A culture medium for lung cancer epithelial cells, a culture method and its application

Abstract

The present invention provides a primary cell culture medium for culturing primary lung cancer epithelial cells, comprising an MST1 / 2 kinase inhibitor, a ROCK kinase inhibitor, a fibroblast growth factor, at least one additive selected from the group consisting of a B27 additive and an N2 additive, epidermal growth factor, transferrin, gastrin, and a TGFβ type I receptor inhibitor. The present invention also relates to a culture method using the primary cell culture medium and its use in drug efficacy evaluation and screening. The culture method uses the primary cell culture medium to culture primary lung cancer cells on a culture vessel coated with extracellular matrix gel, resulting in rapid proliferation of the primary lung cancer cells. The cell model obtained using the primary cell culture medium and primary cell culture method of the present invention can be used for drug efficacy evaluation and screening.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a culture medium and a culture method for culturing or amplifying primary lung cancer epithelial cells in vitro, and also relates to a method and application of the cultured cells in drug efficacy evaluation and screening. Background Art

[0002] Lung cancer is currently the most common respiratory cancer worldwide, with over 1.8 million new lung cancer cases worldwide each year. As the most common malignant tumor in clinical practice, lung cancer is primarily treated with surgery, chemotherapy, radiotherapy, molecularly targeted therapy, and immunotherapy. Surgery, chemotherapy, and radiotherapy are the most commonly used approaches, but these treatments are limited in the populations suitable for these therapies. In recent years, driven by the rise and development of molecular biology, cancer drug treatments have diversified. Molecularly targeted drugs, due to their strong targeting and safety profile, have become a hot topic in clinical lung cancer treatment. However, with so many treatment options available, choosing the right treatment for each patient is crucial. While genetic testing can be used as an indicator, some patients may not have a genetic mutation, or even if they have a specific mutation, multiple targeted drugs are available for that mutation. This makes determining a treatment plan clinically challenging. In addition to genetic sequencing, in vitro primary cell culture from lung cancer patient samples has become an important tool for predicting therapeutic efficacy and guiding clinical drug use. However, rapidly generating primary lung cancer cells in vitro remains a pressing technical challenge.

[0003] Functional testing refers to methods for detecting the sensitivity of cancer patient cells to anti-tumor drugs in vitro. The key to applying this method is to develop tumor cell models with a short growth cycle and that can represent the biological characteristics of lung cancer patients themselves. In addition, the cell model should be easy to operate and able to quickly and efficiently predict the efficacy of clinical drugs, thereby providing timely and precise medication guidance to cancer patients. However, the success rate of establishing cell models in vitro using primary tumor cells obtained from cancer patients is often very low, the growth cycle is long, and there are problems such as excessive proliferation of stromal cells such as fibroblasts, which restrict the development of this field. Currently, there are two relatively mature technologies for culturing primary epithelial cells / stem cells in the field of functional testing of tumor cells. One is the use of irradiated feeder cells and the ROCK kinase inhibitor Y27632 to promote the growth of primary epithelial cells to examine the drug sensitivity of individual patients, namely cell conditional reprogramming technology (Liu et al., Am J Pathol, 180:599-607, 2012). Another technology is to culture adult stem cells in 3D in vitro to obtain organoids similar to tissues and organs (Hans Clevers et al., Cell, 11, 172(1-2): 373-386, 2018).

[0004] However, both technologies have certain limitations. Cell reprogramming technology is a technology that co-cultures the patient's autologous primary epithelial cells with mouse-derived feeder cells. When the patient's primary cells are tested for drug sensitivity, the presence of these mouse-derived cells will interfere with the drug sensitivity test results of the patient's autologous primary cells; but if the mouse-derived feeder cells are removed, the patient's autologous primary cells will be separated from the reprogramming environment, and the cell proliferation rate and intracellular signaling pathways will undergo significant changes (Liu et al., Am J Pathol, 183(6): 1862-1870, 2013; Liu et al., Cell Death Dis., 9(7): 750, 2018), thereby greatly affecting the response of the patient's autologous primary cells to drugs. Organoid technology is a technology that embeds the patient's autologous primary epithelial cells in an extracellular matrix for in vitro three-dimensional culture. This technology does not require feeder cells, so there is no interference problem with mouse-derived feeder cells. However, the culture medium of organoid technology requires the addition of a variety of specific growth factors (such as Wnt protein and R-spondin family protein), which is expensive and not suitable for widespread clinical application. In addition, the cells of organoids need to be embedded in extracellular matrix gel throughout the culture process. The cell inoculation, passage and plating steps of drug sensitivity testing are cumbersome and time-consuming compared to 2D culture operations. Moreover, the size of the organoids formed by this technology is difficult to control, and some organoids are prone to grow too large and cause internal necrosis. Therefore, compared with 2D culture technology, organoid technology is less operable and applicable, requires professional technicians to operate, and is not suitable for large-scale and widespread application in clinical in vitro drug sensitivity testing (Nick Barker, Nat Cell Biol, 18(3): 246-54, 2016).

[0005] Given the limitations of these technologies, clinical applications call for the development of a primary lung cancer epithelial cell culture technology with a short culture cycle, manageable costs, convenient operation, and no interference from exogenous cells. When this technology is applied to construct primary lung cancer tumor cell models, the cultured lung cancer tumor cells can represent the biological characteristics of the lung cancer patient. By evaluating the sensitivity of anti-tumor drugs in vitro in cell models derived from different individual cancer patients, the response rate of clinical anti-tumor drugs can be improved, reducing the pain caused to patients and the waste of medical resources caused by inappropriate drugs. Summary of the Invention

[0006] The present invention aims to address the deficiencies of the existing technology by providing a culture medium for culturing primary lung cancer epithelial cells and a method for culturing primary lung cancer epithelial cells using the culture medium. Using the culture medium and culture method for primary lung cancer epithelial cells of the present invention for cell culture can achieve the goals of a short in vitro culture cycle, controllable costs, convenient operation, and no interference from exogenous cells. When this technology is applied to construct a primary lung cancer tumor cell model, primary lung cancer tumor cells with the biological characteristics of lung cancer patients can be obtained, and can be used for new drug screening and in vitro drug sensitivity testing.

[0007] One aspect of the present invention is to provide a primary cell culture medium for culturing primary lung cancer epithelial cells, which contains an MST1 / 2 kinase inhibitor; a ROCK kinase inhibitor selected from at least one of Y27632, fasudil, and H-1152; a fibroblast growth factor (FGF7); an additive selected from at least one of a B27 additive and an N2 additive; an epidermal growth factor (EGF); transferrin (IGF-1); gastrin (IL-6); and a TGFβ type I receptor inhibitor selected from at least one of A83-01, SB431542, Repsox, SB505124, SB525334, SD208, LY36494, and SJN2511, wherein the MST1 / 2 kinase inhibitor includes a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof.

[0008]

[0009] in,

[0010] 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.);

[0011] R2 and R3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;

[0012] 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.);

[0013] 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).

[0014] In a preferred embodiment, the MST1 / 2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt or solvate thereof,

[0015]

[0016] in,

[0017] 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;

[0018] R5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R5 is more preferably hydrogen;

[0019] R6 are each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, and R6 is more preferably fluorine, methyl or trifluoromethyl.

[0020] Preferably, the MST1 / 2 inhibitor is at least one selected from the following compounds or pharmaceutically acceptable salts or solvates thereof.

[0021]

[0022]

[0023]

[0024]

[0025]

[0026] Most preferably, the MST1 / 2 kinase inhibitor of the present invention is Compound 1.

[0027] In an embodiment of the present invention, the content of the MST1 / 2 kinase inhibitor in the culture medium is generally 2.5 μM to 15 μM, preferably 2.5 μM to 10 μM.

[0028] In another embodiment, the ROCK kinase inhibitor is preferably Y27632. In another embodiment, the content of the ROCK kinase inhibitor in the culture medium is generally 2.5 μM to 18 μM, preferably 5 μM to 15 μM.

[0029] In a preferred embodiment, the content of the fibroblast growth factor is 2.5ng / ml~80ng / ml, more preferably 5ng / ml~40ng / ml; the volume concentration of the B27 additive or the N2 additive in the culture medium is 1:25~1:800, more preferably 1:25~1:200; the content of the epidermal growth factor is 2.5ng / ml~80ng / ml, more preferably 10ng / ml~40ng / ml; the content of the transferrin is 2.5ng / ml~80ng / ml, more preferably 5ng / ml~80ng / ml; the content of the gastrin is 2.5ng / ml~80ng / ml, more preferably 5ng / ml~40ng / ml; the TGFβ type I receptor inhibitor is preferably A83-01, and the content of the TGFβ type I receptor inhibitor is 62.5nM~800nM, preferably 125nM~500nM.

[0030] The culture medium formulation of the present invention further comprises 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 some embodiments, the initial culture medium is preferably DMEM / F12, and the antibiotic is preferably primocin. In further preferred embodiments, the primocin content in the culture medium is 25 to 400 μg / mL, preferably 50 to 200 μg / mL.

[0031] Compared with the components of cell-conditioned reprogramming medium and lung cancer epithelial cell organoid culture medium, the culture medium formula of the present invention adds MST1 / 2 kinase inhibitors, does not contain uncertain components such as serum and bovine pituitary extract, does not contain niche factors necessary for organoid culture such as Wnt agonists, R-spondin family proteins, and BMP inhibitors, and does not contain nicotinamide and N-acetylcysteine. Therefore, the cost of the culture medium is greatly reduced, the operation process of preparing the culture medium is simplified, and the in vitro culture of primary lung cancer epithelial cells with controllable cost and convenient operation is achieved.

[0032] In the present invention, the primary lung cancer epithelial cells can be lung cancer tumor cells, normal lung cancer epithelial cells, or lung cancer epithelial stem cells.

[0033] One aspect of the present invention is to provide a method for culturing primary lung cancer epithelial cells, comprising the following steps:

[0034] (1) Prepare the primary cell culture medium of the present invention according to the above formula.

[0035] (2) Coat the culture vessel with extracellular matrix gel dilution solution.

[0036] Specifically, the extracellular matrix glue uses a low growth factor type extracellular matrix glue, for example, commercially available Matrigel (purchased from Corning) or BME (purchased from Trevigen) can be used. More specifically, the extracellular matrix glue is diluted with serum-free culture medium, and the culture medium can be DMEM / F12 (purchased from Corning). The dilution ratio of the extracellular matrix glue is 1:50-1:400, preferably 1:100-1:200. The coating method is to add the diluted extracellular matrix glue to the culture vessel so that it completely covers the bottom of the culture vessel, and let it stand for more than 30 minutes, preferably at 37°C. The coating time is preferably 30 to 60 minutes. After the coating is completed, the excess extracellular matrix glue dilution is discarded and the culture vessel is ready for use.

[0037] (3) Primary lung cancer epithelial cells were isolated from lung cancer tissue.

[0038] Primary lung cancer epithelial cells can be derived from, for example, lung cancer surgical specimens and biopsy puncture specimens. Lung cancer tissue samples can be derived from, for example, cancer tissue samples obtained from surgically removed lung cancer patients who have given instructions and obtained consent. Biopsy puncture specimens are collected from lung lesions under ultrasound guidance. The above tissue samples are collected within half an hour after the patient's surgical resection or biopsy. More specifically, under a sterile environment, tissue samples are cut from non-necrotic areas with a volume of 5 mm 3 The above is placed in pre-chilled tissue transport medium in a sterile plastic centrifuge tube with a lid, and transported on ice to the laboratory. The tissue transport medium contains DMEM / F12, an MST1 / 2 kinase inhibitor of the present invention (e.g., Compound 1), and 0.2-0.4% primocin by volume. The concentration of the MST1 / 2 kinase inhibitor of the present invention ranges from 0.3 μM to 10 μM, preferably from 2 μM to 5 μM, and more preferably from 3 μM. The concentration of primocin ranges from 25 to 400 μg / mL, preferably from 50 to 200 μg / mL, and more preferably from 100 μg / mL.

[0039] In the biosafety cabinet, transfer the tissue sample to a cell culture dish, rinse the tissue sample with tissue transport fluid, and wash away the blood cells on the surface of the tissue sample. Transfer the rinsed tissue sample to another new culture dish, add 1-3mL of tissue transport fluid, and use sterile surgical blades and surgical forceps to cut the tissue sample into pieces smaller than 3mm. 3 of tissue fragments.

[0040] Transfer the tissue sample fragments into a centrifuge tube and centrifuge at 1000-3000 rpm for 3-5 minutes using a tabletop centrifuge (Sigma 3-18K). Discard the supernatant and add tissue transport solution and tissue digestion solution in a 1:1 ratio (the usage amount is about 5 mL of tissue digestion solution for every 10 mg of tissue. The preparation method of tissue digestion solution is as follows: 1-2 mg / mL collagenase II, 1-2 mg / mL collagenase IV, 50-100 U / mL deoxyribonucleic acid I, 0.5-1 mg / mL hyaluronidase, 0.1-0.5mg / mL calcium chloride, 5-10mg / mL bovine serum albumin dissolved in HBSS and RPMI-1640 with a volume ratio of 1: 1), mark the sample number, seal the tube with a sealing film, and digest it at 37 ° C, 200-300 rpm constant temperature shaker (Zhichu Instrument ZQLY-180N). Observe whether the digestion is complete every 1 hour; if no obvious tissue block is seen, the digestion can be terminated, otherwise the digestion is continued until the digestion is sufficient. The digestion time range is 4-8 hours. After the digestion is completed, the cell strainer (cell sieve pore size is, for example, 70 μm) is used to filter out the undigested tissue agglomerates, the tissue agglomerates on the filter are rinsed with tissue transport fluid, and the residual cells are flushed into a centrifuge tube and centrifuged at 1000-3000 rpm for 3-5 minutes using a desktop centrifuge. The supernatant was discarded and the remaining cell mass was observed to see if it contained blood cells. If so, 3-5 mL of blood cell lysis buffer (purchased from Sigma) was added, mixed, and lysed at 4°C for 10-20 minutes, shaking and mixing once every 5 minutes. After lysis, the cells were removed and centrifuged at 1000-3000 rpm for 3-5 minutes. The supernatant was discarded, the primary cell culture medium of the present invention was added and resuspended, and the total number of cells was obtained by counting using a flow cytometer (Jiangsu Zhuo Microbiology Technology Co., Ltd. JIMBIO FIL).

[0041] (4) The primary lung cancer epithelial cells isolated in step (3) are inoculated into the coated culture vessel and cultured using the primary cell culture medium in step (1).

[0042] More specifically, 2 × 10 4 ~8×10 4 pieces / cm 2 (For example 4×10 4 pieces / cm 2 ) and inoculate primary lung cancer tumor cells at a density of 1 mL / well. Add an appropriate amount, e.g., 2-3 mL, of primary epithelial cell culture medium. Culture the cells in a cell culture incubator at, e.g., 37° C., 5% CO 2 for 8-16 days, replacing the culture medium with fresh primary cell culture medium every 4 days. Digestion and passage are performed when the primary lung cancer epithelial cells grow to a cell density that occupies approximately 80% to 90% of the bottom area of ​​the multiwell plate.

[0043] This inoculation step does not require the use of feeder cells, and compared to cell conditional reprogramming technology, it eliminates the steps of culturing and irradiating feeder cells. Compared to organoid technology, this step also does not require mixing primary cells and matrix gel on ice to form gel droplets, and then waiting for the gel droplets to solidify before adding culture medium. Pre-coated culture vessels can be directly used for primary cell inoculation. In addition, only a small amount of diluted extracellular matrix gel is required to coat the culture vessels. Compared to organoid technology, this saves the use of expensive extracellular matrix gel and simplifies the operation steps.

[0044] Optionally, after culturing the inoculated primary lung cancer epithelial cells for 8 to 16 days, when the cell clones formed in the culture container are confluent to 80% of the bottom area, the supernatant is discarded, and 0.5 to 2 mL of 0.05% trypsin (purchased from Thermo Fisher) is added for cell digestion, and the cells are incubated at room temperature for 5 to 20 minutes; then, the digested cells are resuspended in 1 to 4 mL of DMEM / F12 culture medium containing, for example, 5% (v / v) fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and the cells are centrifuged at 1000 to 3000 rpm for 3 to 5 minutes, and the digested single cells are resuspended in the primary cell culture medium of the present invention, and the resulting cell suspension is placed in a T25 cell culture flask coated with extracellular matrix glue for further expansion and culture. The coating operation of the T25 cell culture flask is the same as step (2).

[0045] The expanded lung cancer epithelial cells grow in 2D, avoiding the uneven size of organoids and internal necrosis of overgrown organoids that occur when organoid technology is expanded.

[0046] On the other hand, lung cancer epithelial cells, particularly lung cancer tumor cells, cultured by the primary lung cancer epithelial cell culture method of the present invention can be used for drug efficacy evaluation and screening, and the method comprises the following steps:

[0047] (1) obtaining primary lung cancer epithelial cells, preferably obtaining a cancer tissue sample or a biopsy cancer tissue sample from a lung cancer patient, isolating the primary lung cancer epithelial cells, and culturing and expanding the primary lung cancer epithelial cells (especially primary lung cancer tumor cells) according to the above-described primary lung cancer epithelial cell culture method for at least 10 5 Order of magnitude, preferably at least 10 6 Order of magnitude of cell number.

[0048] (2) Select the drugs to be tested.

[0049] (3) The drug is at its maximum plasma concentration C max For reference, 2-5 times C max As the starting concentration, multiple drug concentration gradients, such as 5-10, preferably 6-8 drug concentration gradients, are diluted.

[0050] (4) Digesting the lung cancer epithelial cells cultured in step (1) into a single cell suspension, counting them using a flow cytometer, diluting the single cell suspension with the primary cell culture medium of the present invention, and evenly adding the diluted cell suspension to a multi-well plate at a density of 2000-4000 cells per well, for example, 50 μL of cell dilution per well, and allowing them to adhere overnight.

[0051] This step avoids the problem of feeder cells interfering with primary cell counts and subsequent primary cell viability testing, which is common in cell reprogramming techniques. It also eliminates the tedious steps of mixing the cell suspension with Matrigel on ice, embedding it, and then plating it, as is required with organoid technology. This greatly simplifies the operational process and enhances the operability and practicality of the technology. Because the seeded cells are a single-cell suspension rather than a 3D structure like organoids, this technology achieves a more uniform cell number when plated, with less variability between wells, compared to organoid technology, making it more suitable for subsequent high-throughput drug screening.

[0052] (5) Using a high-throughput automated workstation, the adherent cells obtained in step (4) are treated by adding gradiently diluted candidate drugs such as traditional chemotherapy drugs, targeted drugs, antibody drugs, or combinations of several drugs.

[0053] (6) After several hours of drug treatment, for example, 72 hours, the survival rate of lung cancer epithelial cells is detected using the Cell-Titer Glo luminescent cell viability detection kit (purchased from Promega) to screen drug activity.

[0054] Specifically, for example, 10 μL of Cell Titer-Glo reagent (purchased from Promega) is added to each well and shaken evenly. The chemiluminescence intensity of each well is measured using a fluorescence microplate reader. Based on the measured values, a drug dose-effect curve is plotted using GraphPad Prism software with drug concentration as the horizontal axis and fluorescence intensity as the vertical axis to calculate the inhibitory intensity of each drug on the proliferation of the tested cells.

[0055] In the application of primary lung cancer cells in drug screening and in vitro drug sensitivity testing, since the cells are not co-cultured, the feeder cells used in cell reprogramming techniques do not interfere with the test results. Because the cells grow in a 2D environment, the drug interaction time is also shorter than that of organoid-based drug testing (the average drug administration time in organoids is 6 days).

[0056] The beneficial effects of the present invention also include:

[0057] (1) Improve the success rate of primary lung cancer epithelial cell culture to over 80%;

[0058] (2) Ensure that primary lung cancer epithelial cells cultured in vitro can maintain the pathological phenotype and heterogeneity of the patient from whom the primary cells were derived;

[0059] (3) The cultured primary lung cancer epithelial cells are not interfered with by fibroblasts, and purified lung cancer epithelial cells can be obtained;

[0060] (4) The culture medium does not contain serum and is therefore not affected by the quality and quantity of serum from different batches;

[0061] (5) The efficiency of amplifying lung cancer epithelial cells is high, as long as there are 10 4 The number of cells can be successfully expanded to 10 in about two weeks. 6 The number of lung cancer epithelial cells is several orders of magnitude, and the expanded lung cancer epithelial cells can be continuously passaged;

[0062] (6) The cell culture process does not require ice operation or dissociation of the matrix gel, and the cell digestion and cell culture can be completed within 10-15 minutes;

[0063] (7) Controllable culture costs: The primary lung cancer cell culture medium does not require the addition of expensive Wnt agonists, R-spondin family proteins, BMP inhibitors and other factors. It is a simplification and improvement of the existing primary lung cancer epithelial cell organoid culture medium. Cell inoculation does not require the use of a high concentration of extracellular matrix to mix with primary cells to form gel droplets. Instead, only a small amount of diluent prepared with extracellular matrix glue is needed, saving the amount of expensive extracellular matrix.

[0064] (8) Easy operation. Compared with conditional reprogramming technology, this technology does not require the culture of feeder cells and the radiation of feeder cells, thus avoiding the problem that the quality and quantity of different batches of feeder cells affect the efficiency of primary cell culture. The objects of drug screening and detection are only primary lung cancer epithelial cells, without interference from feeder cells in the co-culture system required by cell conditional reprogramming technology. Compared with organoid technology, the extracellular matrix gel coating method adopted in the present invention allows the culture vessels to be prepared in advance, without the need to embed cells in matrix gel as in organoid technology. The technical operation steps are simple and easy.

[0065] (9) The lung cancer epithelial cells cultured using the technology are large in number and highly homogenized, making them suitable for high-throughput screening of new candidate compounds and providing patients with high-throughput in vitro drug sensitivity functional testing.

[0066] The cell culture medium of this embodiment can be used to culture lung cancer epithelial cells derived from humans or other mammals, including lung cancer tumor cells, normal lung epithelial cells, lung cancer epithelial stem cells, or tissues containing at least one of these cells. The culture medium of the present invention can also be used to develop a kit for in vitro expansion and culture of primary lung cancer cells.

[0067] Furthermore, cells obtained by the culture method of this embodiment can be applied to regenerative medicine, basic medical research on lung cancer epithelial cells, screening of drug responses, and development of new drugs for lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figures 1A-1D The figure shows the effects of different factors in the culture medium on the proliferation of primary lung cancer cells.

[0069] Figure 2 It is a graph showing the effect of increasing amounts of different factors in the culture medium on the proliferation of primary lung cancer cells.

[0070] Figures 3A-3H This is a graph showing the effect of the concentration of each added factor on the proliferation of primary lung cancer cells.

[0071] Figure 4A and 4B The photographs are taken under an inverted microscope of lung cancer tumor cells isolated from a clinical lung cancer tissue sample and cultured using the culture medium FLM of the present invention for 4 days and 12 days respectively.

[0072] Figures 5A-5D These are photos taken under an inverted microscope after cells isolated from a lung cancer surgical resection specimen were cultured under four different culture medium conditions for 12 days.

[0073] Figure 6 This is a comparison of the cell proliferation effects of cells isolated from 10 lung cancer surgical resection specimens after culturing for 14 days under four different culture medium conditions.

[0074] Figure 7 This is a comparison chart of cell growth curves obtained by culturing cells isolated from a clinical tissue sample of lung cancer using four different culture medium conditions.

[0075] Figure 8 The figure is a comparison of the immunohistochemical results of a lung cancer surgical resection sample and the lung cancer tumor cells obtained by culturing the cells separated from the sample with the culture medium FLM of the present invention.

[0076] Figure 9The figure is a comparison chart of the growth curves of cells isolated from a clinical tissue sample of lung cancer cultured in the culture medium FLM of the present invention and cultured in a culture medium obtained by deleting different components.

[0077] Figure 10 The results are obtained by analyzing the signaling pathways involved in cell proliferation after culturing cells in FLM medium and medium without Compound 1.

[0078] Figure 11A and 11B The figures respectively represent the dose-effect curves of primary lung cancer tumor cells cultured in the culture medium FLM of the present invention to different chemotherapeutic drugs and targeted drugs from surgically resected cancer tissue samples of two different lung cancer patients. DETAILED DESCRIPTION

[0079] In this specification, epithelial cells include differentiated epithelial cells and epithelial stem cells obtained from epithelial tissue." epithelial stem cells" refers to cells with long-term self-renewal ability and differentiation into epithelial cells, and refers to stem cells derived from epithelial tissue. As epithelial tissue, for example cornea, oral mucosa, skin, conjunctiva, bladder, renal tubules, kidney, digestive organs (esophagus, stomach, duodenum, small intestine (including jejunum and ileum), large intestine (including colon)), liver, pancreas, mammary gland, salivary gland, lacrimal gland, prostate, hair root, trachea, lung etc. can be cited. Wherein, the cell culture medium of the present embodiment is preferably a culture medium for cultivating pulmonary epithelial cells.

[0080] In addition, in this specification, "epithelial tumor cells" refer to cells derived from the above-mentioned epithelial tissues that have become tumors.

[0081] As used herein, “organoid” refers to a three-dimensional, organ-like cellular tissue formed by spontaneously organizing and aggregating cells at high density in a controlled space.

[0082] [Preparation Example of MST1 / 2 Kinase Inhibitor]

[0083] 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.

[0084] 1. Preparation of MST1 / 2 kinase inhibitor compound 1

[0085] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzene Sulfonamide 1

[0086]

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 2. Preparation of other MST1 / 2 inhibitor compounds of the present invention

[0093] 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.

[0094]

[0095]

[0096]

[0097]

[0098]

[0099] [Example 1]

[0100] Isolation of primary human lung cancer epithelial cells

[0101] Lung cancer tissue samples were obtained from surgically removed lung cancer tissue samples from patients who had provided their consent. The following example uses one of these samples (number B4) for illustration.

[0102] The above tissue samples were collected within half an hour after surgical resection. More specifically, tissue samples were collected from non-necrotic areas under a sterile environment with a volume of 0.5 cm 3 The above tissues were placed in 4 mL of pre-cooled tissue transport solution (see Table 1 for specific preparation), and the transport solution was placed in a 5 mL plastic sterile capped cryotube (purchased from Guangzhou Jiete Biotechnology) and transported to the laboratory under cold chain (0-10°C).

[0103] Table 1 Tissue transport fluid formula

[0104] Tissue transport fluid components supplier Final concentration DMEM / F12 Corning 97.8% by volume Primocin Invivogen 0.2 volume % (commercial product concentration 50 mg / ml) Compound 1 self made 3μM

[0105] Table 2 Tissue digestion solution formula

[0106] Tissue digestive fluid components supplier Final concentration HBSS Gibco 50% (volume) RPMI-1640 Corning 50% (volume) Collagenase II Sigma 2mg / mL Collagenase IV Sigma 2mg / mL DNA I Sigma 50 U / mL Hyaluronidase Sigma 0.5mg / mL calcium chloride Shanghai Bioengineering 0.33 mg / mL Bovine serum albumin Shanghai Bioengineering 10 mg / mL

[0107] In a biosafety cabinet, transfer the tissue sample (No. B4) to a 100mm cell culture dish (purchased from NEST). Rinse the tissue sample with tissue transport fluid to remove residual blood on the surface of the tissue sample and remove excess tissue such as fat on the surface of the tissue sample. Transfer the rinsed tissue sample to another new 100mm culture dish, add 2mL of transport fluid, and use a sterile surgical blade and surgical forceps to cut the tissue sample into pieces smaller than 3mm. 3 of tissue fragments.

[0108] Transfer the tissue sample fragments to a 15 mL centrifuge tube and centrifuge at 1500 rpm for 4 minutes using a tabletop centrifuge (Sigma 3-18K). Discard the supernatant and add tissue transport solution and tissue digestion solution in a 1:1 ratio (approximately 5 mL of tissue digestion solution per 10 mg of tissue; see Table 2 for the specific preparation). Label the sample number, seal with sealing film, and digest in a thermostatic shaker (Zhichu Instrument ZQLY-180N) at 37°C and 300 rpm. Observe the digestion completion every 1 hour.

[0109] After digestion, undigested tissue clumps were filtered out through a 70 μm filter. The tissue clumps on the filter were rinsed with tissue transport fluid, and the remaining cells were flushed into a centrifuge tube and centrifuged at 1500 rpm for 4 minutes.

[0110] The supernatant was discarded and the remaining cell mass was observed to see if it contained blood cells. If so, 3 mL of blood cell lysis solution (purchased from Sigma) was added, mixed, and lysed at 4 ° C for 15 minutes, and shaken for 5 minutes to mix once. After the lysis was completed, the cells were taken out and centrifuged at 1500 rpm for 4 minutes. The supernatant was discarded to obtain the primary lung cancer cells after digestion and separation, and basal medium (BM) was added to resuspend the cells, wherein the basal medium was commercially available DMEM / F-12 medium and 0.2% by volume of Primocin (purchased from Invivogen at a concentration of 50 mg / mL) was added to obtain a final concentration of 100 μg / mL. Flow imaging counter (Jiangsu Zhuo Microbial Technology Co., Ltd. JIMBIO FIL) was used for counting, and the total number of cells was 1.02 million.

[0111] [Example 2]

[0112] Optimization of culture medium for primary lung cancer epithelial cells

[0113] (1) The effects of different factors

[0114] Dilute extracellular matrix gel (Matrigel®) (Corning Incorporated) at a ratio of 1:100 using serum-free DMEM / F12 medium to prepare extracellular matrix diluent. Add 500 μl / well of the extracellular matrix diluent to a 48-well culture plate, completely covering the bottom of the wells. Incubate in a 37°C incubator for 1 hour. After 1 hour, remove the extracellular matrix diluent to obtain a Matrigel-coated culture plate.

[0115] Preparation of basal medium (abbreviated as BM): 0.2% by volume of Primocin (purchased from Invivogen, concentration of 50 mg / mL) was added to commercially available DMEM / F-12 medium to obtain a final concentration of 100 μg / mL to prepare BM.

[0116] Next, different types and concentrations of additive factors (Table 3) were added to the basal medium (BM) to prepare lung cancer epithelial cell culture media containing different additive components.

[0117] Table 3 Preparation of different components of culture medium (concentration is final concentration)

[0118]

[0119]

[0120] The culture medium with different components was added into a 48-well plate coated with extracellular matrix gel (Matrigel) at a volume of 500 μl / well. The lung cancer tumor cells (number B18) isolated from lung cancer tissue according to the same method as in Example 1 were cultured at a rate of 2×10 4 pieces / cm 2 The cell density was inoculated into a 48-well culture plate coated with Matrigel, and after surface disinfection, it was placed in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific). The same number of freshly isolated lung cancer tumor cells (No. B18) were cultured under different culture medium formulations. After the start of culture, the culture medium was replaced every 4 days. After 12 days of culture, the cells were counted. Among them, as an experimental control, the basal medium (BM) without any additive factors was used. The results are shown in Figure 1A -D. The ordinate in the figure represents the ratio of the number of cells obtained after culture in different culture media to the number of cells obtained after culture in the basal culture medium BM. As shown in the figure, the addition of different factors in different concentrations in Table 3 on the basis of BM all produced varying degrees of cell proliferation. Specifically, within a specific concentration range, B27 additive, N2 additive, fibroblast growth factor, gastrin, epidermal growth factor, transferrin, compound 1, Y27632 and A83-01 all had a certain promoting effect on cell proliferation.

[0121] (3) Effects of increasing the number of different factors in the culture medium on the proliferation of primary lung cancer cells obtained by this patented method

[0122] Dilute extracellular matrix gel (MatrigelR○) at a ratio of 1:100 in serum-free DMEM / F12 medium to prepare extracellular matrix diluent. Add 500 μl / well of the extracellular matrix diluent to a 48-well culture plate, completely covering the bottom of the wells. Incubate in a 37°C incubator for 1 hour. After 1 hour, remove the extracellular matrix diluent to obtain a Matrigel-coated culture plate.

[0123] Different additive factors (Table 4) were added sequentially to the basal culture medium BM to prepare lung cancer epithelial cell culture medium containing different additive components.

[0124] Table 4 Preparation of different components of culture medium (concentration is final concentration)

[0125] Culture medium No. Components No.1 BM + 10 μM Y27632 NO.2 NO.1+5μM Compound 1 NO.3 NO.2+1:50B27 additive NO.4 NO.3+40ng / mL fibroblast growth factor NO.5 NO.4+40ng / mL epidermal growth factor No. 6 NO.5+40ng / mL transferrin No.7 NO.6+500nM A83-01 No.8 NO.7+20ng / mL gastrin

[0126] Different culture media were added to a 48-well plate coated with extracellular matrix (Matrigel) at a volume of 500 μl / well. BM culture medium was used as an experimental control. Lung cancer tumor cells (number B22) isolated from lung cancer tissue according to the method of Example 1 were cultured at a rate of 2×10 4 pieces / cm 2 The cells were seeded at a density of 100 μg / mL in a 48-well culture plate coated with Matrigel. After surface disinfection, the plate was placed in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific). The same number of freshly isolated lung cancer tumor cells (code B22) were cultured under different culture medium formulations. After 10 days of culture, the cells were counted. The experimental results are shown in Figure 2 As shown in the figure, with the continuous addition of new additive factors, the cell proliferation effect of the culture medium formula continued to improve, and the formula of the No. 8 culture medium was finally determined to be the most preferred culture medium formula for culturing and expanding primary lung cancer cells in this patent.

[0127] (4) Effects of different concentrations of added factors on the proliferation of primary lung cancer cells obtained in this patent

[0128] Dilute extracellular matrix gel (MatrigelR○) in serum-free DMEM / F12 medium at a ratio of 1:100 to prepare extracellular matrix diluent. Add 200 μl / well of extracellular matrix diluent to a 48-well culture plate, completely covering the bottom of the wells. Incubate in a 37°C incubator for 1 hour. After 1 hour, remove the extracellular matrix diluent to obtain a Matrigel-coated culture plate.

[0129] Prepare No. 8 primary lung cancer epithelial cell culture medium.

[0130] The same method as in Example 1 was used to isolate lung cancer epithelial cells from the cancer tissue of a lung cancer patient (No. B26). Next, the lung cancer epithelial cells from the cancer tissue were counted using a flow cytometer (Jiangsu Zhuowei Biotechnology Co., Ltd. JIMBIO FIL) to obtain the total number of cells. Then, 4×10 4 pieces / cm 2 Cells were seeded at a high density into Matrigel®-coated 48-well plates. 2 mL of prepared No. 8 primary lung cancer epithelial cell culture medium was added to the 48-well plates and cultured in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific). When the cells reached approximately 80% of the plate's bottom surface area, the supernatant was discarded and the cells were digested with 500 μL of 0.05% trypsin (purchased from Gibco). The cells were incubated at 37°C for 10 minutes until complete digestion was observed under a microscope (Invitrogen EVOS M500). The digestion was terminated with 1 mL of DMEM / F12 medium containing 5% (v / v) fetal bovine serum (purchased from Ecosine), 100 U / mL penicillin (purchased from Corning), and 100 μg / mL streptomycin (purchased from Corning). The cells were collected into a 15 mL centrifuge tube and centrifuged at 1500 rpm for 4 minutes, after which the supernatant was discarded. The cell pellet after centrifugation was resuspended in basal medium (BM) and counted using a flow cytometer (Jiangsu Zhuowei Microbiology Technology Co., Ltd., JIMBIO FIL) to obtain the total cell count. The resulting cells were used in the following culture experiments.

[0131] Next, the following 8 culture media were prepared for the experiment:

[0132] Formulation 1: No. 8 medium components without B27 additive;

[0133] Formulation 2: No. 8 culture medium components do not contain fibroblast growth factor;

[0134] Formulation 3: No. 8 medium components do not contain transferrin;

[0135] Recipe 4: No. 8 culture medium components do not contain epidermal growth factor;

[0136] Formulation 5: No. 8 medium components do not contain Y27632;

[0137] Formulation 6: No. 8 culture medium components do not contain compound 1;

[0138] Formulation 7: No. 8 medium components do not contain A83-01;

[0139] Formulation 8: No. 8 culture medium components do not contain gastrin.

[0140] The digested cell suspension was diluted using the above formulas 1 to 8, and seeded into a 48-well plate at a concentration of 10,000 cells per well in a volume of 250 μl.

[0141] When using the culture medium of Formula 1, 250 μL of the prepared B27 additive was added to each well of a 48-well plate seeded with primary cells. The final concentrations of the B27 additive were 1:800, 1:400, 1:200, 1:100, 1:50, and 1:25, respectively. Control wells (BC) were set up using the culture medium of Formula 1.

[0142] When using the culture medium of Formula 2, 250 μL of the prepared fibroblast growth factor was added to each well of a 48-well plate seeded with primary cells. The final concentrations of the fibroblast growth factor were 80 ng / mL, 40 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, and 2.5 ng / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 2.

[0143] When using the culture medium of Formula 3, 250 μL of prepared transferrin was added to each well of a 48-well plate seeded with primary cells. The final concentrations of transferrin were 80 ng / mL, 40 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, and 2.5 ng / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 3.

[0144] When using the culture medium of Formula 4, 250 μL of the prepared epidermal growth factor was added to each well of a 48-well plate seeded with primary cells. The final concentrations of the epidermal growth factor were 80 ng / mL, 40 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, and 2.5 ng / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 4.

[0145] When using the culture medium of Formula 5, 250 μL of the prepared Y27632 was added to each well of a 48-well plate seeded with primary cells. The final concentrations of Y27632 were 20 μM, 18 μM, 15 μM, 12.5 μM, 10 μM, 5 μM, and 2.5 μM, respectively; and control wells (BC) were set using the culture medium of Formula 5.

[0146] When using the culture medium of Formula 6, 250 μL of the prepared compound 1 was added to each well of a 48-well plate seeded with primary cells, and the final concentrations of compound 1 were 20 μM, 15 μM, 10 μM, 7.5 μM, 5 μM, 2.5 μM, and 1.25 μM, respectively; and control wells (BC) were set using the culture medium of Formula 6.

[0147] When using the culture medium of Formula 7, 250 μL of prepared A83-01 was added to each well of a 48-well plate seeded with primary cells. The final concentrations of A83-01 were 2000 nM, 1000 nM, 800 nM, 500 nM, 250 nM, 125 nM, and 62.5 nM, respectively. Control wells (BC) were set using the culture medium of Formula 7.

[0148] When using the culture medium of Formula 8, 250 μL of prepared gastrin was added to each well of a 48-well plate seeded with primary cells. The final concentrations of gastrin were 80 ng / mL, 40 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, and 2.5 ng / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 8.

[0149] When the cells are expanded to about 85% of the 48 wells, the digestion counts are calculated and the ratios are calculated with reference to the cell numbers in the control wells (BC). The results are shown in Figures 3A to 3H . Figures 3A to 3H The ratio is the ratio of the number of cells cultured in each culture medium for one generation to the number of cells cultured in the corresponding control well for one generation. A ratio greater than 1 indicates that the culture medium containing the factor or small molecule compound at different concentrations promotes cell proliferation more effectively than the culture medium in the control well. A ratio less than 1 indicates that the culture medium containing the factor or small molecule compound at different concentrations promotes cell proliferation less effectively than the culture medium in the control well.

[0150] according to Figures 3A to 3HAs a result, the volume concentration of B27 additive in the culture medium is preferably 1:25 to 1:800, more preferably 1:25 to 1:200, and most preferably 1:50; the content of fibroblast growth factor is preferably 2.5 ng / ml to 80 ng / ml, more preferably 5 ng / ml to 40 ng / ml, and most preferably 10 ng / ml; the content of transferrin is preferably 2.5 ng / ml to 80 ng / ml, more preferably 5 ng / ml to 80 ng / ml, and most preferably 20 ng / ml; the content of epidermal growth factor is preferably 2.5 ng / ml to 80 ng / ml, more preferably 10 ng / ml to 40 ng / ml, most preferably 20ng / ml; the content of Y27632 is preferably 2.5μM~18μM, more preferably 5μM~15μM, most preferably 10μM; the content of MST1 / 2 kinase inhibitor compound 1 is preferably 2.5μM~15μM, more preferably 2.5μM~10μM, most preferably 5μM; the content of A83-01 is preferably 62.5nM~800nM, more preferably 125nM~500nM, most preferably 500nM; the content of gastrin is preferably 2.5ng / ml~80ng / ml, more preferably 5ng / ml~40ng / ml, most preferably 10ng / ml.

[0151] The optimal concentration combination of each additive factor is used as the most preferred culture medium formula (hereinafter abbreviated as FLM) for culturing and expanding primary lung cancer cells of the present invention: that is, basal medium (BM) + 10 ng / ml fibroblast growth factor (FGF) + 20 ng / ml epidermal growth factor (EGF) + 20 ng / ml transferrin + 1:50 volume ratio B27 additive + 5 μM compound 1 + 10 μM Y27632 + 500 nM A83-01 + 10 ng / mL gastrin.

[0152] [Example 3]

[0153] Culture of primary lung cancer cells derived from lung cancer tissue

[0154] The same method as in Example 1 was used to isolate lung cancer epithelial cells from the cancer tissue of a lung cancer patient (sample number B21). The lung cancer epithelial cells were then counted using a flow cytometer (Jiangsu Zhuowei Biotechnology Co., Ltd., JIMBIO FIL) to obtain the total number of cells. The cells were then counted using a 4×10 4 pieces / cm 2 The cells were seeded at a high density into a 12-well plate coated with Matrigel® (purchased from BD Biosciences). 2 mL of the prepared primary lung cancer epithelial cell culture medium FLM was added to the 12-well plate and cultured in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific).

[0155] Figure 4A Is based on 4×10 4 pieces / cm 2 Microscopic photographs (40x inverted phase contrast microscope) of cells seeded at high density into Matrigel-coated 12-well plates on day 4 of culture from initiation of inoculation. Microscopic observations revealed that the cultured primary lung cancer cells, derived from cancerous tissue, were of high purity and lacked fibroblasts. Figure 4B This is a photo taken on the 12th day after inoculation (photographed with a 40x inverted phase contrast microscope). Figure 4A and 4B As can be seen from the two figures, after the primary lung cancer cells were isolated, obvious clone formation was observed under the microscope after 4 days of in vitro culture, and after 12 days of expansion, the cell number was significantly expanded, indicating that the culture medium of the present invention is a highly efficient culture medium for in vitro expansion of lung cancer epithelial cells.

[0156] [Example 4]

[0157] Effects of different culture media on the proliferation of primary lung cancer cells derived from lung cancer tissue

[0158] (1) Comparison of the effects of different culture media on primary cell clone formation and proliferation

[0159] The same method as in Example 2 was used to prepare the primary lung cancer epithelial cell culture medium FLM and the basal culture medium BM as a control. In addition, the cell-conditioned reprogramming medium FM was prepared as another control example. The preparation steps are described in (Liu et al., Nat Protoc., 12(2):439-451, 2017). The culture medium formula is shown in Table 5. In addition, as another control example, the commercial culture medium EpiMCult was purchased from a stem cell company. TM Plus Medium, hereinafter also referred to as "EpiM medium", the medium formula is shown in Table 6.

[0160] Table 5 Cell Conditioned Reprogramming Medium (FM) Composition

[0161] Culture medium components supplier Final concentration DMEM medium Corning 65% by volume Fetal bovine serum Gibico 10% by volume Ham's F12 Nutrient Solution Gibico 25% by volume hydrocortisone Sigma-Aldrich 25ng / ml Epidermal Growth Factor R&D 0.125ng / ml insulin Sigma-Aldrich 5 μg / ml Amphotericin B Sigma-Aldrich 250ng / ml Gentamycin Gibico 10 μg / ml Cholera toxin Sigma-Aldrich 0.1nM Y27632 Enzo 10 μM

[0162] Table 6 Commercial culture medium EpiMCult TM Plus Medium (EpiM) ingredients

[0163] Culture medium components supplier Final concentration <![CDATA[EpiMCult TM Plus Basal Medium]]> stem cell 98% by volume <![CDATA[EpiMCult TM Plus Supplement]]> stem cell 2% by volume hydrocortisone stem cell 480ng / ml

[0164] Primary lung cancer tumor cells (No. B8) were obtained from lung cancer tissue using the same method as in Example 1. 4 pieces / cm2 ) were cultured under the following five culture conditions:

[0165] A. Press 4×10 4 pieces / cm 2 Seeding density: Primary lung cancer tumor cells were seeded into a 24-well plate coated with Matrigel® and cultured using 2 mL of the primary lung cancer epithelial cell culture medium FLM of the present invention;

[0166] B. Press 4×10 4 pieces / cm 2 Seeding density: Primary lung cancer tumor cells were inoculated onto a plate containing γ-ray irradiated mouse fibroblast J2 cells (purchased from Kerafast) and cultured in a 24-well plate using cell conditional reprogramming medium FM (for details, see Liu et al., Am J Pathol, 183(6):1862-1870, 2013).

[0167] C. Press 4×10 4 pieces / cm 2 Seeding density: Primary lung cancer tumor cells were seeded into Matrigel®-coated 24-well plates and cultured in 2 mL of commercial EpiM culture medium.

[0168] D. Press 4×10 4 pieces / cm 2 Seeding Density: Primary lung cancer tumor cells were seeded into a 24-well plate coated with Matrigel® and cultured in 2 mL of basal medium BM.

[0169] In the four culture conditions, the medium was changed every 4 days. The clone formation and cell proliferation of cells in each culture medium in the 24-well plate were observed, and the cell growth status was recorded using a microscope (Invitrogen EVO SM500).

[0170] For primary lung cancer tumor cells (number B8) cultured using the technology of the present invention, when the cell growth in the culture plate reached about 80% of the bottom area, the culture supernatant in the 24-well plate was discarded, 500 μL of 0.05% trypsin (purchased from GIBCO) was added to digest the cells, and incubated at 37°C for 10 minutes until the cells could be observed to be completely digested under a microscope (Invitrogen EVOS M500). The digestion was terminated with 1 mL of DMEM / F12 culture medium containing 5% (v / v) fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and the cells were collected into a 15 mL centrifuge tube and centrifuged at 1500 rpm for 4 minutes, and the supernatant was discarded. The cell pellet after centrifugation was resuspended using the culture medium of the present invention and counted using a flow cytometer (Jiangsu Zhuo Microbiology Technology Co., Ltd. JIMBIO FIL), resulting in a total cell count of 464,000. The cells cultured under the other three culture conditions were digested and counted using the same procedures as described above. The total number of cells obtained by culturing using culture media FM, EpiM, and BM was 350,000, 110,000, and 68,000, respectively.

[0171] Figures 5A-5D The cell photos are taken under a microscope (40 times inverted phase contrast microscope) of sample number B8 on the 12th day after culture under four different culture conditions: Figure 5A This is a microscopic photograph of B8 cultured in basal medium BM on day 12; Figure 5B This is a microscopic photograph of B8 cultured on day 12 using our patented culture medium FLM. Figure 5C This is a microscopic photograph of B8 cultured on day 12 using the commercial culture medium EpiM; Figure 5D This is a microscopic photograph of B8 cultured in the conditional reprogramming medium FM on day 12. As can be seen from the figure, sample B8 was cultured in the basic medium BM ( Figure 5A ) could not form cell clones after 12 days of culture; using EpiM ( Figure 5C ) cultured for 12 days, only a small number of cell clones were formed, and the cell state was poor; using conditional reprogramming medium FM ( Figure 5D ) After 12 days of culture, the cells expanded to a certain extent, but the cell density and cell number were not as good as those of the patented medium FLM ( Figure 5B ) The effect of cultivation is obvious.

[0172] Figure 6 This is a comparison of the cell proliferation effects of primary lung cancer cells obtained from 10 lung cancer patient samples according to Example 1, after culturing for 14 days under the above four different culture medium conditions. √ represents general clone formation ability and proliferation promoting effect, √√ represents relatively obvious clone formation ability and proliferation promoting effect, √√√ represents relatively strong clone formation ability and proliferation promoting effect, and × represents no clone formation. Figure 6 It can be confirmed that the FLM culture medium of the present invention has obvious advantages over the other three culture conditions in terms of clone formation ability, cell proliferation promoting effect, and culture success rate when culturing primary cells obtained from lung cancer tissue.

[0173] (2) Continuous culture of primary lung cancer cells using different culture media and plotting of growth curves

[0174] The same method as in Example (1) was used to obtain the primary lung cancer epithelial cell culture medium FLM, as well as the culture media BM, FM and EpiM as controls.

[0175] Primary lung cancer tumor cells (No. B16) derived from lung cancer tissue were cultured under four different culture medium conditions using the same method as in Example (1), and digested, passaged, and counted.

[0176] When the cells after passage grow to about 80% of the bottom area of ​​the culture plate again, the cells obtained by culture are digested and collected again according to the above operation method and counted. 4 The cells were seeded at a density of 100 cells / well and cultured continuously.

[0177] The following is the calculation formula for the population doubling of primary lung cancer epithelial cells under different culture conditions:

[0178] Population Doubling(PD)=3.32*log 10 (Total number of cells after digestion / initial number of cells seeded), the formula is shown in (Chapman et al. Stem Cell Research & Therapy 2014, 5: 60).

[0179] Figure 7 These are growth curves of B16 cells plotted using Graphpad Prism software under four different culture conditions. The horizontal axis represents the number of days in cell culture, while the vertical axis represents the cumulative cell proliferation factor, which indicates the number of times the cells expanded during the culture cycle. A larger value indicates a greater number of cell proliferations within a given cycle, i.e., a greater number of cells expanded. The slope represents the rate of cell expansion. The graph confirms that the proliferation rate of lung cancer epithelial cells cultured in the FLM culture medium of the present invention is superior to that of the other four culture conditions. It also confirms that the culture medium of the present invention can sustain the continuous culture of primary lung cancer epithelial cells, maintaining a constant proliferation rate for more than 20 days.

[0180] [Example 5]

[0181] Immunohistochemical identification of primary lung cancer tissue and subcultured lung cancer cells

[0182] A mung bean-sized piece of cancerous tissue (sample number B16) was removed from a clinical surgical resection specimen from a lung cancer patient and fixed in 1 mL of 4% paraformaldehyde. Lung cancer epithelial cells (sample number B16) were obtained from the remaining cancerous tissue using the same method as in Example 1. Sample B16 was cultured using the FLM medium of the present invention using the method of Example 3 until the fourth passage.

[0183] Immunohistochemistry was used to detect the expression of important lung cancer-related biomarkers in the original B16 tissue and primary cells obtained by continuous culture to the fourth generation. The tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm thick tissue sections using a microtome. Conventional immunohistochemistry was then performed (for specific steps, see Li et al., Nature Communications, (2018) 9:2983). The primary antibodies used were P63 antibody (purchased from CST) and Ki67 antibody (purchased from R&D).

[0184] Depend on Figure 8 It was confirmed that the expression of lung cancer-related biomarkers in lung cancer tumor cells (sample number B16) cultured in the medium of the present invention at passage 4 was essentially consistent with that in the original tissue sections from which the cells were derived. This indicates that cells cultured in the medium of the present invention retain the original pathological characteristics of lung cancer tissue from lung cancer patients.

[0185] [Example 6]

[0186] Effects of removing a single factor from the culture medium on the sustained proliferation of primary lung cancer cells

[0187] A culture medium FLM for primary lung cancer epithelial cells was prepared using the same method as in Example 2. As a control, a basal culture medium BM was prepared using the same method as in Example 2. In addition, eight other culture media were prepared according to Table 7.

[0188] Table 7 Different culture medium components (concentrations are final concentrations)

[0189]

[0190] The same method as in Example 1 was used to obtain a sample of primary lung cancer tumor cells (No. B18) derived from lung cancer tissue. 4×10 4 pieces / cm 2 Seeding Density Primary lung cancer tumor cells were seeded into a 48-well plate coated with MatrigelR○ and cultured in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific) using 2 mL of the primary lung cancer epithelial cell culture medium (FLM) of the present invention.

[0191] When the cell growth in the culture plate reaches about 80% of the bottom area, the culture supernatant in the 48-well plate is discarded, and 200 μL of 0.05% trypsin (purchased from GIBCO) is added to digest the cells. The cells are incubated at 37°C for 10 minutes until the cells can be observed to be completely digested under a microscope (Invitrogen EVOS M500). The digestion is terminated with 800 μL of DMEM / F12 culture medium containing 5% (v / v) fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, and the cells are collected into a 15 mL centrifuge tube and centrifuged at 1500 rpm for 4 minutes, and the supernatant is discarded. The cell pellet after centrifugation is resuspended using the culture medium of the present invention and counted using a flow cytometer (Jiangsu Zhuo Microbiology Technology Co., Ltd. JIMBIO FIL) to obtain the total number of cells. According to 2×10 4 pieces / cm 2 The cells were seeded at a certain density into another 48-well culture plate coated with extracellular matrix glue and continued to be cultured.

[0192] Cells cultured under the other 8 culture medium conditions and BM culture medium conditions were digested, passaged, and counted using the same operation as above, and cultured using different culture media.

[0193] When the cells after passage grow to about 80% of the bottom area of ​​the culture plate again, the cells obtained by culture are digested and collected again according to the above operation method and counted. 4 pieces / cm 2 Density inoculation and continuous cultivation.

[0194] The following is the calculation formula for the population doubling of primary lung cancer epithelial cells under different culture medium conditions:

[0195] Population Doubling(PD)=3.32*log 10 (Total number of cells after digestion / initial number of cells seeded), the formula is shown in (Chapman et al. Stem Cell Research & Therapy 2014, 5: 60).

[0196] Figure 9 These are cell growth curves plotted using Graphpad Prism software under ten different culture medium conditions. The horizontal axis represents the number of days in cell culture, and the vertical axis represents the cumulative cell proliferation factor, which indicates the number of times the cells expanded during the culture cycle. A larger value indicates a greater number of cell proliferations within a given cycle, which means a greater number of cells were expanded. The slope represents the rate of cell proliferation.

[0197] Depend on Figure 9The results show that after removing various small molecules and additive factors from the culture medium of the present invention, the cell proliferation effect is weakened to a certain extent.

[0198] [Example 7]

[0199] Freshly isolated primary lung cancer epithelial cell samples (No. B16) were obtained according to the steps in embodiment 1. Then, the primary epithelial cells were inoculated onto a plate coated with Matrigel. TM 6-well plates. 3 mL of FLM medium and 3 mL of medium containing compound 1 in FLM were added to the wells seeded with the above-mentioned epithelial cells. The cells were cultured in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific). The culture medium was changed every 4 days after the start of culture. After 12 days of culture, the cells in each group were collected, and the expression of Hippo pathway-related proteins YAP and TAZ in the cell nucleus by compound 1 was detected by recognized immunoblotting. Yes-associated protein (YAP) and its homologous transcriptional coactivator PDZ-binding motif (TAZ) are key effectors of the Hippo pathway that control cell growth and organ size. Their dysregulation can lead to tumorigenesis or hypertrophy. After activation, YAP / TAZ translocate to the cell nucleus and bind to TEAD transcription factors to promote cell proliferation or cell specification transcriptional programs. Immediate early genes represented by the AP-1 complex are rapidly induced and control late transcriptional programs, playing a key role in tumorigenesis and organ maintenance. This result suggests that compound 1 can maintain the stemness and proliferation characteristics of lung cancer cells by inhibiting the MST1 / 2-mediated signaling pathway in breast tumor cells, and play a role in continuously promoting the proliferation of lung cancer cells in vitro.

[0200] Figure 10 The test results showed that the expression of nuclear YAP and TAZ proteins in FLM culture medium supplemented with compound 1 was significantly increased compared with that in culture medium without compound 1. This indicates that compound 1 can activate the Hippo pathway, causing YAP / TAZ to translocate to the cell nucleus and bind to TEAD transcription factors, promoting continuous cell proliferation.

[0201] [Example 8]

[0202] Xenograft tumor formation experiment of primary lung cancer cells derived from cancer tissue in mice

[0203] The same method as in Example 1 was used to isolate lung cancer cells (numbered B16) from a patient with pathologically diagnosed lung cancer tissue. B16 was cultured using the medium FLM of the present invention according to the method of Example 3. When the number of lung cancer cells reached 1×10 7The lung cancer cells were digested and collected using the method of Example 4. The lung cancer cell culture medium FLM of the present invention and MatrigelR○ were mixed in a ratio of 1:1, and 100 μL of the culture medium mixed with Matrigel was taken to 5×10 6 The lung cancer tumor cells were resuspended and injected into the lung fat pad and the right forelimb axilla of 6-week-old female highly immunodeficient mice (NCG) (purchased from Nanjing Institute of Model Animals). The volume and growth rate of the tumors formed by the lung cancer tumor cells in the mice were observed every three days.

[0204] On day 15 after tumor cell inoculation, tumors were observed at both inoculation sites in the mice. From day 15 to day 30, tumor proliferation was evident in the mice. This demonstrates that the lung cancer cells derived from cancerous tissue cultured using the present method are tumorigenic in mice.

[0205] [Example 9]

[0206] Functional testing of drug sensitivity of lung cancer cells derived from cancer tissue

[0207] The following uses surgical resection samples from lung cancer patients as an example to illustrate that lung cancer tumor cells cultured from patient-derived lung cancer tumor samples can be used to detect the sensitivity of patient tumor cells to different drugs.

[0208] 1. Plating of primary lung cancer cells: The cell suspension of isolated lung cancer cells (numbered B25 and B26) obtained according to the method in Example 1 was plated at 4×10 4 pieces / cm 2Density seeding was performed in Matrigel®-coated 12-well plates. 2 mL of prepared FLM culture medium for primary lung cancer epithelial cells was added to the 12-well plates and cultured in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific). When cell growth reached approximately 80% of the bottom area of ​​the culture plate, the supernatant from the 12-well plate was discarded and 500 μL of 0.05% trypsin (purchased from Gibco) was added to digest the cells. The cells were incubated at 37°C for 10 minutes until complete digestion could be observed under a microscope (Invitrogen EVOS M500). Digestion was then terminated with 1 mL of DMEM / F12 culture medium containing 5% (v / v) fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The cells were collected in a 15 mL centrifuge tube and centrifuged at 1500 rpm for 4 minutes, after which the supernatant was discarded. The centrifuged cell pellet was resuspended in FLM medium and counted using a flow cytometer (Jiangsu Zhuowei Microbiology Technology Co., Ltd., JIMBIO FIL), yielding a total cell count of 880,000 and 680,000, respectively. Cells were seeded at a density of 2,000 to 4,000 cells / well in 384-well plates and allowed to adhere overnight.

[0209] 2. Drug gradient experiment:

[0210] (1) Prepare drug storage plates using the concentration gradient dilution method: draw 10 μL of the drug stock solution to be tested (the concentration of the drug stock solution is 2 times the maximum blood concentration of the drug in the human body, C max Preparation), added to a 0.5mL EP tube containing 20μL of DMSO, and then 10μL was drawn from the above EP tube into a second 0.5mL EP tube containing 20μL of DMSO, that is, the drug was diluted at a ratio of 1:3. Repeat the above method, dilute in sequence, and finally obtain 8 concentrations required for dosing. Drugs of different concentrations were added to a 384-well drug storage plate. An equal volume of DMSO was added to each well of the solvent control group as a control. In this embodiment, the drugs to be tested are paclitaxel (purchased from MCE), gemcitabine (purchased from MCE), afatinib (purchased from MCE) and erlotinib (purchased from MCE).

[0211] (2) Using a high-throughput automated workstation (Perkin Elmer JANUS), different concentrations of drug and solvent controls from a 384-well drug reservoir plate were added to a 384-well cell culture plate containing lung cancer cells. Three replicate wells were set up for each drug group and solvent control group. The volume of drug added to each well was 100 nL.

[0212] (3) Cell viability detection: 72 hours after drug administration, the chemiluminescence value of the cells after drug addition and culture was detected using Cell Titer-Glo detection reagent (purchased from Promega). The size of the chemiluminescence value reflects the cell viability and the effect of the drug on cell viability. 10 μL of the prepared Cell Titer-Glo detection solution was added to each well, mixed, and the chemiluminescence value was detected using a microplate reader (Perkin Elmer Envision).

[0213] (4) Cell viability assay: The cell viability after different drugs were treated was calculated according to the formula: cell viability (%) = chemiluminescence value of drug-treated wells / chemiluminescence value of control wells * 100%. Graphpad Prism software was used to plot and calculate the half inhibition rate (IC). 50 .

[0214] (5) Drug sensitivity test results such as Figure 11A and 11B shown.

[0215] Figure 11A and 11B The figures represent the sensitivity of lung cancer cells cultured from surgically removed cancer tissue samples (numbers B25 and B26) from two different lung cancer patients to two chemotherapy drugs, paclitaxel and gemcitabine, and to two targeted drugs, afatinib and erlotinib. Figure 11A Results of sensitivity tests of lung cancer cells cultured from sample B25 to four drugs; Figure 11B Results of a sensitivity test of lung cancer cells cultured from sample B26 to four drugs. The results showed that cells from the same patient had different sensitivities to different drugs, and cells from different patients also had different sensitivities to the same drug.

[0216] Industrial Applicability

[0217] The present invention provides a culture medium and a culture method for culturing primary lung cancer epithelial cells. The cultured lung cancer epithelial cells can be used for evaluating and screening drug efficacy. Therefore, the present invention is suitable for industrial application.

[0218] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications or improvements may be made based on this description. Therefore, such modifications or improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A primary cell culture medium for culturing primary lung cancer epithelial cells, characterized in that: Made with the following ingredients: MST1 / 2 kinase inhibitor; Y27632; fibroblast growth factor; B27 supplement; epidermal growth factor; transferrin; gastrin; A83-01; initial culture medium and antibiotics, Wherein, the MST1 / 2 kinase inhibitor is compound 1 or a pharmaceutically acceptable salt or solvate thereof, in, The content of the MST1 / 2 kinase inhibitor is 2.5 μM to 15 μM; The content of Y27632 is 2.5 μM to 18 μM; The content of the fibroblast growth factor is 2.5ng / ml to 80ng / ml; The volume concentration of the B27 additive in the primary cell culture medium is 1:25 to 1:800; The content of the epidermal growth factor is 2.5ng / ml to 80ng / ml; The content of transferrin is 2.5ng / ml to 80ng / ml; The gastrin content is 2.5ng / ml to 80ng / ml; The content of A83-01 is 62.5nM to 800nM.

2. The primary cell culture medium according to claim 1, wherein: The content of the MST1 / 2 kinase inhibitor is 2.5 μM to 10 μM.

3. The primary cell culture medium according to claim 1, wherein The primary cell culture medium satisfies any one, multiple or all of the following: The content of Y27632 is 5 μM to 15 μM; The content of the fibroblast growth factor is 5ng / ml to 40ng / ml; The volume concentration of the B27 additive in the primary cell culture medium is 1:25 to 1:200; The content of the epidermal growth factor is 10ng / ml to 40ng / ml; The content of transferrin is 5ng / ml to 80ng / ml; The gastrin content is 5ng / ml to 40ng / ml; The content of A83-01 is 125nM to 500nM.

4. The primary cell culture medium according to claim 1, wherein The initial culture medium is selected from DMEM / F12, DMEM, F12 or RPMI-1640; and The antibiotic is selected from one or more of streptomycin / penicillin, amphotericin B and primocin.

5. The primary cell culture medium according to claim 1, wherein: The primary lung cancer epithelial cells are selected from lung cancer epithelial stem cells.

6. A method for culturing primary lung cancer epithelial cells, characterized in that: The following steps are involved: (1) preparing a primary cell culture medium according to any one of claims 1 to 5; (2) Coating the culture vessel with extracellular matrix gel dilution solution; (3) Primary lung cancer epithelial cells isolated from lung cancer tissue were inoculated into a culture vessel coated with extracellular matrix gel and cultured using the primary cell culture medium described in step (1).

7. A method for screening drugs for treating lung cancer, characterized in that: The following steps are involved: (1) Lung cancer epithelial cells are obtained by culturing according to the culturing method of claim 6; (2) Select the drug to be tested and dilute it into different drug concentration gradients; (3) Adding the gradient diluted drug to the lung cancer epithelial cells cultured in step (1), and performing cell viability detection.

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