to the 96-well plate inoculated with organoids, and the final concentrations of KGF are 2 ng/mL, 10 ng/mL, and 40 ng respectively. /mL; and set up control wells (BC) using the medium of formula 8.

When using the medium of Formula 9, add 200 μL per well of GlutaMAX prepared on the basis of Formula 9 to the 96-well plate inoculated with organoids, and the final concentrations of GlutaMAX are 1:200, 1:100, 1 : 50; and use the medium of formula 9 to set up control wells (BC).

When the medium of Formula 10 was used, 200 μL of Compound 1 prepared on the basis of Formula 10 was added to the 96-well plate seeded with organoids, and the final concentrations of Compound 1 were 2.5 μM, 5 μM, and 10 μM; And set up control wells (BC) using the medium of formulation 10.

When using the medium of Formula 11, add 200 μL of prepared nicotinamide to each well of the 96-well plate inoculated with organoids on the basis of Formula 11, and the final concentrations of nicotinamide are 1 mM, 2.5 mM, and 10 mM respectively; And set up control wells (BC) using the medium of formulation 11.

After 12 days, the cultured organoids were photographed, and the diameters of the organoids were measured and counted, and the promotion effect of each factor concentration on the proliferation of esophageal cancer organoids was compared. The data collected from the two samples are summarized in Figures 1A-1K. In Figures 1A-1K, the ratios are the diameters of the organoids cultured for 12 days using each medium to the diameter of the organoids cultured for 12 days in the corresponding BC control wells. A ratio greater than 1 indicates that the prepared medium containing different concentrations of factors or small molecular compounds has a better effect on promoting proliferation than the culture medium of the control well; a ratio less than 1 indicates that the prepared medium containing different concentrations of factors or small molecular compounds promotes proliferation The effect was weaker than that of the culture medium in the control well.

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

Example 3 Esophageal cancer organoid culture and identification

The esophageal cancer primary cells (OE1, OE2, OE4) obtained according to the method described in (2) of Example 1 were resuspended and counted with the esophageal cancer organoid medium EOM of the present invention, and the cell density was diluted to 5-10 × ¹⁰ cells/mL, take 400 μL of the diluted cell suspension and add it to an equal volume of Matrigel (Corning) to mix gently, and then inoculate the mixture in a 24-well plate at 40 μL/well. Put the inoculated culture plate into the incubator for 30 minutes, wait until the Matrigel is completely solidified, then add the esophageal cancer organoid medium EOM that has been returned to room temperature in advance, 500 μL per well, and expand the culture by replacing the medium every five days.

On days 0-15, the cultured esophageal cancer organoids were observed using a microscope (EVOS M500 from Invitrogen). Figures 2A-2D are microscopic photographs of samples OE1 (day 0), OE1 (day 14), OE2 (day 15, photographed with a 10x mirror), and OE2 (day 15, photographed with a mirror of 20x) obtained after culture Photograph of esophageal cancer organoids. As shown, organoids grow in size during culture; different types of organoids can be formed from the same sample, which can mimic tumor heterogeneity in vitro.

Pathological and immunohistochemical identifications were performed on the cultured esophageal cancer organoids, and the corresponding original tissue samples were also identified pathologically and immunohistochemically to compare the consistency of organoids and histopathological indicators.

Figure 3 is a graph showing the pathological and immunohistochemical identification results of the original esophageal cancer tissue sample OE4 and the esophageal cancer organoid obtained after OE4 cultured in vitro, all of which were photographed under a 20x objective lens. As shown in the figure, the result shows that the structure and morphology of the organoids are cancerous tissue morphology; according to the immunohistochemical indicators, it is judged that the cells obtained after culturing the organoids in this case are esophageal cancer cells. The results show that the diagnosis results of the esophageal cancer organoid cultured with the medium EOM of the present invention are consistent with those of the esophageal cancer tissue before culture.

Embodiment 4 and the comparison of existing culture medium effect

(1) Preparation of control medium

Prepare the medium used in the literature (Yuta Kasagi et al., (Cell Mol Gastroenterol Hepatol 2018; 5:333–352), the formula of which is Advanced DMEM/F12 medium (purchased from Invitrogen)+1:100 Penicillin/Streptomycin (purchased from Corning)+1mM L-glutamine (purchased from Corning)+10mM HEPES (purchased from Thermo Fisher)+1:50B27 (purchased from Gibco)+1:100N2 (purchased from Gibco)+1mmol/L N -Acetylcysteine (purchased from MCE Company)+10mmol/L Niacinamide (purchased from MCE Company)+100ng/mL R-Spondin 1 (purchased from Sino Biological Company)+100ng/mL wnt3A (purchased from RD Company) +25ng/mL gastrin (purchased from MCE company)+10ng/mL epidermal growth factor (purchased from sino biological company)+100ng/ml Noggin (purchased from sino biologicala company)+500nmol/L SB202190 (purchased from MCE company )+500nmol/L A8301 (purchased from MCE Company)+10 μmol/L Y27632 (purchased from MCE Company)+1mmol/L calcium chloride (purchased from Shanghai Sangong Company). Hereinafter referred to as ROM medium.

(2) Esophageal cancer organoid culture

According to the method of (2) of Example 1, primary esophageal cancer cells were obtained from the intraoperative tissue sample OE8, and the organoid culture was carried out according to the method of Example 3 with EOM medium and ROM medium respectively.

On the 15th day of culture, the cultured esophageal cancer organoids were observed using a microscope (EVOS M500 from Invitrogen Company). Figures 4A and 4B are photographs of organoids cultured in EOM medium and ROM medium for 15 days respectively under a 4x objective lens.

According to the results in Figures 4A and 4B, compared with ROM medium, EOM medium can significantly promote the formation and expansion of esophageal cancer organoids.

Example 5 Esophageal cancer organoids amplified using the medium of the present invention are used for drug screening

(1) Esophageal cancer organoid culture

Primary esophageal cancer cells were isolated from the intraoperative esophageal cancer sample (OE6) according to the method of Example 1 (2), and organoid culture was performed using EOM medium, and drug screening was performed when the diameter of the esophageal cancer organoid exceeded 50 μm.

(2) Screen drug preparation

Two drugs (hydroxycamptothecin and sorafenib; both purchased from MCE Company) with 10 concentration gradients were prepared and stored for later use.

Preparation of different concentrations of hydroxycamptothecin and sorafenib drug additive solution: hydroxycamptothecin and sorafenib were prepared into 10 stock solutions with different concentrations, the highest concentration was 20000μM, and then diluted by 2 times, The stock solutions with different concentrations of 10000μM, 5000μM, 2500μM, 1250μM, 625μM, 312.5μM, 156.25μM, 78.13μM, 39.06μM and 19.53μM were obtained.

(3) Dosing

Take out the prepared drug storage solution, place it at room temperature, and dilute the drug 1000 times with EOM medium to obtain 20000nM, 10000nM, 5000nM, 2500nM, 1250nM, 625nM, 312.5nM, 156.25nM, 78.13nM and 39.06nM. Take out the organoids obtained by culturing according to step (1) from the incubator, remove the medium in the culture wells, and slowly pour the drug-containing medium into the 96-well transparent culture plate along the well wall at 100 μL per well. After the drug addition, the surface of the 96-well plate was sterilized and moved to the incubator to continue culturing, and the viability of the organoids was measured 7 days later.

(4) Organoid Viability Test

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

(5) Data processing

According to the formula drug inhibition rate (%)=100%-(the chemiluminescence value _{drug treatment group} of the culture well on the 7th day/the chemiluminescence value _{drug treatment group of the culture} well on the 0th day)/(the chemiluminescence value _DMSO of the culture well on the 7th day/day The chemiluminescent value of the zero-day culture wells _(DMSO )*100% was calculated to obtain the inhibition rates of different drugs, and the results are shown in FIGS. 5A-5D . Figures 5A and 5B are photos of the growth of organoids in the non-medicated treatment group taken under a microscope (Invitrogen EVOS M500) under a 4x objective lens, and the photos of organoid growth after 7 days of drug treatment with hydroxycamptothecin and sorafenib, respectively. Photo, Figures 5C and 5D are the inhibition rate curves of different concentrations of test drugs inhibiting the growth of esophageal cancer organoids.

It can be confirmed from Figures 5A and 5B that the organoids cultured using the esophageal cancer organoid medium of the present invention are in good growth state, and the growth inhibition of organoids after treatment with hydroxycamptothecin and sorafenib is concentration-dependent. After high-concentration drug treatment, the growth of organoids was significantly inhibited, and the cells were observed to shrink, become smaller or even lyse under the microscope. Figures 5C and 5D are the inhibition rate curves of the two tested drugs at different concentrations inhibiting the growth of esophageal cancer organoids. Among the two antitumor drugs, the inhibitory effects of different concentrations of hydroxycamptothecin and sorafenib were different in a dose-dependent manner, which indicated that organoids from the same patient had different effectiveness and sensitivity to different drugs. According to the results, the effectiveness and the effective dosage of the drug can be judged when the esophagus cancer patients are used clinically.

Industrial applicability

The present invention provides a medium and a culture method for culturing organoids of esophageal cancer, and the cultured organoids can be applied to the efficacy evaluation and screening of drugs. Thus, the present invention is suitable for industrial applications.

Although the present invention has been described in detail herein, the present invention is not limited thereto, and those skilled in the art can make modifications according to the principle of the present invention. Therefore, all modifications made according to the principle of the present invention should be understood as falling within protection scope of the present invention.

Claims (10)

1. A culture medium for esophageal cancer organoids, characterized by comprising MST1/2 kinase inhibitors, at least one cell culture additive selected from N2 and B27, hepatocyte growth factor, SB202190, Y27632, A83-01, epidermis Cell Growth Factor, Gastrin, Keratinocyte Growth Factor, GlutaMAX, and Niacinamide,

   Wherein, the MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof,

   

   in,

   R ₁ is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted by _1-2 independent R Base, aryl C1-C6 alkyl and heteroaryl;

   R ₂ and R ₃ are each independently selected from C1-C6 alkyl;

   R ₄ and R ₅ are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxyl, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclyl C1-C6 alkyl;

   R ₆ is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.
2. culture medium as claimed in claim 1, wherein

   _R is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and benzene optionally substituted by _1-2 independently R phenyl, naphthyl, benzyl and thienyl;

   R ₂ and R ₃ are each independently selected from C1-C3 alkyl;

   R ₄ and R ₅ are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxyl, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, piperidinyl C1-C6 alkyl, and tetrahydropyranyl C1-C6 alkyl;

   R ₆ is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.
3. The culture medium according to claim 1, wherein the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt thereof, or a solvate,

   

   in,

   _R is selected from C1-C6 alkyl, phenyl optionally substituted by 1-2 independently _R6 , thienyl optionally substituted by 1-2 independently _R6 , and optionally substituted by 1 -2 independently _R6 substituted benzyl groups;

   R _is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl;

   Each R ₆ is independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl.
4. culture medium as claimed in claim 3, wherein

   R ₁ is phenyl optionally substituted by 1-2 independently R ₆ ;

   _R is hydrogen;

   _R6 is preferably fluoro, methyl or trifluoromethyl.
5. The culture medium according to claim 1, wherein the MST1/2 kinase inhibitor is selected from at least one of the following compounds or pharmaceutically acceptable salts thereof:

   

   

   

   

   
6. The culture medium according to any one of claims 1 to 5, characterized in that the content of each component in the culture medium satisfies any one or more or all of the following:

   The concentration of the MST1/2 kinase inhibitor is 2.5-10 μM;

   The volume ratio of the B27 or N2 cell culture supplement to the medium is 1:25 to 1:100;

   The concentration of the hepatocyte growth factor is 5-40 ng/mL;

   The concentration of the SB202190 is 200-1000nM;

   The concentration of Y27632 is 2.5-10 μM;

   The concentration of the A83-01 is 200-1000nM;

   The concentration of the epidermal growth factor is 1-40 ng/mL;

   The concentration of the gastrin is 5-20 ng/mL;

   The concentration of the keratinocyte growth factor is 2-40 ng/mL;

   The volume ratio of the GlutaMAX to the medium is 1:50 to 1:200;

   The concentration of the nicotinamide is 1-10mM.
7. The culture medium according to any one of claims 1 to 6, further comprising:

   An initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and

   One or more antibiotics selected from streptomycin/penicillin, amphotericin B and Primocin.
8. The medium according to any one of claims 1 to 7, characterized in that the medium does not contain Wnt agonists, R-spondin family proteins, Noggin proteins, or BMP inhibitors.
9. A method for culturing esophageal cancer organoids, characterized in that it comprises the following steps:

   (1) Separating samples from esophageal cancer solid tumor tissues to obtain primary esophageal cancer cells;

   (2) preparing a culture medium for the esophageal cancer organoid according to any one of claims 1 to 8, and performing organoid culture on the primary esophageal cancer cells obtained in step (1).
10. A method for evaluating or screening a drug for treating esophageal cancer, comprising the following steps:

    (1) using the culture method of esophageal cancer organoids according to claim 9 to cultivate esophageal cancer organoids;

    (2) Select the drug to be detected and dilute it according to the required concentration gradient;

    (3) adding the diluted drug to the organoid cultured in (1);

    (4) Perform organoid size or organoid viability detection.

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