Culture medium, culture method and application of lung cancer pleural effusion-derived organoids

Abstract

The present invention relates to a culture medium for culturing lung cancer organoids, comprising an MST1 / 2 kinase inhibitor, at least one cell culture additive selected from N2 and B27, fibroblast growth factor 10, SB202190, Y27632, A83-01, neuregulin 1, insulin-like growth factor-1, keratinocyte growth factor, GlutaMAX, nicotinamide, and HEPES. The present invention also relates to a culture method for lung cancer pleural effusion-derived organoids and its use. By using the lung cancer organoid culture medium of the present invention, effective and rapid expansion of lung cancer pleural effusion organoids can be achieved. The organoids obtained by such expansion maintain the patient's pathological characteristics, improve the culture success rate and expansion rate of lung 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 specifically relates to a culture medium for culturing lung cancer organoids, a method for culturing lung cancer pleural effusion-derived organoids using the culture medium, and an application of the culture medium in drug efficacy evaluation and screening. Background Art

[0002] Lung cancer is a common malignant tumor of the lung and one of the most threatening to human health and life. Clinical studies have confirmed that malignant pleural effusion (MPE) often develops in advanced lung cancer. The presence of MPE signals the end of surgical treatment and indicates a poor prognosis. High-throughput drug sensitivity testing requires the in vitro isolation and purification of sufficient numbers of autologous tumor cells. However, in vitro culture of tumor cells has been a bottleneck in drug sensitivity testing. Because patients with advanced lung cancer and associated pleural effusion often harbor high-purity tumor cells, high-throughput drug sensitivity testing is suitable for screening optimal treatment options. Traditional clinical drug sensitivity testing mostly utilizes two-dimensional cell culture. However, two-dimensional cell culture only simulates tissue physiological conditions to a limited extent and lacks the authentic in vivo tissue structure, which can lead to low differentiation levels and loss of cellular physiological function. This, in turn, makes experimental results difficult to predict clinical outcomes.

[0003] Organoids are three-dimensional (3D) cell cultures 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, playing a crucial role in maintaining their functional morphology. Under certain induction 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 grown in the laboratory using primary tumors taken from patients. Tumor organoids closely mimic the characteristics of the original tumor tissue, preserving inter-individual tumor heterogeneity and enabling functional testing such as high-throughput drug screening and personalized precision medicine. Currently, lung cancer organoid culture methods often utilize expensive protein factors such as R-spondin-1, WNT3A, and Noggin, resulting in high costs. Furthermore, the complex and technically challenging nature of this technology has limited its large-scale commercial application. Therefore, there is a need for a low-cost, simple, and highly successful organoid culture method and culture medium. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a culture medium and a culture method for rapidly expanding lung cancer organoids in vitro.

[0005] One aspect of the present invention is to provide a culture medium for lung cancer organoids, comprising an MST1 / 2 kinase inhibitor, at least one cell culture additive selected from N2 and B27, fibroblast growth factor 10, SB202190, Y27632, A83-01, neuregulin 1, insulin-like growth factor 1, keratinocyte growth factor, GlutaMAX, nicotinamide, and HEPES. The MST1 / 2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof.

[0006]

[0007] in,

[0008] 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.);

[0009] R2 and R3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;

[0010] 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.);

[0011] 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).

[0012] In a preferred embodiment, the MST1 / 2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt or solvate thereof,

[0013]

[0014] in,

[0015] 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;

[0016] R5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R5 is more preferably hydrogen;

[0017] R6 are each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, and R6 is more preferably fluorine, methyl or trifluoromethyl.

[0018] Preferably, the MST1 / 2 inhibitor is at least one selected from the following compounds or pharmaceutically acceptable salts or solvates thereof.

[0019]

[0020]

[0021]

[0022]

[0023]

[0024] Most preferably, the MST1 / 2 kinase inhibitor of the present invention is Compound 1.

[0025] 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:

[0026] (1) The concentration of MST1 / 2 kinase inhibitor is preferably 2.5-10 μM;

[0027] (2) The volume ratio of B27 or N2 cell culture additive to culture medium is preferably 1:25 to 1:100;

[0028] (3) The concentration of fibroblast growth factor 10 is preferably 25 to 200 ng / mL;

[0029] (4) The concentration of SB202190 is preferably 200-1000 nM;

[0030] (5) The concentration of Y27632 is preferably 2.5 to 10 μM;

[0031] (6) The concentration of A83-01 is preferably 200 to 1000 nM;

[0032] (7) The concentration of neuregulin 1 is preferably 1 to 40 ng / mL;

[0033] (8) The concentration of insulin-like growth factor 1 is preferably 2 to 40 ng / mL;

[0034] (9) The concentration of keratinocyte growth factor is preferably 2 to 40 ng / mL;

[0035] (10) The volume ratio of GlutaMAX to culture medium is preferably 1:50 to 1:200;

[0036] (11) The concentration of nicotinamide is preferably 1 to 10 mM;

[0037] (12) The concentration of HEPES 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 lung cancer organoids. In the method for culturing lung cancer organoids of the present invention, primary cells derived from lung cancer pleural effusion tissue are cultured using the lung cancer organoid culture medium of the present invention.

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

[0042] 1. Isolate samples from lung cancer pleural effusion tissue to obtain primary lung cancer cells. The processing process includes the following steps:

[0043] (1) Isolate lung cancer pleural effusion tissue samples, transfer the collected lung cancer pleural effusion into a centrifuge tube, centrifuge at a speed of 1800-2200 rpm, and centrifuge for 8-12 minutes;

[0044] (2) After centrifugation, discard the supernatant, add basal medium to resuspend, and then sieve the collected cell suspension and centrifuge at a speed of 1200-1600 rpm for 3-5 minutes;

[0045] (3) Observe the cell pellet. If it contains red blood cells, add 3-5 ml of red blood cell lysis buffer and lyse on ice for 5-10 minutes. After complete lysis, centrifuge at a speed of 1200-1600 rpm for 3-5 minutes to obtain a cell pellet for use.

[0046] The basic culture medium formula 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.

[0047] 2. Prepare the lung cancer organoid culture medium of the present invention and culture the primary lung cancer cells obtained in the above steps.

[0048] The primary lung cancer cells obtained in step 1 were resuspended in the lung cancer organoid culture medium of the present invention and counted, and the cell density was diluted to 5-10×10 4 The cell suspension was added to an ultra-low attachment culture plate or culture flask for expansion culture.

[0049] The present invention also provides a method for evaluating or screening drugs for treating lung cancer, comprising the following steps:

[0050] (1) Cultivating lung cancer organoids using the lung cancer organoid culture method of the present invention;

[0051] (2) Select the drug to be tested and dilute it according to the required concentration gradient;

[0052] (3) adding the diluted drug to the organoid cultured in (1);

[0053] (4) Conduct organoid size or organoid viability tests.

[0054] The beneficial effects of the present invention include:

[0055] (1) Improve the success rate of culturing organoids derived from lung cancer tissue to over 85%;

[0056] (2) Ensure that primary cultured lung cancer organoids in vitro can maintain the patient's pathological characteristics;

[0057] (3) High amplification efficiency, capable of rapidly culturing lung cancer organoids, and the amplified lung cancer organoids can be continuously passaged;

[0058] (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;

[0059] (5) The technology can produce a large number of lung 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

[0060] Figures 1A-1LThis is a graph showing the effects of different concentrations of factors added to the lung cancer organoid culture medium of the present invention on the proliferation of lung cancer organoids.

[0061] Figures 2A-2D The photograph of the lung cancer pleural effusion organoids cultured using the lung cancer organoid culture medium of the present invention is observed under a microscope. Figure 2A A photograph showing organoids obtained from sample OPB1 after 5 days of culture; Figure 2B Photographs showing organoids obtained from sample OPB1 after 10 days of culture; Figure 2C Shows a photo of organoids obtained from sample OPB2 after 16 days of culture (photographed at 4x magnification); Figure 2D A photograph showing organoids obtained from sample OPB2 after 16 days of culture (photographed at 10x magnification).

[0062] Figure 3A and 3B To compare the pathological and immunohistochemical identification results of the original pleural effusion cell sample OPB1 and the lung cancer organoids obtained by culturing the sample OPB1 using the lung cancer organoid culture medium of the present invention.

[0063] Figure 4A and 4B The results of electron microscopic analysis of lung cancer organoids obtained by culturing sample OPB3 using the lung cancer organoid culture medium of the present invention are as follows: Figure 4B for Figure 4A A partial enlarged view of .

[0064] Figure 5A and 5B The comparison results of culturing lung cancer organoids using the lung cancer organoid culture medium of the present invention and the existing culture medium are shown in FIG. Figure 5A A photograph showing cells cultured with the LPOM medium of the present invention for 14 days; Figure 5B The photograph shows the cells after 14 days of culture using the literature medium ROM.

[0065] Figure 6A and 6B The results of drug concentration sensitivity testing of lung cancer organoids obtained by culturing using the lung cancer organoid culture medium of the present invention are shown. DETAILED DESCRIPTION

[0066] 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.

[0067] [Preparation Example of MST1 / 2 Kinase Inhibitor]

[0068] 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.

[0069] 1. Preparation of MST1 / 2 kinase inhibitor compound 1

[0070] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzene Sulfonamide 1

[0071]

[0072] 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). Then, add thionyl chloride (1.2 mL) dropwise under an ice bath. The reaction mixture is allowed to react at 85°C overnight. After completion of the reaction, the solvent is evaporated under reduced pressure to yield a white solid, which is used directly in the next step.

[0073] 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 system 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.

[0074] 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.

[0075] 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, water was added to quench the reaction, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, and 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.

[0076] 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.

[0077] 2. Preparation of other MST1 / 2 inhibitor compounds of the present invention

[0078] 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.

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] Example 1 Effects of Various Added Factors in Lung Cancer Organoid Culture Medium on the Proliferation of Lung Cancer Organoids

[0085] (1) Preparation of lung cancer organoid culture medium

[0086] First, prepare a basal medium containing an initial culture medium. The initial culture medium can be selected from DMEM / F12, DMEM / F12, or RPMI-1640, commonly used in the art. In this example, the basal medium formulation is: DMEM / F12 (purchased from Corning) + 100 μg / mL Primocin (purchased from InvivoGen, 0.2% (v / v), commercially available at a concentration of 50 mg / ml).

[0087] Different types of additives (see Table 1) were added to the basal culture medium to prepare lung cancer organoid culture medium containing different additives.

[0088] (2) Isolation and processing of primary lung cancer cells

[0089] 1. Sample selection

[0090] Pleural effusion samples for lung cancer were obtained from the patient's chest cavity by professional medical staff at a specialized medical institution, with all patients providing signed informed consent. Pleural effusion volumes ranged from 200-500 ml and were stored and transported using a cold chain.

[0091] 2. Material Preparation

[0092] After surface disinfection, sterile 15mL centrifuge tubes, pipettes, 10mL pipettes, and sterile pipette tips should be placed in a clean bench and exposed to UV light for 30 minutes. Remove the basal culture medium from the 4°C refrigerator 30 minutes in advance.

[0093] 3. Sample separation

[0094] 3.1 The collected pleural effusion was divided into 50 ml centrifuge tubes and centrifuged at 2000 rpm for 10 minutes at room temperature;

[0095] 3.2 Remove the centrifuged cells, discard the supernatant, and resuspend in 30 ml of basal medium; pass through a 40-μm sieve, turn the sieve upside down, and rinse the sieve repeatedly with 20 ml of basal medium to ensure that as many cells as possible on the sieve are collected in a 50 ml centrifuge tube;

[0096] 3.3 Take the collected cell suspension and centrifuge it at 1500 rpm for 5 minutes at room temperature. If it contains red blood cells, add 3-5 ml of red blood cell lysis buffer and lyse it on ice for 5-10 minutes. After complete lysis, centrifuge it at 1500 rpm for 5 minutes at room temperature to obtain a cell pellet for later use.

[0097] 4. Wright-Giemsa staining identification of primary lung cancer cells

[0098] 4.1 Pipette 10 μl of the cells obtained in 3.3 for cell smear, let it dry at room temperature, then add 1 drop of Wright-Giemsa solution A, followed by 3 drops of Wright-Giemsa solution B, mix well, and stain for 3 minutes;

[0099] 4.2 Rinse with running water (do not pour out the dye solution first when rinsing, but rinse it with running water to prevent sediment from settling on the specimen);

[0100] 4.3 Dry, observe and take pictures under the microscope, first take pictures with a low-power microscope (10x) and then with a high-power microscope (40x).

[0101] 5. Cell Counting and Processing

[0102] 5.1 Microscopic observation: 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).

[0103] 5.2 Viable Cell Counting: Take 12 μL of the resuspended cell suspension and add 12 μL of trypan blue dye (manufacturer: Sangon Biotech (Shanghai) Co., Ltd.) to the suspension. Mix thoroughly and then take 20 μL of the suspension into a cell counting plate (manufacturer: Countstar, specification: 50 plates / box). Calculate the percentage of viable large cells (cell size >10 μm) using a cell counter (Countstar, IC1000) as follows: number of viable cells / total number of cells*100%.

[0104] (3) Culture of lung cancer organoids

[0105] The primary lung cancer cells obtained in the above steps were resuspended in basal medium and counted, and the cell density was diluted to 5-10×10 4 Cells / mL were plated in an ultra-low adsorption round-bottom 96-well plate (purchased from Corning) at a density of 10,000 cells / well, and 200 microliters of culture medium with different additives were added to each well (see Table 1 for the type and concentration of additives). The inoculated culture plate was placed in an incubator for culture. After 10 days, the cultured organoids were photographed, and the diameter of the organoids was measured and statistically analyzed to compare the promoting effect of each factor on the proliferation of lung cancer organoids. As an experimental control, a basal culture medium without any additives was used, and the experimental results are shown in Table 1.

[0106] Table 1 Additives in culture medium and their effects on promoting organoid proliferation

[0107] Serial number Types of culture medium additives supplier Final concentration Grading of proliferation degree 1 N2 Gibco 1:50 + 2 HEPES Corning 10mM + 3 R-spondin1 Beijing Yiqiao 20 ng / mL ○ 4 Gastrin MCE 10nM ○ 5 B27 Gibco 1:50 + 6 A8301 MCE 100nM + 7 SB202190 MCE 200nM + 8 Fibroblast Growth Factor / FGF Beijing Yiqiao 10 ng / mL ○ 9 Fibroblast Growth Factor 10 / FGF10 Beijing Yiqiao 50 ng / mL + 10 Noggin Beijing Yiqiao 50 ng / mL ○ 11 Fetal bovine serum / FBS Excell 5％ + 12 Insulin-like growth factor 1 / IGF-1 Beijing Yiqiao 10 ng / mL + 13 Keratinocyte Growth Factor / KGF Beijing Yiqiao 25 ng / mL + 14 GlutaMAX Gibco 1:100 + 15 Niacinamide MCE 2.5mM + 16 Neuregulin 1 / NRG1 Beijing Yiqiao 10 ng / mL + 17 Y27632 MCE 10 μM + 18 ITS Cell Culture Supplement Gibco 1:100 ○ 19 Compound 1 Preparation Example 2.5 μM + 20 CHIR99021 MCE 2.5 μM - 21 Hepatocyte Growth Factor / HGF Beijing Yiqiao 10 ng / mL ○

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

[0109] Based on the above results, factors such as compound 1, Y27632, SB202190, keratinocyte growth factor (KGF), fibroblast growth factor 10 (FGF10), A83-01, B27, GlutaMAX, insulin-like growth factor-1 (IGF-1), nicotinamide, neuregulin 1 (NRG1) and HEPES were selected for further culture experiments.

[0110] Example 2 Effects of different concentrations of culture medium additives on the proliferation of lung cancer organoids

[0111] Primary lung cancer cells were obtained from pleural effusion samples (numbered OPB1 and OPB2) according to the method of Example 1 (2), 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 LPOM medium. The following experiments for Recipes 1-12 were performed in the same manner and are not further detailed here.

[0115] When using the medium of Formula 2, 200 μL of prepared FGF10 was added to each well of the 96-well plate seeded with organoids based on Formula 2. The final concentrations of FGF10 were 200 ng / mL, 100 ng / mL, and 25 ng / mL, respectively. Control wells (BC) were set up using the medium of Formula 2.

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

[0117] When using the culture medium of Formula 4, 200 μL of Y27632 prepared in Formula 4 was added to each well of a 96-well plate seeded with organoids. 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. 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, 200 μL of the prepared NRG1 was added to each well of the 96-well plate seeded with organoids, with final NRG1 concentrations of 1 ng / mL, 5 ng / mL, and 40 ng / mL, respectively; and control wells (BC) were set up using the culture medium of Formula 6.

[0120] When using the medium of Formula 7, 200 μL of prepared IGF-1 was added to each well of a 96-well plate seeded with organoids. The final concentrations of IGF-1 were 2 ng / mL, 10 ng / mL, and 40 ng / mL, respectively. Control wells (BC) were set up using the medium of Formula 7.

[0121] When using the medium of Formula 8, add 200 μL of prepared KGF to each well of a 96-well plate seeded with organoids based on Formula 8. The final concentrations of KGF are 2 ng / mL, 10 ng / mL, and 40 ng / mL, respectively. Set up control wells (BC) using the 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. The final concentrations of GlutaMAX are 1:200, 1:100, and 1:50, respectively. 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 the prepared nicotinamide based on Formula 11 was added to each well of a 96-well plate seeded with organoids. The final concentrations of nicotinamide were 1 mM, 2.5 mM, and 10 mM, respectively. Control wells (BC) were set up using the culture medium of Formula 12.

[0125] When using the medium of Formula 12, add 200 μL of prepared HEPES per well to the 96-well plate seeded with organoids based on Formula 12, with final HEPES concentrations of 1 mM, 2.5 mM, and 10 mM, respectively; and set up control wells (BC) using the medium of Formula 12.

[0126] After 12 days, the cultured organoids were photographed, and the diameter of the organoids was measured and statistically analyzed to compare the promoting effect of each factor concentration on the proliferation of lung cancer organoids. The data collected from the two samples are summarized and shown in Figures 1A to 1L . Figures 1A to 1L 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.

[0127] according to Figures 1A to 1L According to the results, the volume concentration of B27 is preferably 1:25 to 1:100; the content of fibroblast growth factor 10 is preferably 25 to 200 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 neuregulin 1 is preferably 1 to 40 ng / mL; the content of IGF-1 is preferably 2 to 40 ng / 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; the content of nicotinamide is preferably 1 to 10 mM; and the content of HEPES is preferably 1 to 10 mM.

[0128] Example 3 Lung cancer organoid culture and identification

[0129] The primary lung cancer cells (OPB1, OPB2, OPB3) obtained according to the method described in Example 1 (2) were resuspended in the lung cancer organoid culture medium LPOM of the present invention and counted. The cell density was diluted to 5-10×10 4 Cells were seeded into ultra-low adsorption 24-well plates at a concentration of 500 mL / well and cultured in an incubator. LPOM medium was replenished every 5 days with 100 μL added each time.

[0130] On days 5-16, the cultured lung cancer organoids were observed using a microscope (Invitrogen EVOS M500). Figures 2A-2D The images below show microscopic images of lung cancer organoids from samples OPB1 (day 5, 4x magnification), OPB1 (day 10, 4x magnification), OPB2 (day 16, 4x magnification), and OPB2 (day 16, 10x magnification) after culture. As shown, the organoids continuously increase in size and form microscopic tissue-like structures during the culture process. Sample OPB2 develops a variety of structures after 16 days of culture, demonstrating the ability to mimic in vitro tumor heterogeneity.

[0131] The cultured lung cancer organoids were pathologically and immunohistochemically identified, and the corresponding original pleural effusion cells were also pathologically and immunohistochemically identified to compare the consistency of the pathological indicators of the organoids and the original pleural effusion.

[0132] The expanded lung cancer organoids were collected and analyzed by electron microscopy according to standard procedures.

[0133] Figure 3A and 3B The images below show the results of pathological and immunohistochemical characterization of lung cancer organoids obtained from sample OPB3 cultured in vitro, taken under a 20x objective. As shown, the results demonstrate that the organoids' structural morphology resembles that of cancerous tissue. Immunohistochemical markers confirm that the cells obtained from this sample are lung cancer cells, and the pathological markers are consistent with those of pleural effusion cells. This indicates that the lung cancer organoids cultured using the culture medium LPOM presented herein have consistent diagnostic results with those of the pre-culture lung cancer pleural effusion tissue sample. Furthermore, the resulting organoids are structurally more complex than pleural effusion cells, better reflecting the complex microenvironment in the body.

[0134] Figure 4A and 4B The results of electron microscopy examination of lung cancer organoids obtained by in vitro culture of sample OPB3 are shown in Figure 2. Figure 4A and 4B As shown, under different magnifications, it can be observed that the organoids have the pulmonary cilia structure unique to lung tissue, which proves that the organoids cultured in the culture medium of the present invention have certain physiological functions.

[0135] Comparison of Example 4 with existing culture medium culture effects

[0136] (1) Preparation of control culture medium

[0137] The culture medium used in the preparation literature (Norman Sachs et al., The EMBO Journal (2019) e100300) was prepared, and its formula was Advanced DMEM / F12 medium (purchased from Invitrogen) + 1:100 Penicillin / Streptomycin (purchased from Corning) + 50 μg / mL Primocin (purchased from Invivogen) + 1:100 GlutaMAX (purchased from Corning) + 10 mM HEPES (purchased from Thermo Fisher Scientific) + 1:50 B27 (purchased from Gibco) + 1.25 mmol / L N-acetylcysteine ​​(purchased from MCE) + 5 mmol / L nicotinamide (purchased from MCE) + 500 ng / mL R-Spondin 1 (purchased from Sino Biological) + 25 ng / mL keratinocyte growth factor (purchased from Sinobiological) + 100 ng / mL fibroblast growth factor 10 (purchased from Sino Biological Company) + 100ng / ml Noggin (purchased from Sino Biologica Company) + 500nmol / L SB202190 (purchased from MCE Company) + 500nmol / L A8301 (purchased from MCE Company) + 5μmol / L Y27632 (purchased from MCE Company). Hereinafter referred to as ROM medium.

[0138] (2) Lung cancer organoid culture

[0139] Primary lung cancer cells were obtained from the intraoperative tissue sample OPB8 according to the method of Example 1 (2), and organoid culture was performed using LPOM medium and ROM medium according to the method of Example 3, respectively.

[0140] On the 14th day of culture, the cultured lung cancer organoids were observed using a microscope (Invitrogen EVOS M500). Figure 5A and 5B These are photos of organoids obtained by culturing in LPOM medium and ROM medium for 14 days, taken under a 4x objective lens.

[0141] according to Figure 5A and 5B The results showed that compared with ROM medium, LPOM medium can significantly promote the formation and expansion culture of lung cancer organoids.

[0142] Example 5: Lung cancer organoids expanded using the culture medium of the present invention for drug screening

[0143] (1) Lung cancer organoid culture

[0144] Primary lung cancer cells were isolated from an intraoperative lung cancer sample (OPB6) according to the method of Example 1 (2), and organoids were cultured using LPOM medium, with 10,000 cells / 100 μL medium per well in an ultra-low adsorption 96-well plate. Drug screening was performed when the diameter of the lung cancer organoids exceeded 50 μm.

[0145] (2) Screening drug preparation

[0146] Two drugs (bortezomib and afatinib; both purchased from MCE) with 10 concentration gradients were prepared as follows and stored for future use.

[0147] Preparation of bortezomib and afatinib drug additive solutions at different concentrations: Bortezomib and afatinib were prepared into 10 stock solutions with the highest concentration being 20,000 μM. The solutions were then diluted at a 2-fold 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, 78.125 μM, and 39.0625 μM.

[0148] (3) Dosing

[0149] The prepared drug storage solution was taken out and placed at room temperature. The drug was diluted 500 times with LPOM medium and then used. The organoids obtained by culture according to step (1) were taken out from the incubator, and the medium containing the drug was slowly poured into the 96-well ultra-low adsorption culture plate along the well wall at a rate of 100 μL per well. The final test drug concentrations were 20000 nM, 10000 nM, 5000 nM, 2500 nM, 1250 nM, 625 nM, 312.5 nM, 156.25 nM, 78.13 nM and 39.06 nM. 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 5 days.

[0150] (4) Organoid viability test

[0151] Remove CellTiter-Glo luminescent reagent (purchased from Promega) from a 4°C refrigerator, take 10 ml of the reagent into the sample reservoir, remove 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, pipette 100 μL into another white 96-well plate, and use a multi-function microplate reader (Perkin Elmer Envision) for detection.

[0152] (5) Data processing

[0153] According to the formula, drug inhibition rate (%) = 100% - (chemiluminescence value of the fifth day culture well drug treatment group / chemiluminescence value of the zero day culture well drug treatment group) / (chemiluminescence value of the fifth day culture well drug treatment group) DMSO / Chemiluminescence value of culture wells on day 0 DMSO )*100%, and the inhibition rates of different drugs were calculated. The results are shown in FIG6 . Figure 6A and 6B Figure 2 shows the inhibition rate curves for lung cancer organoid growth at different concentrations of the test drugs. Of the two anti-cancer drugs, bortezomib exhibited a strong inhibitory effect on organoid growth at all 10 concentrations. Afatinib exhibited varying inhibitory effects at different concentrations, demonstrating a dose-dependent pattern. This suggests that organoids from the same patient may 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 settings for lung cancer patients.

[0154] Industrial Applicability

[0155] The present invention provides a culture medium and method for culturing lung cancer organoids. The cultured organoids can be used for evaluating and screening drug efficacy. Therefore, the present invention is suitable for industrial applications.

[0156] 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 lung cancer organoids, characterized in that Made with the following ingredients: MST1 / 2 kinase inhibitor, B27, fibroblast growth factor 10, SB202190, Y27632, A83-01, neuregulin 1, insulin-like growth factor 1, keratinocyte growth factor, GlutaMAX, nicotinamide, HEPES, an initial culture medium selected from DMEM / F12, DMEM, F12, or RPMI-1640; and antibiotic; 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 B27 to culture medium is 1:25 to 1:100; The concentration of the fibroblast growth factor 10 is 25 to 200 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 neuregulin 1 is 1 to 40 ng / mL; The concentration of the insulin-like growth factor 1 is 2 to 40 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 is 1:50 to 1:200; The concentration of nicotinamide is 1 to 10 mM; The concentration of the HEPES is 1-10 mM.

2. The culture medium according to claim 1, wherein The antibiotic is selected from one or more of streptomycin / penicillin, amphotericin B and primocin.

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

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

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