CN115960815A - Culture medium, culture method and application of organoids derived from pleural effusion of lung cancer - Google Patents

Abstract

The invention relates to a culture medium for lung cancer organoid culture, 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 invention also relates to a culture method and application of the lung cancer pleural effusion source organoid. By using the lung cancer organoid culture medium, the lung cancer pleural effusion organoid can be effectively and rapidly amplified, so that the amplified organoid keeps the pathological characteristics of a patient, the culture success rate and the amplification rate of the lung cancer organoid are improved, and a research basis can be provided for the personalized treatment of the patient.

Description

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

Technical Field

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 evaluating and screening drug efficacy.

Background Art

Lung cancer is a common malignant tumor of the lungs and one of the most threatening malignant tumors to human health and life. Clinical studies have confirmed that malignant pleural effusion (MPE) will occur in advanced lung cancer. The appearance of MPE indicates that the opportunity for surgical treatment has been lost, indicating that the patient has a poor prognosis. High-throughput drug sensitivity testing requires the separation and purification of a sufficient number of patient autologous tumor cells in vitro, and the in vitro culture of tumor cells has always been a bottleneck problem that hinders the drug sensitivity testing of tumor cells. Since patients with advanced lung cancer and pleural effusion often have high-purity tumor cells in their pleural effusion, it is suitable to use high-throughput drug sensitivity testing technology to screen the best treatment plan for patients. Traditional clinical drug sensitivity testing mostly uses two-dimensional cell culture. However, cells cultured in two dimensions only simulate tissue physiological conditions to a limited extent and lack the real tissue structure in vivo, which can easily lead to low differentiation levels and loss of cell physiological functions, and thus make it difficult for the obtained experimental results to predict the actual clinical results.

Organoids are three-dimensional (3D) cell cultures that are mainly derived from embryonic stem cells, induced pluripotent stem cells, and adult stem cells with differentiation ability in the human body. Endogenous tissue stem cells exist in different tissues and organs, and play an important role in maintaining the functional morphology of each organ. Under certain induction conditions in vitro, these stem cells can self-organize into a miniature structure with a diameter of only a few millimeters. Tumor organoids are microscopic 3D tumor cell models cultured in the laboratory from primary tumors taken from patients. Tumor organoids highly simulate the characteristics of the source tumor tissue, retain the tumor heterogeneity between individuals, and can be used for functional testing, such as high-throughput drug screening and personalized precision treatment. At present, the culture method of lung cancer organoids mostly uses expensive protein factors such as R-spondin-1, WNT3A, and Noggin, resulting in a high cost of organoid culture; and this technology is complex to operate and technically difficult, which limits its large-scale commercial application. Therefore, it is necessary to develop a low-cost, simple, and high-success rate organoid culture method and culture medium.

Summary of the invention

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.

One aspect of the present invention is to provide a culture medium for lung cancer organoids, the culture medium 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,

in,

R ₁ is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and aryl (e.g. phenyl and naphthyl, etc.), aryl C1-C6 alkyl (e.g. benzyl, etc.) and heteroaryl (e.g. thienyl, etc.) optionally substituted by 1-2 independently R ₆ ;

_R2 and _R3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;

_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.);

R ₆ 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).

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 ₁ is selected from C1-C6 alkyl, phenyl optionally substituted by 1-2 independently R ₆ , thienyl optionally substituted by 1-2 independently R ₆ , and benzyl optionally substituted by 1-2 independently R ₆ , and R ₁ is more preferably phenyl optionally substituted by 1-2 independently R ₆ ;

R ₅ is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, and 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 MST1/2 kinase inhibitor is preferably 2.5-10 μM;

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

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

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

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

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

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

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

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

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

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

(12) The concentration of HEPES is preferably 1 to 10 mM.

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.

In a preferred embodiment, when the antibiotic is selected from streptomycin/penicillin, the concentration range of streptomycin is 25-400 μg/mL, the concentration range of penicillin is 25-400 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 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.

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

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

(1) Separating lung cancer pleural effusion tissue samples, transferring the collected lung cancer pleural effusion into a centrifuge tube, centrifuging at a speed of 1800 to 2200 rpm for 8 to 12 minutes;

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

(3) Observe the cell pellet. If it contains red blood cells, add 3-5 ml of red blood cell lysis buffer and lyse the cells 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 later use.

The basal 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.

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

The primary lung cancer cells obtained in the above step 1 are resuspended and counted in the lung cancer organoid culture medium of the present invention, and the cell density is diluted to 5 to 10×10 ⁴ cells/mL. The cell suspension is added to an ultra-low adsorption culture plate or culture bottle for expansion culture.

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

(1) culturing lung cancer organoids using the lung cancer organoid culturing method of the present invention;

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

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

(4) Conduct organoid size or organoid viability tests.

The beneficial effects of the present invention include:

(1) Improve the success rate of culturing organoids derived from lung cancer tissue to more than 85%;

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

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

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

(5) The technology can generate 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

Figures 1A-1L are graphs 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.

Figures 2A-2D are photographs of lung cancer pleural effusion organoids obtained by culture using the lung cancer organoid culture medium of the present invention observed under a microscope, wherein Figure 2A shows a photograph of the organoid obtained from sample OPB1 after 5 days of culture; Figure 2B shows a photograph of the organoid obtained from sample OPB1 after 10 days of culture; Figure 2C shows a photograph of the organoid obtained from sample OPB2 after 16 days of culture (photographed with a 4x microscope); Figure 2D shows a photograph of the organoid obtained from sample OPB2 after 16 days of culture (photographed with a 10x microscope).

3A and 3B are comparisons of 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.

Figures 4A and 4B are the results of electron microscopy analysis of lung cancer organoids obtained by culturing sample OPB3 using the lung cancer organoid culture medium of the present invention, and Figure 4B is a partial enlarged view of Figure 4A.

Figures 5A and 5B are comparison results of culturing lung cancer organoids using the lung cancer organoid culture medium of the present invention and the existing culture medium, respectively, wherein Figure 5A shows a photograph after 14 days of culture using the LPOM culture medium of the present invention; Figure 5B shows a photograph after 14 days of culture using the literature culture medium ROM.

Figures 6A and 6B show 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.

DETAILED DESCRIPTION

In order to better understand the present invention, the present invention is further described below in conjunction with the embodiments and drawings. The following embodiments are only for illustrating the present invention but not for limiting it.

[Preparation Example of MST1/2 Kinase Inhibitor]

In this specification, MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1/2 signal transduction. Generally speaking, MST1/2 kinase inhibitors, for example, bind to MST1/2 kinases and reduce their activity. Since MST1 and MST2 have similar structures, MST1/2 kinase inhibitors can also be 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): Add 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) to a round-bottom flask, then add methanol (30 ml), and then add dichlorothionyl (1.2 ml) dropwise under ice bath. The reaction system is reacted at 85°C overnight. After the reaction is completed, the solvent is evaporated under reduced pressure, and the obtained white solid is directly used in the next step.

2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetic acid methyl ester (A3): Add 2-amino-2-(2,6-difluorophenyl)acetic acid methyl ester (2 g) to a round-bottom flask, then add acetone (30 ml) and potassium carbonate (2.2 g), then cool the system to -10°C with an ice-salt bath, and then slowly add 2,4-dichloro-5-nitropyrimidine (3.1 g) in acetone. The reaction system is stirred at room temperature overnight. After the reaction is completed, the reaction is filtered, the filtrate is freed from the solvent under reduced pressure, and the residue is 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-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetic acid methyl ester (2.5 g) to a round-bottom flask, then add 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 under reduced pressure to dryness, and the obtained product was neutralized with saturated sodium bicarbonate to alkalinity. The organic phase was extracted with ethyl acetate, and the organic phase was washed with water and saturated brine, respectively, and then 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) to a round-bottom flask, cool to -35°C, add iodomethane (0.9 ml), then add sodium hydride (615 mg), and continue stirring the reaction system for two hours. After the reaction is completed, water is added to quench the reaction, and ethyl acetate is used for extraction. The organic phase is washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate. The organic phase is filtered and evaporated to dryness under reduced pressure to obtain a crude product. The crude product is 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)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-butyl alcohol (5 ml) were added to a round-bottom flask. The reaction system was stirred at 120°C overnight. After the reaction was completed, the mixture was filtered and washed 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 in a similar manner to compound 1, and their structures and mass spectrometry data are shown in the following table.

Example 1 Effects of various added factors in lung cancer organoid culture medium on the proliferation of lung cancer organoids

(1) Preparation of lung cancer organoid culture medium

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 commonly used in the art. In this embodiment, the formula of the basal culture medium is: DMEM/F12 culture medium (purchased from Corning) + 100 μg/mL Primocin (purchased from InvivoGen, 0.2% (v/v), commercially available product concentration 50 mg/ml).

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

(2) Isolation and processing of primary lung cancer cells

1. Sample selection

Pleural effusion samples of lung cancer were obtained from the chest cavity of patients by professional medical staff of professional medical institutions, and all patients signed informed consent forms. The volume of pleural effusion was 200-500 ml and was stored and transported under cold chain.

2. Material preparation

After disinfecting the surfaces of 15mL sterile centrifuge tubes, pipettes, 10mL pipettes, sterile pipette tips, etc., place them in a clean bench and irradiate them with UV light for 30 minutes. Take out the basal culture medium from the 4℃ refrigerator 30 minutes in advance.

3. Sample separation

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

3.2 Take out the centrifuged cells, discard the supernatant, and resuspend them in 30 ml of basal culture medium; Pass them through a 40-micron sieve, turn the sieve upside down, and repeatedly rinse the sieve with 20 ml of basal culture medium to ensure that as many cells as possible on the sieve are collected in a 50 ml centrifuge tube;

3.3 Take the collected cell suspension and centrifuge it at 1500rpm 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 lysis is complete, centrifuge it at 1500rpm for 5 minutes at room temperature to obtain a cell pellet for later use.

4. Wright-Giemsa staining of primary lung cancer cells

4.1 Take 10 μl of the cells obtained in 3.3 for cell smear, dry at room temperature, add 1 drop of Wright-Giemsa A solution, then add 3 drops of Wright-Giemsa B solution, mix well and stain for 3 minutes;

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);

4.3 Dry, observe and take pictures under the microscope, first use low power microscope (10 times) and then use high power microscope (40 times).

5. Cell Counting and Processing

5.1 Observation under microscope: Take a small amount of resuspended cells and spread them on a culture dish, and observe the density and morphology of cancer cells under a microscope (CNOPTEC, BDS400);

5.2 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). Calculate the percentage of live large cells (cell size>10 μm) using a cell counter (Countstar, IC1000) = number of live cells/total number of cells*100%.

(3) Culture of lung cancer organoids

The primary lung cancer cells obtained in the above steps were resuspended and counted with basal culture medium, and the cell density was diluted to 5-10×10 ⁴ cells/mL. They were inoculated in an ultra-low adsorption round-bottom 96-well plate (purchased from Corning) at 10,000 cells/well, and then 200 microliters of culture medium with different additives were added to each well (additive types and concentrations are shown in Table 1). 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 measured 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.

Table 1 Additives in the culture medium and their effects on promoting organoid proliferation

Serial number Types of culture medium additives supplier Final concentration Proliferation degree grading 1 N2 Gibco 1:50 + 2 HEPES Corning 10mM + 3 R-spondin1 Sino Biological 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 Sino Biological 10 ng/mL ○ 9 Fibroblast Growth Factor 10/FGF10 Sino Biological 50ng/mL + 10 Noggin Sino Biological 50ng/mL ○ 11 Fetal Bovine Serum/FBS Excell 5% + 12 Insulin-like growth factor 1/IGF-1 Sino Biological 10 ng/mL + 13 Keratinocyte Growth Factor/KGF Sino Biological 25 ng/mL + 14 GlutaMAX Gibco 1:100 + 15 Niacinamide MCE 2.5mM + 16 Neuregulin 1/NRG1 Sino Biological 10 ng/mL + 17 Y27632 MCE 10μM + 18 ITS Cell Culture Supplement Gibco 1:100 ○ 19 Compound 1 Preparation Example 2.5μM + 20 CHIR99021 MCE 2.5μM - twenty one Hepatocyte Growth Factor/HGF Sino Biological 10 ng/mL ○

Among them, "+" indicates that compared with the basic culture medium, the culture medium with the addition of the additive has a proliferation-promoting effect on at least two of the lung cancer organoids isolated from lung cancer tissue; "-" indicates that the culture medium with the addition of the additive shows an inhibitory effect on at least one of the lung cancer organoids 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 of the lung cancer organoids isolated from lung cancer tissue.

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 are planned to be selected for further culture experiments.

Example 2 Effects of different concentrations of culture medium added factors on the proliferation of lung cancer organoids

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.

Table 2 Culture medium formula (concentration is final concentration)

When using the medium of Formula 1, 200 μL of prepared B27 was added to each well of the 96-well plate seeded with organoids on the basis of Formula 1, and the final concentrations of B27 were 1:25, 1:50, and 1:100, respectively; and the control well (BC) was set up using the medium of Formula 1. The final concentrations of other added factors in this series of culture media are the same as those of LPOM culture media. The experiments of the following formulas 1-12 were also carried out in the same way and will not be repeated here.

When using the culture medium of Formula 2, add 200 μL of prepared FGF10 to each well of the 96-well plate seeded with organoids based on Formula 2. The final concentrations of FGF10 are 200 ng/mL, 100 ng/mL, and 25 ng/mL, respectively; and set up control wells (BC) using the culture medium of Formula 2.

When using the culture medium of Formula 3, 200 μL of the prepared SB202190 cell culture supplement was added to each well of the 96-well plate seeded with organoids based on Formula 3. The final concentrations of SB202190 cell culture supplement were 200 nM, 500 nM, and 1000 nM, respectively; and control wells (BC) were set up using the culture medium of Formula 3.

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

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

When using the culture medium of Formula 6, 200 μL of prepared NRG1 was added to each well of the 96-well plate seeded with organoids based on Formula 6, and the final concentrations of NRG1 were 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.

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; and set up control wells (BC) using the culture medium of Formula 7.

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, and the final concentrations of KGF were 2 ng/mL, 10 ng/mL, and 40 ng/mL, respectively; and control wells (BC) were set up using the culture medium of Formula 8.

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

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 the 96-well plate seeded with organoids, and the final concentrations of compound 1 were 2.5 μM, 5 μM, and 10 μM, respectively; and control wells (BC) were set using the culture medium of Formula 10.

When using the culture medium of Formula 11, add 200 μL of prepared nicotinamide to each well of the 96-well plate seeded with organoids based on Formula 11, and the final concentrations of nicotinamide are 1 mM, 2.5 mM, and 10 mM, respectively; and use the culture medium of Formula 12 to set up control wells (BC).

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

After 12 days, the cultured organoids were photographed, and the diameter of the organoids was measured and counted 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 in Figures 1A to 1L. In Figures 1A to 1L, the ratio is the ratio of the organoid diameter obtained by culturing for 12 days using each culture medium to the organoid diameter obtained by culturing for 12 days in the corresponding BC control well. A ratio greater than 1 indicates that the culture medium containing different concentrations of factors or small molecule compounds has a better proliferation promoting effect than the control well culture medium; a ratio less than 1 indicates that the culture medium containing different concentrations of factors or small molecule compounds has a weaker proliferation promoting effect than the control well culture medium.

According to the results of Figures 1A to 1L, 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.

Example 3 Lung cancer organoid culture and identification

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

On days 5-16, the cultured lung cancer organoids were observed using a microscope (Invitrogen EVOS M500). Figures 2A-2D are photos of the lung cancer organoids obtained after culture of samples OPB1 (day 5, 4x), OPB1 (day 10, 4x), OPB2 (day 16, 4x), and OPB2 (day 16, 10x) under a microscope. As shown in the figure, the volume of the organoids continued to increase during the culture process, and micro-tissue structures were continuously formed; sample OPB2 could form various types of structures after 16 days of culture, indicating that the heterogeneity of in vivo tumors can be simulated in vitro.

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 pathological indicators between the organoids and the original pleural effusion.

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

Figures 3A and 3B are the results of pathological and immunohistochemical identification of lung cancer organoids obtained after in vitro culture of sample OPB3, and are pictures taken under a 20x objective lens. As shown in the figure, the results show that the structural morphology of the organoids is the morphology of cancer tissue; according to the immunohistochemical indexes, the cells obtained after the culture of the sample organoids are lung cancer cells, and the pathological indexes are consistent with the pleural effusion cell indexes, indicating that the lung cancer organoids cultured using the culture medium LPOM of the present invention are consistent with the diagnosis results of the lung cancer pleural effusion tissue sample before culture. At the same time, the organoids obtained by culture are more complex in structure than pleural effusion cells, and can better reflect the complex microenvironment in the body.

Figures 4A and 4B are the results of electron microscopic examination of lung cancer organoids obtained after in vitro culture of sample OPB3. As shown in Figures 4A and 4B, it can be observed at different magnifications that the organoids have lung cilia structures unique to lung tissue, proving that the organoids cultured in the culture medium of the present invention have certain physiological functions.

Comparison of Example 4 with existing culture medium culture effects

(1) Preparation of control culture medium

The culture medium used in the preparation literature (Norman Sachs et al., The EMBO Journal (2019) e100300) was prepared as follows: Advanced DMEM/F12 culture 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 Company) + 100ng/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.

(2) Lung cancer organoid culture

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.

On the 14th day of culture, the cultured lung cancer organoids were observed using a microscope (EVOS M500, Invitrogen). Figures 5A and 5B are photographs of organoids cultured in LPOM medium and ROM medium for 14 days, respectively, taken under a 4x objective lens.

According to the results of Figures 5A and 5B, compared with ROM medium, LPOM medium can significantly promote the formation and expansion culture of lung cancer organoids.

Example 5 Lung cancer organoids obtained by expansion using the culture medium of the present invention are used for drug screening

(1) Lung cancer organoid culture

Primary lung cancer cells were isolated from lung cancer intraoperative samples (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.

(2) Screening of drug preparation

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

Preparation of different concentrations of bortezomib and afatinib drug additive solutions: Bortezomib and afatinib were prepared into 10 stock solutions of different concentrations, with the highest concentration being 20,000 μM, and then diluted at a 2-fold dilution ratio to obtain stock solutions of 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.

(3) Dosing

Take out the prepared drug storage solution, place it at room temperature, and dilute the drug 500 times with LPOM medium for later use. Take out the organoids cultured according to step (1) from the incubator, and slowly add 100 μL of the medium containing the drug to each well along the well wall into the 96-well ultra-low adsorption culture plate, and finally obtain the test drug concentrations of 20000nM, 10000nM, 5000nM, 2500nM, 1250nM, 625nM, 312.5nM, 156.25nM, 78.13nM and 39.06nM. 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.

(4) Organoid Viability Test

Take out CellTiter-Glo luminescent reagent (purchased from Promega) from the 4℃ 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, observe the cell status in the 96-well plate after standing for 30 minutes, 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 (Envision, Perkin Elmer) for detection.

(5) Data processing

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 _DMSO / chemiluminescence value of the zero day culture well _DMSO ) * 100%, the inhibition rate of different drugs was calculated, and the results are shown in Figure 6. Figures 6A and 6B are the inhibition rate curves of different concentrations of test drugs inhibiting the growth of lung cancer organoids. Among the two anti-tumor drugs, bortezomib has a strong effect of inhibiting the growth of organoids at 10 concentrations, and the inhibitory effects of different concentrations of afatinib have certain differences and are dose-dependent, which shows that the effectiveness and sensitivity of the organoids of the same patient to different drugs are different. According to the results, the effectiveness and effective dosage of this drug in clinical use of lung cancer patients can be judged.

Industrial Applicability

The present invention provides a culture medium and a culture method for culturing lung cancer organoids, and the cultured organoids can be used for evaluating and screening the efficacy of drugs. Therefore, the present invention is suitable for industrial application.

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

Claims (10)

1. A culture medium for lung cancer organoids, characterized by 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, Wherein, the MST1/2 kinase inhibitor includes 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 aryl, aryl C1-C6 alkyl and heteroaryl optionally substituted by 1-2 independently R ₆ ; _R2 and _R3 are each independently selected from C1-C6 alkyl; _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; _R6 is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl. 2. The culture medium of claim 1, wherein R ₁ is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and phenyl, naphthyl, benzyl and thienyl optionally substituted by 1-2 independently R ₆ ; _R2 and _R3 are each independently selected from C1-C3 alkyl; _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, piperidinylC1-C6 alkyl, and tetrahydropyranylC1-C6 alkyl; _R6 is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl. 3. The culture medium of claim 1, wherein the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt or solvate thereof,

in, R ₁ is selected from C1-C6 alkyl, phenyl optionally substituted by 1-2 independently R ₆ , thienyl optionally substituted by 1-2 independently R ₆ , and benzyl optionally substituted by 1-2 independently R ₆ ; _R5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl; R ₆ is each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl. 4. The culture medium as claimed in claim 3, wherein R ₁ is phenyl optionally substituted by 1-2 independently R ₆ ; _R5 is hydrogen; _R6 is preferably fluorine, 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 to 10 μM; The volume ratio of the B27 or N2 cell culture additive to the culture medium is 1:25 to 1:100; The concentration of the fibroblast growth factor 10 is 25-200 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 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. 7. The culture medium according to any one of claims 1 to 5, further comprising: An initial culture medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and An antibiotic selected from one or more of Streptomycin/Penicillin, Amphotericin B and Primocin. 8. The culture medium according to any one of claims 1 to 5, characterized in that the culture medium does not contain Wnt agonists, R-spondin family proteins, Noggin proteins, or BMP inhibitors. 9. A method for culturing lung cancer organoids, characterized by comprising the following steps: (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 any one of claims 1 to 8, and performing organoid culture on the primary lung cancer cells obtained in step (1). 10. A method for evaluating or screening drugs for treating lung cancer, comprising the following steps: (1) culturing lung cancer organoids using the lung cancer organoid culture method according to claim 9; (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) Conducting organoid size or organoid viability testing.

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