Culture medium, culture method and application of lung cancer organoids

Abstract

The present invention relates to 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, and nicotinamide. The present invention also relates to a culture method for lung cancer tissue-derived organoids and its use. By using the lung cancer tissue-derived organoid culture medium of the present invention, effective and rapid expansion of lung cancer organoids can be achieved. The expanded organoids retain 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 tissue-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 malignant tumor originating from epithelial tissue, ranking first in both morbidity and mortality among various malignant tumors in my country. In recent years, targeted therapy for lung cancer has become a major treatment option. However, the specific targeted drug used for lung cancer patients depends on the results of genetic sequencing. Not all patients with gene mutations experience effective remission after targeted drug treatment, necessitating new research models for clinical drug treatment and the development of novel lung cancer treatment drugs. However, traditional lung cancer cell lines no longer meet this need, and an increasing number of studies are using primary cells derived from patient tissue for various studies.

[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, lung 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, there is a need to develop a low-cost, simple, and highly successful organoid culture method and culture medium. 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 lung cancer organoids in vitro.

[0006] 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, 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 the MST1 / 2 kinase inhibitor is preferably 2.5 to 10 μM, more preferably 5 to 10 μM;

[0028] (2) The volume ratio of B27 or N2 cell culture additive to culture medium is preferably 1:25 to 1:100, more preferably 1:50 to 1:100;

[0029] (3) The concentration of fibroblast growth factor 10 is preferably 25 to 200 ng / mL, more preferably 25 to 100 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 neuregulin 1 is preferably 1 to 40 ng / mL, more preferably 5 to 40 ng / mL;

[0034] (8) The concentration of insulin-like growth factor-1 is preferably 2 to 40 ng / mL, more preferably 10 to 40 ng / mL;

[0035] (9) The concentration of keratinocyte growth factor is preferably 2 to 40 ng / mL, more preferably 10 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, more preferably 2.5 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, the lung cancer organoids 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 solid lung cancer tissue to obtain primary lung cancer cells. The process includes the following steps:

[0043] (1) Lung cancer tissue samples were isolated and added with basal culture medium and tissue digestion solution in a 1:1 ratio (about 10 mL of tissue digestion solution was added per 1 g of tumor tissue) and placed 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 hours;

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

[0047] The primary lung cancer cells obtained in step 1 above were resuspended in the initial culture medium, basal culture medium or lung 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 evenly, then inoculate the mixture into a multi-well plate, place the inoculated multi-well plate in an incubator for 30 to 60 minutes, wait for the Matrigel to completely solidify, and then add the lung cancer organoid culture medium of the present invention for expansion culture.

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

[0049] (1) Cultivating lung cancer organoids using the lung cancer organoid culture 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 organoid 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 lung cancer tissue to over 85%;

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

[0056] (3) High amplification efficiency, capable of rapidly culturing lung cancer organoids, and the amplified lung cancer organoids can 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 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

[0059] Figures 1A-1K This 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.

[0060] Figures 2A-2F This is a photograph of lung cancer organoids cultured using the lung cancer organoid culture medium of the present invention observed under a microscope, wherein Figure 2A Photographs showing organoids obtained from sample OB1 after 5 days of culture; Figure 2B Photographs showing organoids obtained from sample OB1 after 12 days of culture; Figure 2C Photographs showing organoids obtained from sample OB2 after 7 days of culture; Figure 2D Photographs showing organoids obtained from sample OB2 after 15 days of culture; Figure 2E and 2F Photographs showing different fields of view of organoids obtained from sample OB3 after 21 days of culture.

[0061] Figure 3A Results of pathological and immunohistochemical identification of the tissue-derived sample OB3 and the lung cancer organoids obtained by culturing the sample OB3 using the lung cancer organoid culture medium of the present invention; Figure 3B These are the results of pathological and immunohistochemical identification of the tissue-derived sample OB4 and the lung cancer organoids obtained by culturing the sample OB4 using the lung cancer organoid culture medium of the present invention.

[0062] Figure 4A and 4B These are the results of electron microscopic analysis of lung cancer organoids obtained by culturing sample OB4 using the lung cancer organoid culture medium of the present invention.

[0063] Figure 5A and 5B Figure 2 is a comparison of the results of culturing lung cancer organoids using the lung cancer organoid culture medium of the present invention and the existing culture medium, wherein Figure 5A A photograph showing the cultured cells after 15 days using the LOM medium of the present invention; Figure 5B The photograph shows the cells after 15 days of culture using the literature medium ROM.

[0064] Figure 6A and 6BThe results of drug concentration sensitivity testing of lung cancer organoids obtained by culture using the lung cancer organoid culture medium of the present invention are shown. DETAILED DESCRIPTION

[0065] 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.

[0066] [Preparation Example of MST1 / 2 Kinase Inhibitor]

[0067] 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.

[0068] 1. Preparation of MST1 / 2 kinase inhibitor compound 1

[0069] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzene Sulfonamide 1

[0070]

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 2. Preparation of other MST1 / 2 inhibitor compounds of the present invention

[0077] 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.

[0078]

[0079]

[0080]

[0081]

[0082]

[0083] Example 1 Effects of Various Added Factors in Lung Cancer Organoid Culture Medium on the Proliferation of Lung Cancer Organoids

[0084] (1) Preparation of lung cancer organoid culture medium

[0085] 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).

[0086] Different types of additives (see Table 1) were added to the basal culture medium to prepare lung cancer organoid culture medium containing different additives.

[0087] (2) Isolation and processing of primary lung cancer cells

[0088] 1. Sample selection

[0089] Lung 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.

[0090] 2. Material Preparation

[0091] 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.

[0092] 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).

[0093] 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.

[0094] 3. Sample separation

[0095] 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;

[0096] 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;

[0097] 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.

[0098] 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.

[0099] 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;

[0100] 3.6 Discard the supernatant and add 2 mL of basal culture medium to resuspend the cells for later use.

[0101] 4. Cell Counting and Processing

[0102] 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).

[0103] 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%.

[0104] (3) Culture of lung cancer organoids

[0105] The primary lung 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 lung 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.

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

[0107]

[0108]

[0109] 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.

[0110] 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, and neuregulin 1 (NRG1) were selected for further culture experiments.

[0111] Example 2 Effects of different concentrations of culture medium additives on the proliferation of lung cancer organoids

[0112] Primary lung cancer cells were obtained from intraoperative tissue samples (numbered OB1 and OB2) according to the method of Example 1 (2), and organoid culture was performed using the culture medium formula in Table 2 below.

[0113] Table 2 Culture medium formula (concentration is final concentration)

[0114]

[0115]

[0116] When using medium from Recipe 1, 200 μL of B27 prepared in Recipe 1 was added to each well of a 96-well plate seeded with organoids, with final B27 concentrations of 1:25, 1:50, and 1:100, respectively. Control wells (BC) were also prepared using medium from Recipe 1. The final concentrations of other added factors in this series of media were the same as those in LOM medium. The following experiments with Recipes 1-12 were performed in the same manner and are not further detailed here.

[0117] When using the culture 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 culture medium of Formula 2.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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 based on Formula 6, 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.

[0122] When using the culture medium of Formula 7, add 200 μL of prepared IGF-1 to each well of the 96-well plate seeded with organoids based on Formula 7. The final concentrations of IGF-1 are 2 ng / mL, 10 ng / mL, and 40 ng / mL, respectively. Set up control wells (BC) using the culture medium of Formula 7.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] After 10 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 lung 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 15 days of culture using each medium to the diameter of the organoid obtained after 15 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.

[0128] according to Figures 1A to 1KThe results show that the volume concentration of B27 is preferably 1:25 to 1:100, more preferably 1:50 to 1:100; the content of fibroblast growth factor 10 is preferably 25 to 200 ng / mL, more preferably 25 to 100 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; and the content of neuregulin 1 is preferably 1 to 40 ng / mL. , more preferably 5-40 ng / mL; the content of insulin-like growth factor-1 is preferably 2-40 ng / mL, more preferably 10-40 ng / mL; the content of keratinocyte growth factor is preferably 2-40 ng / mL, more preferably 10-40 ng / mL; the volume concentration of GlutaMAX is preferably 1:50-1:200; the content of MST1 / 2 kinase inhibitor is preferably 2.5-10 μM; the content of nicotinamide is preferably 1-10 mM, more preferably 2.5-10 mM.

[0129] The optimal concentrations of the above-mentioned supplementary factors were used to prepare the lung cancer organoid culture medium LOM used in the following examples of the present invention, which contained: basal medium BM, 1:50 B27 supplement, 100 ng / mL FGF10, 500 nM SB202190, 10 μM Y27632, 500 nM A83-01, 5 ng / mL NRG1, 10 ng / mL IGF-1, 10 ng / mL KGF, 1:100 GlutaMAX, 5 μM compound 1, and 2.5 mM nicotinamide.

[0130] Example 3 Lung cancer organoid culture and identification

[0131] The primary lung cancer cells (OB1, OB2, OB3) obtained according to the method described in Example 1 (2) were resuspended in the lung cancer organoid culture medium LOM 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 lung cancer organoid culture medium (LOM), previously brought to room temperature, was added to each well. The culture was expanded by replacing the medium every five days.

[0132] On days 5-21, the cultured lung cancer organoids were observed using a microscope (Invitrogen EVOS M500). Figures 2A-2FImages of lung cancer organoids from samples OB1 (days 5 and 12), OB2 (days 7 and 15), and OB3 (day 21, different fields of view) were taken under a 4x objective. As shown, the organoids continuously increase in size during culture; sample OB3 reaches a maximum diameter of 1800μM after 21 days of culture. The ability to generate different organoid types from the same sample allows for in vitro simulation of tumor heterogeneity.

[0133] The cultured lung 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.

[0134] The expanded lung cancer organoids were collected and analyzed by electron microscopy according to standard procedures.

[0135] Figure 3A Results of pathological and immunohistochemical identification of the tissue-derived sample OB3 and the lung cancer organoids obtained by culturing the sample OB3 using the lung cancer organoid culture medium of the present invention; Figure 3B Images taken at a 20x objective lens show the results of pathological and immunohistochemical characterization of tissue-derived sample OB4 and lung cancer organoids obtained by culturing sample OB4 using the lung cancer organoid culture medium of the present invention. As shown in the figures, the organoids' structural morphology resembles that of cancerous tissue; immunohistochemical markers confirm that the cells obtained from these two organoid cultures are lung cancer cells. The results demonstrate that the lung cancer organoids cultured using the LOM culture medium of the present invention produce consistent diagnostic results with those of the pre-cultured lung cancer tissue.

[0136] Figure 4A and 4B The results of electron microscopy examination of lung cancer organoids obtained from sample OB4 cultured in vitro. Figure 4A and 4B As shown, at magnifications of 1500 times and 6000 times, respectively, it can be observed that the organoids have the pulmonary cilia structure unique to lung tissue, proving that the organoids cultured with the patented culture medium of the present invention have certain physiological functions.

[0137] Comparison of Example 4 with existing culture medium culture effects

[0138] (1) Preparation of control culture medium

[0139] 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 LA8301 (purchased from MCE Company) + 5μmol / L Y27632 (purchased from MCE Company). Hereinafter referred to as ROM medium.

[0140] (2) Lung cancer organoid culture

[0141] Primary lung cancer cells were obtained from the intraoperative tissue sample OB8 according to the method of Example 1 (2), and organoid culture was performed using LOM medium and ROM medium according to the method of Example 3, respectively.

[0142] On the 15th 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 LOM medium and ROM medium for 15 days, taken under a 4x objective lens.

[0143] according to Figure 5A and 5B The results showed that compared with ROM medium, LOM medium can significantly promote the formation and expansion culture of lung cancer organoids.

[0144] Example 5: Lung cancer organoids expanded using the culture medium of the present invention for drug screening

[0145] (1) Lung cancer organoid culture

[0146] Primary lung cancer cells were isolated from an intraoperative lung cancer sample (OB6) according to the method of Example 1 (2), and organoids were cultured using LOM medium. Drug screening was performed when the diameter of the lung cancer organoids exceeded 50 μm.

[0147] (2) Screening drug preparation

[0148] According to the table below, 10 concentration gradients of two drugs (bortezomib and afatinib; both purchased from MCE) were prepared and stored for use.

[0149] Preparation of bortezomib and afatinib drug additive solutions at different concentrations: Bortezomib and afatinib 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, 78.125 μM, and 39.0625 μM.

[0150] (3) Dosing

[0151] The prepared drug storage solution was taken out and placed at room temperature. The drug was diluted 1000 times with LOM 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 5 days.

[0152] (4) Organoid viability test

[0153] 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.

[0154] (5) Data processing

[0155] 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%, calculate the inhibition rate of different drugs, and show the results in Figure 6A and 6B . 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.

[0156] Industrial Applicability

[0157] 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.

[0158] 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: MST1 / 2 kinase inhibitor, B27, fibroblast growth factor 10, SB202190, Y27632, A83-01, neuregulin 1, insulin-like growth factor-1, keratinocyte growth factor, GlutaMAX, niacinamide, and 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 the nicotinamide is 1-10 mM.

2. The culture medium according to claim 1, wherein The content of each component in the culture medium is: The concentration of the MST1 / 2 kinase inhibitor is 5 to 10 μM; The volume ratio of B27 to culture medium is 1:50 to 1:100; The concentration of the fibroblast growth factor 10 is 25 to 100 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 5 to 40 ng / mL; The concentration of the insulin-like growth factor-1 is 10 to 40 ng / mL; The concentration of the keratinocyte growth factor is 10 to 40 ng / mL; The volume ratio of the GlutaMAX to the culture medium is 1:50 to 1:200; The concentration of the nicotinamide is 2.5-10 mM.

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

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

5. 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 4; (2) Select the drug to be tested and dilute it according to the required concentration gradient; (3) adding the diluted drug to the organoid cultured in (1); (4) Conduct organoid size or organoid viability testing.

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