WO2023060684A1 - 肺癌类器官的培养基、培养方法及其应用 - Google Patents

肺癌类器官的培养基、培养方法及其应用 Download PDF

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WO2023060684A1
WO2023060684A1 PCT/CN2021/130659 CN2021130659W WO2023060684A1 WO 2023060684 A1 WO2023060684 A1 WO 2023060684A1 CN 2021130659 W CN2021130659 W CN 2021130659W WO 2023060684 A1 WO2023060684 A1 WO 2023060684A1
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lung cancer
concentration
alkyl
medium
organoids
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刘青松
胡洁
黄涛
陈程
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合肥中科普瑞昇生物医药科技有限公司
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  • the invention belongs to the field of biotechnology, and in particular relates to a medium for culturing lung cancer organoids, a method for cultivating lung cancer tissue-derived organoids using the medium, and its application in drug efficacy evaluation and screening.
  • Lung cancer is a malignant tumor originating from epithelial tissue, and its morbidity and mortality rank first among various malignant tumors in my country.
  • targeted therapy for lung cancer has become the main treatment method, but which targeted drug is used for lung cancer patients depends on the results of gene sequencing, not all patients with gene mutations can get effective treatment after targeted drug therapy.
  • Remission which requires new research models for clinical drug treatment, and new models for the development of new drugs for the treatment of lung cancer.
  • traditional lung cancer cell lines can no longer meet this need. More and more studies are using primary cells obtained from patients' own tissues for various studies.
  • Organoids which belong to three-dimensional (3D) cell cultures, are mainly derived from human embryonic stem cells, induced pluripotent stem cells and adult stem cells with differentiation ability. Endogenous tissue stem cells exist in different tissues and organs, and play an important role in maintaining the functional morphology of various organs. Under certain induction conditions in vitro, these stem cells can self-organize to form a miniature structure with a diameter of only a few millimeters.
  • Tumor organoids are obtained from primary tumors in patients, and some miniature 3D tumor cell models are cultivated in the laboratory. Tumor organoids highly simulate the characteristics of the source tumor tissue, retain the tumor heterogeneity among individuals, and can be used for functional testing, such as high-throughput drug screening and individualized precision therapy.
  • 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 medium for lung cancer organoids, said medium comprising MST1/2 kinase inhibitors, at least one cell culture additive selected from N2 and B27, fibroblast growth factor 10, SB202190, Y27632, A83-01, neuregulin 1, insulin-like growth factor-1, keratinocyte growth factor, GlutaMAX, and nicotinamide.
  • the MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof,
  • R 1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted by 1-2 independent R (such as phenyl and naphthyl, etc.), aryl C1-C6 alkyl (such as benzyl, etc.) and heteroaryl (such as thienyl, etc.);
  • R 2 and R 3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;
  • R 4 and R 5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxyl, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclyl C1-C6 alkyl (the heterocyclyl is selected from, for example, piperidinyl, tetrahydropyran base, etc.);
  • R is selected from halogen (preferably fluorine and chlorine, more preferably fluorine), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy), and C1-C6 haloalkyl (preferably trifluoro methyl).
  • halogen preferably fluorine and chlorine, more preferably fluorine
  • C1-C6 alkyl preferably methyl
  • C1-C6 alkoxy preferably methoxy
  • C1-C6 haloalkyl preferably trifluoro methyl
  • the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt thereof, or a solvate,
  • R is selected from C1-C6 alkyl, phenyl optionally substituted by 1-2 independently R6 , thienyl optionally substituted by 1-2 independently R6 , and optionally substituted by 1 -2 independently R6 substituted benzyl, R1 is more preferably optionally 1-2 independently R6 substituted phenyl;
  • R 5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R 5 is more preferably hydrogen;
  • R 6 is each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, and R 6 is more preferably fluorine, methyl or trifluoromethyl.
  • the MST1/2 inhibitor is at least one selected from the following compounds or pharmaceutically acceptable salts or solvates thereof.
  • the MST1/2 kinase inhibitor of the present invention is Compound 1.
  • the content of each component in the culture medium of the present invention satisfies any one or more or all of the following:
  • the concentration of the MST1/2 kinase inhibitor is preferably 2.5-10 ⁇ M, more preferably 5-10 ⁇ M;
  • the volume ratio of B27 or N2 cell culture supplement to the medium is preferably 1:25 to 1:100, more preferably 1:50 to 1:100;
  • the concentration of fibroblast growth factor 10 is preferably 25-200 ng/mL, more preferably 25-100 ng/mL;
  • the concentration of SB202190 is preferably 200-1000nM
  • the concentration of Y27632 is preferably 2.5-10 ⁇ M
  • the concentration of A83-01 is preferably 200-1000nM
  • the concentration of neuregulin 1 is preferably 1-40 ng/mL, more preferably 5-40 ng/mL;
  • the concentration of insulin-like growth factor-1 is preferably 2-40 ng/mL, more preferably 10-40 ng/mL;
  • the concentration of keratinocyte growth factor is preferably 2-40 ng/mL, more preferably 10-40 ng/mL;
  • the volume ratio of GlutaMAX to the medium is preferably 1:50 to 1:200;
  • the concentration of nicotinamide is preferably 1 to 10 mM, more preferably 2.5 to 10 mM.
  • the medium also contains an initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and one selected from streptomycin/penicillin, amphotericin B and Primocin one or more antibiotics.
  • the streptomycin concentration ranges from 25 to 400 ⁇ g/mL, and the penicillin concentration ranges from 25 to 400 U/mL; when the antibiotic is selected from amphotericin B, The concentration range is 0.25-4 ⁇ g/mL, and when the antibiotic is selected from Primocin, the concentration range is 25-400 ⁇ g/mL.
  • the invention also provides a method for culturing lung cancer organoids.
  • lung cancer organoids 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.
  • the process includes the following steps:
  • tissue samples Separate lung cancer tissue samples, add 1:1 ratio of basal medium and tissue digestion solution (the amount of tissue digestion solution is about 10mL tissue digestion solution per 1g of tumor tissue) and place in a constant temperature shaker for digestion,
  • the digestion temperature is 4-37°C
  • the shaker speed is 200rpm-300rpm
  • the digestion time is 3-6 hours;
  • the basal medium formula includes an initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and one or more antibiotics selected from streptomycin/penicillin, amphotericin B and Primocin.
  • the formula of tissue digestion solution includes 1640 medium, collagenase II (1-2mg/mL), collagenase IV (1-2mg/mL), DNase (50-100U/mL), hyaluronidase (0.5-1mg/mL) mL), calcium chloride (1 ⁇ 5mM), bovine serum albumin BSA (5 ⁇ 10mg/mL).
  • the present invention also provides a method for evaluating or screening a drug for the treatment of lung cancer, comprising the following steps:
  • the amplification efficiency is high, and lung cancer organoids can be rapidly cultured, and the amplified lung cancer organoids can also be continuously passaged;
  • the culture cost is controllable, and the medium does not need to add expensive Wnt agonists, R-spondin family proteins and Noggin proteins;
  • the number of lung cancer organoids obtained by the technique is large, which is suitable for high-throughput screening of candidate compounds and providing patients with high-throughput drug sensitivity functional tests in vitro.
  • 1A-1K are graphs showing the effects of different concentrations of factors added to the lung cancer organoid medium of the present invention on the proliferation of lung cancer organoids.
  • Figures 2A-2F are photographs of lung cancer organoids cultured using the lung cancer organoid medium of the present invention observed under a microscope, wherein Figure 2A shows photos of organoids obtained from sample OB1 after 5 days of culture; Figure 2B shows photos obtained from sample OB1 The photographs of organoids obtained after 12 days of culture; Figure 2C shows the photos of organoids obtained from sample OB2 after 7 days of culture; Figure 2D shows the photos of organoids obtained from sample OB2 after 15 days of culture; Figure 2E and 2F show the photos of organoids obtained from sample OB3 Photographs of different fields of view of organoids cultured for 21 days.
  • Figure 3A is the results of pathological and immunohistochemical identification of tissue-derived sample OB3 and lung cancer organoids obtained by culturing sample OB3 using the lung cancer organoid medium of the present invention
  • Figure 3B is the results of tissue-derived samples OB4 and Results of pathological and immunohistochemical identification of lung cancer organoids obtained by culturing sample OB4 using the lung cancer organoid medium of the present invention.
  • 4A and 4B are the electron microscope analysis results of the lung cancer organoids cultured in the lung cancer organoid medium of the present invention in the sample OB4.
  • Figures 5A and 5B are graphs comparing the results of culturing lung cancer organoids using the lung cancer organoid medium of the present invention and the existing medium, respectively, wherein Figure 5A shows a photograph of culturing with the LOM medium of the present invention for 15 days; Figure 5B Photographs are shown after 15 days of culture with literature medium ROM.
  • 6A and 6B show the results of drug concentration sensitivity testing of lung cancer organoids cultured using the lung cancer organoid medium of the present invention.
  • an MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1/2 signal transduction.
  • MST1/2 kinase inhibitors for example, bind to MST1/2 kinase and reduce its activity. Due to the similarity in the structures of MST1 and MST2, MST1/2 kinase inhibitors can also be, for example, compounds that bind to MST1 or MST2 and reduce their activity.
  • 2-Amino-2-(2,6-difluorophenyl)acetic acid methyl ester (A2): In a round bottom flask was added 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) Methanol (30 mL) was then added, followed by the dropwise addition of thionyl chloride (1.2 mL) under ice-cooling. The reaction system was reacted overnight at 85°C. After the reaction, the system was evaporated to dryness under reduced pressure to obtain a white solid, which was directly used in the next step.
  • MST1/2 inhibitor compounds of the present invention were synthesized according to a method similar to compound 1, and their structures and mass spectrometry data are shown in the table below.
  • the initial medium can be selected from DMEM/F12, DMEM, F12 or RPMI-1640 commonly used in the art.
  • the formulation of the basal medium is: DMEM/F12 medium (purchased from Corning Company)+100 ⁇ g/mL Primocin (purchased from InvivoGen Company, 0.2% (v/v), commercially available product concentration 50mg/ml ).
  • Lung cancer solid tumor tissue samples (intraoperative) were obtained from patients by professional medical staff of professional medical institutions, and all patients signed informed consent. Intraoperative samples of 5-10 mm 3 were stored and transported in commercial tissue preservation solution (manufacturer: Miltenyi Biotec).
  • Tissue digestion solution formula 1640 medium (Corning, 10-040-CVR), collagenase II (2mg/mL), collagenase IV (2mg/mL), DNase (50U/mL), hyaluronidase (0.75 mg/mL), calcium chloride (3.3mM), bovine serum albumin BSA (10mg/mL).
  • Collagenase II, collagenase IV, DNase, and hyaluronidase mentioned above were all purchased from Sigma; calcium chloride and BSA were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.
  • “+” means that compared with the basal medium, the medium added with this additive has the effect of promoting the proliferation of at least two cases of lung cancer organoids isolated from lung cancer tissue; “-” means that the medium added with this additive At least one case of lung cancer organoids isolated from lung cancer tissues showed an inhibitory effect on proliferation; “ ⁇ ” indicates that the medium supplemented with this additive has no obvious proliferation of at least two cases of lung cancer organoids isolated from lung cancer tissues Impact.
  • the medium of formula 7 When the medium of formula 7 is used, 200 ⁇ L of IGF-1 prepared on the basis of formula 7 is added to the 96-well plate inoculated with organoids, and the final concentrations of IGF-1 are 2 ng/mL and 10 ng respectively. /mL, 40ng/mL; and use the medium of formula 7 to set up the control well (BC).
  • a ratio greater than 1 indicates that the prepared medium containing different concentrations of factors or small molecular compounds has a better effect on promoting proliferation than the culture medium of the control well; a ratio less than 1 indicates that the prepared medium containing different concentrations of factors or small molecular compounds promotes proliferation The effect was weaker than that of the culture medium in the control well.
  • the volume concentration of B27 is preferably 1:25 to 1:100, more preferably 1:50 to 1:100; the content of fibroblast growth factor 10 is preferably 25 to 200 ng/mL, more preferably The preferred content is 25-100 ng/mL; the content of SB202190 is preferably 200-1000 nM; the content of Y27632 is preferably 2.5-10 ⁇ M; the content of A83-01 is preferably 200-1000 nM; the content of neuregulin 1 is preferably 1-40 ng/mL , more preferably 5-40 ng/mL; the content of insulin-like growth factor-1 is preferably 2-40 ng/mL, more preferably 10-40 ng/mL; the content of keratinocyte growth factor is preferably 2-40 ng/mL, More preferably 10-40ng/mL; the volume concentration of GlutaMAX is preferably 1:50-1:200; the content of MST1/2 kinase inhibitor is preferably 2.5-10 ⁇ M
  • the lung cancer organoid medium LOM used in the following examples of the present invention was prepared by adopting the optimal concentration of the above-mentioned added factors, which included: basal medium BM, 1:50 B27 additive, 100ng/mL FGF10, 500nM SB202190, 10 ⁇ M Y27632 , 500nM A83-01, 5ng/mL NRG1, 10ng/mL IGF-1, 10ng/mL KGF, 1:100 GlutaMAX, 5 ⁇ M compound 1 and 2.5mM nicotinamide.
  • basal medium BM 1:50 B27 additive
  • 100ng/mL FGF10 500nM SB202190
  • 10 ⁇ M Y27632 10 ⁇ M Y27632
  • 500nM A83-01 5ng/mL NRG1, 10ng/mL IGF-1, 10ng/mL KGF, 1:100 GlutaMAX, 5 ⁇ M compound 1 and 2.5mM nicotinamide.
  • the lung cancer primary cells (OB1, OB2, OB3) obtained according to the method described in (2) of Example 1 were resuspended and counted with the lung cancer organoid medium LOM of the present invention, and the cell density was diluted to 5-10 ⁇ 10 5 cells/mL, take 400 ⁇ L of the diluted cell suspension and add it to an equal volume of Matrigel (Corning) to mix gently, and then inoculate the mixture in a 24-well plate at 40 ⁇ L/well. Put the inoculated culture plate into the incubator for 30 minutes, wait until the Matrigel is completely solidified, then add the lung cancer organoid medium LOM that has been returned to room temperature in advance, 500 ⁇ L per well, and expand the culture by replacing the medium every five days.
  • Matrigel Matrigel
  • Figure 2A-2F are the lung cancer organoids obtained after culture of samples OB1 (day 5 and day 12), OB2 (day 7 and day 15), and OB3 (day 21, different fields of view) under a 4x objective lens Photo.
  • the volume of organoids increases continuously during the culture process; sample OB3 can reach a maximum diameter of 1800 ⁇ M after 21 days of culture, and the same sample can form different types of organoids, which can simulate the heterogeneity of tumors in vitro .
  • Pathological and immunohistochemical identifications were performed on the cultured lung cancer organoids, and the corresponding original tissue samples were also identified pathologically and immunohistochemically to compare the consistency of organoids and histopathological indicators.
  • the expanded lung cancer organoids were collected and analyzed by electron microscopy according to standard operations.
  • Figure 3A is the results of pathological and immunohistochemical identification of tissue-derived sample OB3 and lung cancer organoids obtained by culturing sample OB3 using the lung cancer organoid medium of the present invention
  • Figure 3B is the results of tissue-derived samples OB4
  • the pathological and immunohistochemical identification results of lung cancer organoids obtained by culturing sample OB4 with the lung cancer organoid medium of the present invention are pictures taken under a 20 ⁇ objective lens.
  • the results showed that the structural morphology of the organoids was cancerous tissue morphology; according to the immunohistochemical indicators, it was judged that the cells obtained after the cultured organoids of the two samples were lung cancer cells.
  • the results show that the lung cancer organoids cultured using the medium LOM of the present invention are consistent with the diagnostic results of the lung cancer tissue before culture.
  • Figures 4A and 4B are the results of electron microscope detection of lung cancer organoids obtained from sample OB4 cultured in vitro. As shown in Figures 4A and 4B, organoids can be observed at magnifications of 1500 and 6000 times, respectively, to have a lung cilia structure unique to lung tissue, which proves that the organoids cultured in the patent medium of the present invention have certain physiological functions .
  • Figures 5A and 5B are photographs of organoids cultured in LOM medium and ROM medium for 15 days under a 4x objective lens.
  • LOM medium can significantly promote the formation and expansion of lung cancer organoids.
  • Example 5 Lung cancer organoids amplified using the medium of the present invention are used for drug screening
  • Primary lung cancer cells were isolated from the lung cancer intraoperative sample (OB6) according to the method of (2) of Example 1, and organoids were cultured using LOM medium, and drug screening was performed when the diameter of lung cancer organoids exceeded 50 ⁇ m.
  • bortezomib and afatinib drug additive solution Preparation of different concentrations of bortezomib and afatinib drug additive solution: Bortezomib and afatinib were prepared into 10 stock solutions with different concentrations, the highest concentration was 20000 ⁇ M, and then diluted by 2 times to obtain 10000 ⁇ M , 5000 ⁇ M, 2500 ⁇ M, 1250 ⁇ M, 625 ⁇ M, 312.5 ⁇ M, 156.25 ⁇ M, 78.125 ⁇ M and 39.0625 ⁇ M stock solutions with different concentrations.
  • the prepared drug storage solution was taken out, placed at room temperature, and the drug was diluted 1000 times with LOM medium to obtain 20000nM, 10000nM, 5000nM, 2500nM, 1250nM, 625nM, 312.5nM, 156.25nM, 78.13nM and 39.06nM.
  • the drug inhibition rate (%) 100%-(the chemiluminescence value drug treatment group of the culture well on the fifth day/the chemiluminescence value drug treatment group of the culture well on the zero day)/(the chemiluminescence value DMSO of the culture well on the fifth day/the The chemiluminescent value of the zero-day culture wells (DMSO )*100% was calculated to obtain the inhibition rates of different drugs, and the results are shown in FIGS. 6A and 6B . 6A and 6B are the inhibition rate curves of different concentrations of test drugs inhibiting the growth of lung cancer organoids.
  • bortezomib has a strong inhibitory effect on the growth of organoids at 10 concentrations, and the inhibitory effects of different concentrations of afatinib are different in a dose-dependent manner, which indicates that the same Patient organoids vary in their effectiveness and sensitivity to different drugs. According to the results, the effectiveness and effective dosage of the drug can be judged when the lung cancer patients are used clinically.
  • the invention provides a culture medium and a culture method for lung cancer organoids, and the cultured organoids can be applied to the efficacy evaluation and screening of drugs.
  • the present invention is suitable for industrial applications.

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Abstract

一种用于肺癌类器官的培养基，包括MST1/2激酶抑制剂、选自N2和B27的至少一种细胞培养添加剂、成纤维细胞生长因子10、SB202190、Y27632、A83-01、神经调节蛋白1、胰岛素样生长因子-1、角化细胞生长因子、GlutaMAX、和烟酰胺。还涉及肺癌组织来源类器官的培养方法及其用途。通过使用所述肺癌组织来源类器官培养基，能够实现肺癌类器官的有效快速扩增，这样扩增得到的类器官保持了患者的病理特性，提高了肺癌类器官的培养成功率和扩增速率，可以为患者的个性化治疗提供研究基础。

Description

肺癌类器官的培养基、培养方法及其应用 技术领域
本发明属于生物技术领域，具体涉及一种用于肺癌类器官培养的培养基、使用该培养基培养肺癌组织来源类器官的方法、及其在药物的疗效评估和筛选中的应用。
背景技术
肺癌是起源于上皮组织的恶性肿瘤，在我国各种恶性肿瘤中发病率和死亡率均排名第一。近年来，肺癌的靶向治疗成为了主要的治疗手段，但是对于肺癌患者使用何种靶向药物取决于基因测序的结果，并不是所有的基因突变患者使用靶向药物治疗之后都能得到有效的缓解，这就需要新的研究模型来进行临床药物的治疗，同时需要新的模型来开发新型的肺癌治疗的药物。但是传统的肺癌细胞系已经不能满足这种需要，越来越多的研究使用的是从病人自身获取的组织进行的原代细胞进行各种研究。
传统临床药物敏感性检测大多采用二维细胞培养。然而，二维培养的细胞仅在有限程度上模拟组织生理条件，缺乏体内真实的组织结构，易导致低分化水平和细胞生理功能的丢失，进而导致获得的实验结果很难预测临床实际结果。类器官，属于三维(3D)细胞培养物，主要来源于人体具有分化能力的胚胎干细胞、诱导多潜能干细胞和成体干细胞。不同组织器官都存在内源组织干细胞，在维持各器官的功能形态发挥着重要作用。这些干细胞在体外一定的诱导条件下，可以自组织形成一个直径仅为几毫米的迷你结构。肿瘤类器官是用取自患者体内原发性肿瘤，在实验室中培养出一些微型的3D肿瘤细胞模型。肿瘤类器官高度模拟了来源肿瘤组织的特征，保留了个体之间的肿瘤异质性，可用于功能性的测试，如进行高通量药物筛选和个体化精准治疗。
当前，肺癌类器官培养方法多采用R-spondin-1、WNT3A和Noggin等昂贵的蛋白因子，导致类器官培养成本较高；且这项技术操作复杂和技术难度大，导致其大规模商业化应用受到限制。因此，需要开发 一种低成本、简单且成功率高的类器官培养方法和培养基。
发明内容
为了解决上述技术问题，本发明提供了一种用于在体外快速扩增肺癌类器官的培养基及培养方法。
本发明的一个方面在于提供一种肺癌类器官的培养基，所述培养基包含MST1/2激酶抑制剂、选自N2和B27的至少一种细胞培养添加剂、成纤维细胞生长因子10、SB202190、Y27632、A83-01、神经调节蛋白1、胰岛素样生长因子-1、角化细胞生长因子、GlutaMAX、和烟酰胺。其中，所述MST1/2激酶抑制剂包括式(I)的化合物或其药学可接受的盐、或溶剂化物，
Figure PCTCN2021130659-appb-000001
其中，
R 1选自C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C2-C6螺环烷基、以及任选地被1-2个独立地R 6取代的芳基(例如苯基和萘基等)、芳基C1-C6烷基(例如苯甲基等)和杂芳基(例如噻吩基等)；
R 2和R 3各自独立地选自C1-C6烷基，优选C1-C3烷基，更优选甲基；
R 4和R 5各自独立地选自氢、C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C1-C6烷基羟基、C1-C6卤代烷基、C1-C6烷基氨基C1-C6烷基、C1-C6烷氧基C1-C6烷基、和C3-C6杂环基C1-C6烷基(所述杂环基选自例如哌啶基、四氢吡喃基等)；
R 6选自卤素(优选氟和氯，更优选氟)、C1-C6烷基(优选甲基)、C1-C6烷氧基(优选甲氧基)、和C1-C6卤代烷基(优选三氟甲基)。
优选的实施方式中，MST1/2激酶抑制剂包括式(Ia)的化合物或其 药学可接受的盐、或溶剂化物，
Figure PCTCN2021130659-appb-000002
其中，
R 1选自C1-C6烷基、任选地被1-2个独立地R 6取代的苯基、任选地被1-2个独立地R 6取代的噻吩基、和任选地被1-2个独立地R 6取代的苯甲基，R 1更优选为任选地被1-2个独立地R 6取代的苯基；
R 5选自氢、C1-C6烷基、和C3-C6环烷基，R 5更优选为氢；
R 6各自独立地选自卤素、C1-C6烷基、和C1-C6卤代烷基，R 6更优选为氟、甲基或三氟甲基。
优选地，所述MST1/2抑制剂是选自以下化合物或其药学可接受的盐、或溶剂化物中的至少一种。
Figure PCTCN2021130659-appb-000003
Figure PCTCN2021130659-appb-000004
Figure PCTCN2021130659-appb-000005
Figure PCTCN2021130659-appb-000006
Figure PCTCN2021130659-appb-000007
最优选地，本发明的MST1/2激酶抑制剂为化合物1。
在本发明的实施方式中，本发明的培养基中各成分的含量满足以下任意一项或多项或全部满足：
(1)MST1/2激酶抑制剂的浓度优选为2.5～10μM，更优选为5～10μM；
(2)B27或N2细胞培养添加剂相对于培养基的体积比优选为1:25～1:100，更优选为1:50～1:100；
(3)成纤维细胞生长因子10的浓度优选为25～200ng/mL，更优选为25～100ng/mL；
(4)SB202190的浓度优选为200～1000nM；
(5)Y27632的浓度优选为2.5～10μM；
(6)A83-01的浓度优选为200～1000nM；
(7)神经调节蛋白1的浓度优选为1～40ng/mL，更优选为5～40ng/mL；
(8)胰岛素样生长因子-1的浓度优选为2～40ng/mL，更优选为10～40ng/mL；
(9)角化细胞生长因子的浓度优选为2～40ng/mL，更优选为10～40ng/mL；
(10)GlutaMAX相对于培养基的体积比优选为1:50～1:200；
(11)烟酰胺的浓度优选为1～10mM，更优选为2.5～10mM。
在本发明的实施方式中，所述培养基还含有选自DMEM/F12、DMEM、F12或RPMI-1640的初始培养基；和选自链霉素/青霉素、两性霉素B和Primocin中的一种或多种的抗生素。
在优选的实施方式中，当抗生素选自链霉素/青霉素时，链霉素浓度范围为25～400μg/mL，青霉素浓度范围为25～400U/mL，当抗生素选自两性霉素B时，浓度范围为0.25～4μg/mL，当抗生素选自Primocin时，浓度范围为25～400μg/mL。
本发明还提供一种肺癌类器官的培养方法。在本发明的肺癌类器官的培养方法中，使用本发明的肺癌类器官培养基对肺癌类器官进行培养。
本发明的肺癌类器官培养方法包括以下步骤。
1.从肺癌实体瘤组织分离样本，获得肺癌原代细胞。该处理过程包括以下步骤：
(1)分离肺癌组织样本，加入1:1比例的基础培养基和组织消化液(组织消化液的加入量是每1g肿瘤组织使用约10mL组织消化液)中置于恒温摇床中进行消化，消化温度为4～37℃，摇床转速为200rpm～300rpm，消化时间为3～6小时；
(2)消化完成后，离心后弃去上清液，离心转速为1200～1600 rpm，离心时间为2～6分钟。
其中，基础培养基配方包括选自DMEM/F12、DMEM、F12或RPMI-1640的初始培养基；和选自链霉素/青霉素、两性霉素B和Primocin中的一种或多种的抗生素。组织消化液配方包括1640培养基、胶原酶Ⅱ(1～2mg/mL)、胶原酶Ⅳ(1～2mg/mL)、DNA酶(50～100U/mL)、透明质酸酶(0.5～1mg/mL)、氯化钙(1～5mM)、牛血清白蛋白BSA(5～10mg/mL)。
2.配制本发明的肺癌类器官培养基，并对上述步骤获得的肺癌原代细胞进行培养。
将上述步骤1中获得的肺癌原代细胞用本发明的初始培养基、基础培养基或肺癌类器官培养基重悬并计数，将细胞密度稀释为5～10×10 5个/mL，取出稀释后的细胞悬液加入等体积的Matrigel基质胶中混匀，然后将混合物接种于多孔板，将接种好的多孔板放入培养箱30～60分钟，待Matrigel完全凝固，然后加入本发明的肺癌类器官培养基进行扩大培养。
本发明还提供一种用于评估或筛选治疗肺癌疾病的药物的方法，其包括以下步骤：
(1)使用本发明的肺癌类器官的培养方法培养肺癌类器官；
(2)选定需要检测的药物并按照所需浓度梯度进行稀释；
(3)对(1)中培养得到的类器官添加稀释后的所述药物；
(4)进行类器官大小或类器官活力测试。
本发明的有益效果包括：
(1)提高肺癌组织来源类器官培养的成功率，成功率达到85％以上；
(2)保证体外原代培养的肺癌类器官能够保持病人的病理特性；
(3)扩增效率高，能快速培养出肺癌类器官，扩增出的肺癌类器官还可以连续传代；
(4)培养成本可控，培养基无需加入价格昂贵的Wnt激动剂、R-spondin家族蛋白和Noggin蛋白；
(5)所述技术养获得的肺癌类器官数量多，适合高通量筛选候选化合物和为病人提供高通量药物体外敏感性功能测试。
附图说明
图1A-1K为显示本发明的肺癌类器官培养基所添加因子的不同浓度对肺癌类器官增殖的影响的图。
图2A-2F为利用显微镜观察使用本发明的肺癌类器官培养基培养得到的肺癌类器官的照片，其中图2A显示由样本OB1获得的类器官培养5天后的照片；图2B显示由样本OB1获得的类器官培养12天后的照片；图2C显示由样本OB2获得的类器官培养7天后的照片；图2D显示由样本OB2获得的类器官培养15天后的照片；图2E和2F显示由样本OB3获得的类器官培养21天的不同视野的照片。
图3A为分别对组织来源的样本OB3和使用本发明的肺癌类器官培养基培养样本OB3而得到的肺癌类器官进行病理和免疫组化鉴定的结果；图3B为分别对组织来源的样本OB4和使用本发明的肺癌类器官培养基培养样本OB4而得到的肺癌类器官进行病理和免疫组化鉴定的结果。
图4A和4B为对样本OB4使用本发明的肺癌类器官培养基培养得到的肺癌类器官进行电镜分析的结果。
图5A和5B为分别使用本发明的肺癌类器官培养基和现有培养基对肺癌类器官进行培养的比较结果图，其中图5A显示用本发明的LOM培养基培养15天后的照片；图5B显示用文献培养基ROM培养15天后的照片。
图6A和6B显示使用本发明的肺癌类器官培养基培养得到的肺癌类器官进行药物浓度敏感性测试的结果。
具体实施方式
为更好地理解本发明，下面结合实施例及附图对本发明作进一步描述。以下实施例仅是对本发明进行说明而非对其加以限定。
[MST1/2激酶抑制剂的制备实施例]
本说明书中，MST1/2激酶抑制剂是指直接或间接地对MST1/2信号传导进行负调节的任意的抑制剂。一般来说，MST1/2激酶抑制剂例如与MST1/2激酶结合并降低其活性。由于MST1和MST2的结构具 有相似性，MST1/2激酶抑制剂也可以是例如与MST1或MST2结合并降低其活性的化合物。
1.MST1/2激酶抑制剂化合物1的制备
4-((7-(2,6-二氟苯基)-5,8-二甲基-6-氧代-5,6,7,8-四氢蝶啶-2-基)氨基)苯 磺酰胺1
Figure PCTCN2021130659-appb-000008
2-氨基-2-(2,6-二氟苯基)乙酸甲酯(A2)：在圆底烧瓶中加入2-氨基-2-(2,6-二氟苯基)乙酸(2.0克)后加入甲醇(30毫升)，随后冰浴下滴加二氯亚砜(1.2毫升)。反应体系在85℃反应过夜。反应结束后，体系在减压下蒸干溶剂，所得白色固体，直接用于下一步。
2-((2-氯-5-硝基嘧啶-4-基)氨基)-2-(2,6-二氟苯基)乙酸甲酯(A3)：在圆底烧瓶中加入2-氨基-2-(2,6-二氟苯基)乙酸甲酯(2克)后加入丙酮(30毫升)和碳酸钾(2.2克)，然后用冰盐浴使体系冷却到-10℃，接着缓慢加入2,4-二氯-5-硝基嘧啶(3.1克)的丙酮溶液。反应体系在室温搅拌过夜。反应结束后，过滤，滤液在减压下除去溶剂，残留物经加压硅胶柱层析提纯后得化合物A3。LC/MS：M+H 359.0。
2-氯-7-(2,6-二氟苯基)-7,8-二氢蝶啶-6(5H)-酮(A4)：在圆底烧瓶中加入2-((2-氯-5-硝基嘧啶-4-基)氨基)-2-(2,6-二氟苯基)乙酸甲酯(2.5克)后加入醋酸(50毫升)和铁粉(3.9克)。反应体系在60℃搅拌两小时。反应结束后，体系在减压下蒸干溶剂，所得物用饱和碳酸氢钠中和至碱性。乙酸乙酯萃取，有机相分别用水、饱和食盐水洗涤后用无水硫酸钠干燥。有机相经过滤，减压蒸干后得粗品。粗品经乙醚洗涤后得化合物A4。LC/MS：M+H 297.0。
2-氯-7-(2,6-二氟苯基)-5,8-二甲基-7,8-二氢蝶啶-6(5H)-酮(A5)：在圆底烧瓶中加入2-氯-7-(2,6-二氟苯基)-7,8-二氢蝶啶-6(5H)-酮(2克)和N,N-二甲基乙酰胺(10毫升)，冷却至-35℃，加入碘甲烷(0.9毫 升)，随后加入氢化钠(615毫克)，反应体系继续搅拌两小时。反应结束后，加水淬灭，乙酸乙酯萃取，有机相分别用水、饱和食盐水洗涤后用无水硫酸钠干燥。有机相经过滤，减压蒸干后得粗品。粗品经乙醚洗涤后得化合物A5。LC/MS：M+H 325.0。
4-((7-(2,6-二氟苯基)-5,8-二甲基-6-氧代-5,6,7,8-四氢蝶啶-2-基)氨基)苯磺酰胺(1)：在圆底烧瓶中加入2-氯-7-(2,6-二氟苯基)-5,8-二甲基-7,8-二氢蝶啶-6(5H)-酮(100毫克)、磺胺(53毫克)、对甲苯磺酸(53毫克)和仲丁醇(5毫升)。反应体系在120℃搅拌过夜。反应结束后，过滤，甲醇和乙醚洗涤得化合物1。LC/MS：M+H 461.1。
2.本发明的其他MST1/2抑制剂化合物的制备
本发明的其他MST1/2抑制剂化合物按照与化合物1类似的方法合成，其结构及质谱数据如下表所示。
Figure PCTCN2021130659-appb-000009
Figure PCTCN2021130659-appb-000010
Figure PCTCN2021130659-appb-000011
Figure PCTCN2021130659-appb-000012
Figure PCTCN2021130659-appb-000013
实施例1肺癌类器官培养基中各添加因子对肺癌类器官增殖的影响
(1)肺癌类器官培养基的配制
首先配制含有初始培养基的基础培养基。初始培养基可选自本领域常用的DMEM/F12、DMEM、F12或RPMI-1640。在本实施例中，基础培养基的配方为：DMEM/F12培养基(购自Corning公司)+100μg/mL Primocin(购自InvivoGen公司，0.2％(v/v)，市售产品浓度50mg/ml)。
在基础培养基内分别加入不同种类的添加剂(参见表1)配制成含有不同添加成分的肺癌类器官培养基。
(2)肺癌原代细胞的分离和处理
1样品选择
肺癌实体瘤组织样品(术中)由专业医疗机构的专业医务人员从患者体内获取，患者均签署了知情同意书。术中样本5-10mm 3，采用商品化组织保存液(生产厂家：Miltenyi Biotec)存储运输。
2材料准备
15mL无菌离心管、移液枪、10mL移液管、无菌枪头等表面消毒后放入超净工作台中紫外照射30分钟。提前30分钟从4℃冰箱取出基础培养基，提前30分钟从-20℃冰箱取出组织消化液。
组织消化液配方：1640培养基(Corning，10-040-CVR)、胶原酶Ⅱ(2mg/mL)、胶原酶Ⅳ(2mg/mL)、DNA酶(50U/mL)、透明质酸酶(0.75mg/mL)、氯化钙(3.3mM)、牛血清白蛋白BSA(10mg/mL)。
以上提及的胶原酶Ⅱ、胶原酶Ⅳ、DNA酶、透明质酸酶均购自Sigma公司；氯化钙和BSA购自生工生物工程(上海)股份有限公司。
3样品分离
3.1超净台中取组织样品于培养皿中，去除带血液的组织，用基础培养基冲洗2次，将组织转移至另一培养皿中用无菌手术刀进行机械分离，将组织块分割为1*2*1mm 3大小；
3.2将切割后的术中组织吸至15mL离心管中，加入5mL基础培养基，混匀，于1500rpm离心3分钟；
3.3弃上清，加入1:1比例的基础培养基和组织消化液(注：组织消化液的加入量是1g肿瘤组织使用约10mL组织消化液)，标记样品名称及编号，用封口膜密封，在37℃下于300rpm摇床(知楚仪器ZQLY-180N)中进行消化，期间每30分钟观察消化是否完成，判断依据为无肉眼可见的颗粒物；
3.4消化完成后，经70μm滤网过滤掉未消化的组织团块，滤网上的组织团块用基础培养基冲洗入离心管中以减少细胞损失，室温1500rpm离心3分钟；
3.5弃上清，观察是否有血细胞，若有血细胞，加8mL血细胞裂解液(购自Sigma公司)，混匀，4℃裂解20分钟，期间颠倒混匀一次，室温1500rpm离心4分钟；
3.6弃上清，加入2mL基础培养基重悬细胞，备用。
4细胞计数及处理
4.1镜下观察：移取少量重悬细胞平铺于培养皿中，显微镜(CNOPTEC，BDS400)下观察癌细胞密度和形态；
4.2活细胞计数：取重悬的细胞悬液12μL，12μL台盼蓝染液(生产厂家：生工生物工程(上海)股份有限公司)充分混合后，取20μL加入细胞计数板(生产厂家：Countstar，规格：50片/盒)，细胞计数仪(Countstar，IC1000)下计算出活的大细胞(细胞粒径>10μm)百分率＝活细胞数/总细胞数*100％。
(3)肺癌类器官的培养
将上述步骤中获得的肺癌原代细胞用预冷的DMEM/F12重悬并计数，将细胞密度稀释为5～10×10 5个/mL，取出400μL稀释后的细胞悬液加入等体积的Matrigel基质胶(Corning)中轻轻混匀，然后将混合物按照8μL/孔接种于96孔板。将接种好的培养板放入培养箱30分钟，待Matrigel完全凝固，然后分别加入事先恢复到室温的表1所示培养基，按照每五天更换一次培养基进行扩大培养。10天后对所培养的类器官进行拍照，并测量统计类器官的直径大小，比较各因子对肺癌类器官增殖的促进作用。其中，作为实验对照，使用未添加任何添加剂的基础培养基，将实验结果示于表1。
表1培养基中的添加成分及促类器官增殖效果
序号 培养基添加剂种类 供应商 终浓度 促增殖程度分级
1 N2 Gibco 1:50 +
2 R-spondin1 北京义翘 20ng/mL ○
3 胃泌素 MCE 10nM ○
4 B27 Gibco 1:50 +
5 A8301 MCE 100nM +
6 SB202190 MCE 200nM +
7 成纤维细胞生长因子/FGF 北京义翘 10ng/mL ○
8 成纤维细胞生长因子10/FGF10 北京义翘 50ng/mL +
9 Noggin 北京义翘 50ng/mL ○
10 胎牛血清/FBS Excell 5％ +
11 胰岛素样生长因子-1/IGF-1 北京义翘 10ng/mL +
12 角化细胞生长因子/KGF 北京义翘 10ng/mL +
13 GlutaMAX Gibco 1:100 +
14 烟酰胺 MCE 100μM ○
15 神经调节蛋白1/NRG1 北京义翘 10ng/mL +
16 Y27632 MCE 10μM +
17 ITS细胞培养添加剂 Gibco 1:100 ○
18 化合物1 制备例 2.5μM +
19 CHIR99021 MCE 2.5μM -
20 肝细胞生长因子/HGF 北京义翘 10ng/mL ○
其中，“+”表示与基础培养基相比，加入该添加剂的培养基对从肺癌组织分离出的肺癌类器官中的至少两例有促进增殖的作用；“－”表示添加该添加剂的培养基对从肺癌组织分离出的肺癌类器官中的至少一例显示有抑制增殖的作用；“○”表示添加该添加剂的培养基对从肺癌组织分离出的肺癌类器官中的至少两例的增殖没有明显的影响。
根据以上结果，拟选择化合物1、Y27632、SB202190、角化细胞生长因子(KGF)、成纤维细胞生长因子10(FGF10)、A83-01、B27、GlutaMAX、胰岛素样生长因子-1(IGF-1)、烟酰胺、神经调节蛋白1(NRG1)等因子进行进一步培养实验。
实施例2培养基添加因子的不同浓度对肺癌类器官的增殖作用
按照实施例1之(2)的方法从术中组织样本(编号为OB1、OB2)获得肺癌原代细胞，并使用下表2中的培养基配方进行类器官培养。
表2培养基配方(浓度为终浓度)
Figure PCTCN2021130659-appb-000014
在使用配方1的培养基时，在接种有类器官的96孔板中在配方1的基础上分别添加配制好的B27每孔200μL，B27的终浓度分别为1:25、1:50、1:100；并使用配方1的培养基设置对照孔(BC)。该系列的培养基中其他添加因子的终浓度与LOM培养基相同。以下配方1-12的实验也以同样的方式进行，不再赘述。
在使用配方2的培养基时，在接种有类器官的96孔板中在配方2的基础上分别添加配制好的FGF10每孔200μL，FGF10的终浓度分别为200ng/mL、100ng/mL、25ng/mL；并使用配方2的培养基设置对照孔(BC)。
在使用配方3的培养基时，在接种有类器官的96孔板中在配方3的基础上分别添加配制好的SB202190细胞培养添加剂每孔200μL，SB202190细胞培养添加剂终浓度分别为200nM、500nM、1000nM；并使用配方3的培养基设置对照孔(BC)。
在使用配方4的培养基时，在接种有类器官的96孔板中在配方4的基础上分别添加配制好的Y27632每孔200μL，Y27632的终浓度分别为2.5μM、5μM、10μM；并使用配方4的培养基设置对照孔(BC)。
在使用配方5的培养基时，在接种有类器官的96孔板中在配方5的基础上分别添加配制好的A83-01每孔200μL，A83-01的终浓度分别为200nM、500nM、1000nM；并使用配方5的培养基设置对照孔(BC)。
在使用配方6的培养基时，在接种有类器官的96孔板中在配方6的基础上分别添加配制好的NRG1每孔200μL，NRG1终浓度分别为1ng/mL、5ng/mL、40ng/mL；并使用配方6的培养基设置对照孔(BC)。
在使用配方7的培养基时，在接种有类器官的96孔板中在配方7的基础上分别添加配制好的IGF-1每孔200μL，IGF-1的终浓度分别为2ng/mL、10ng/mL、40ng/mL；并使用配方7的培养基设置对照孔(BC)。
在使用配方8的培养基时，在接种有类器官的96孔板中在配方8的基础上分别添加配制好的KGF每孔200μL，KGF的终浓度分别为2ng/mL、10ng/mL、40ng/mL；并使用配方8的培养基设置对照孔(BC)。
在使用配方9的培养基时，在接种有类器官的96孔板中在配方9 的基础上分别添加配制好的GlutaMAX每孔200μL，GlutaMAX的终浓度分别为1:200、1:100、1:50；并使用配方9的培养基设置对照孔(BC)。
在使用配方10的培养基时，在接种有类器官的96孔板中在配方10的基础上分别添加配制好的化合物1每孔200μL，化合物1的终浓度分别为2.5μM、5μM、10μM；并使用配方10的培养基设置对照孔(BC)。
在使用配方11的培养基时，在接种有类器官的96孔板中在配方11的基础上分别添加配制好的烟酰胺每孔200μL，烟酰胺的终浓度分别为1mM、2.5mM、10mM；并使用配方11的培养基设置对照孔(BC)。
10天后对所培养的类器官进行拍照，并测量统计类器官的直径大小，比较各因子浓度对肺癌类器官增殖的促进作用。将2例样本收集的数据汇总示于图1A～1K。图1A～1K中，比值为使用各培养基培养15天得到的类器官直径与对应的BC对照孔培养15天得到的类器官直径的比。比值大于1说明配制的含不同浓度的因子或小分子化合物的培养基促增殖效果优于对照孔培养基；比值小于1，则说明配制的含不同浓度的因子或小分子化合物的培养基促增殖效果较对照孔培养基促增殖效果弱。
根据图1A～1K的结果，B27的体积浓度优选为1:25～1:100，更优选为1:50～1:100；成纤维细胞生长因子10的含量优选为25～200ng/mL，更优选为25～100ng/mL；SB202190的含量优选为200～1000nM；Y27632的含量优选为2.5～10μM；A83-01的含量优选为200～1000nM；神经调节蛋白1的含量优选为1～40ng/mL，更优选为5～40ng/mL；胰岛素样生长因子-1的含量优选为2～40ng/mL，更优选为10～40ng/mL；角化细胞生长因子的含量优选为2～40ng/mL，更优选为10～40ng/mL；GlutaMAX的体积浓度优选为1:50～1:200；MST1/2激酶抑制剂的含量优选为2.5～10μM；烟酰胺的含量优选为1～10mM，更优选为2.5～10mM。
采用上述添加因子中的最优浓度，配制本发明下面实施例中所使用的肺癌类器官培养基LOM，其包含：基础培养基BM、1:50B27添加剂、100ng/mL FGF10、500nM SB202190、10μM Y27632、500nM  A83-01、5ng/mL NRG1、10ng/mL IGF-1、10ng/mL KGF、1:100 GlutaMAX、5μM化合物1和2.5mM烟酰胺。
实施例3肺癌类器官培养及鉴定
将按照实施例1之(2)所述方法获得的肺癌原代细胞(OB1、OB2、OB3)用本发明的肺癌类器官培养基LOM重悬并计数，将细胞密度稀释为5～10×10 5个/mL，取出400μL稀释后的细胞悬液加入等体积的Matrigel基质胶(Corning)中轻轻混匀，然后将混合物按照40μL/孔接种于24孔板。将接种好的培养板放入培养箱30分钟，待Matrigel完全凝固，然后加入事先恢复到室温的肺癌类器官培养基LOM，每孔500μL，按照每五天更换一次培养基进行扩大培养。
在第5-21天，使用显微镜(Invitrogen公司EVOS M500)观察培养得到的肺癌类器官。图2A-2F分别是4倍物镜下拍摄样本OB1(第5天和第12天)、OB2(第7天和第15天)、OB3(第21天，不同视野)培养后得到的肺癌类器官的照片。如图所示，类器官在培养过程中体积是不断增大的；样本OB3在培养21天后直径最大可达1800μM，同一个样本可以形成不同类型的类器官，可以在体外模拟肿瘤的异质性。
对培养得到的肺癌类器官进行病理和免疫组化鉴定，同时将对应的原始组织样本也进行病理和免疫组化鉴定，比较类器官和组织病理指标的一致性。
收集扩增好的肺癌类器官，按照标准的操作进行电镜分析。
图3A为分别对组织来源的样本OB3和使用本发明的肺癌类器官培养基培养样本OB3而得到的肺癌类器官进行病理和免疫组化鉴定的结果；图3B为分别对组织来源的样本OB4和使用本发明的肺癌类器官培养基培养样本OB4而得到的肺癌类器官进行病理和免疫组化鉴定的结果，分别为20倍物镜下拍照的图片。如图所示，结果显示类器官的结构形态为癌组织形态；根据免疫组化指标判断该两例样本类器官培养后得到的细胞为肺癌细胞。结果表明使用本发明的培养基LOM培养的肺癌类器官与培养前的肺癌组织的诊断结果一致。
图4A和4B为由样本OB4体外培养后得到的肺癌类器官进行电镜 检测的结果。如图4A和4B所示，分别在1500倍和6000倍的放大倍数下均能够观察到类器官具有肺组织特有的肺纤毛结构，证明本发明专利培养基培养得到的类器官是具有一定生理功能。
实施例4与现有培养基培养效果的比较
(1)对照培养基的配制
配制文献(Norman Sachs等，The EMBO Journal(2019)e100300)中使用的培养基，其配方为Advanced DMEM/F12培养基(购自Invitrogen公司)+1:100 Penicillin/Streptomycin(购自Corning公司)+50μg/mL Primocin(购自Invivogen公司)+1:100 GlutaMAX(购自Corning公司)+10mM HEPES(购自赛默飞公司)+1:50 B27(购自Gibco公司)+1.25mmol/L N-乙酰半胱氨酸(购自MCE公司)+5mmol/L烟酰胺(购自MCE公司)+500ng/mL R-Spondin 1(购自sino biological公司)+25ng/mL角化细胞生长因子(购自sino biological公司)+100ng/mL成纤维细胞生长因子10(购自sino biological公司)+100ng/ml Noggin(购自sino biologica公司)+500nmol/L SB202190(购自MCE公司)+500nmol/L A8301(购自MCE公司)+5μmol/L Y27632(购自MCE公司)。以下简称为ROM培养基。
(2)肺癌类器官培养
按照实施例1之(2)的方法从术中组织样本OB8获得肺癌原代细胞，并分别用LOM培养基和ROM培养基按照实施例3的方法进行类器官培养。
在培养第15天，使用显微镜(Invitrogen公司EVOS M500)观察培养得到的肺癌类器官。图5A和5B是4倍物镜下拍摄分别由LOM培养基和ROM培养基培养15天得到的类器官的照片。
根据图5A和5B的结果可知，与ROM培养基相比，LOM培养基能显著促进肺癌类器官的形成和扩增培养。
实施例5使用本发明的培养基扩增得到的肺癌类器官用于药物筛选
(1)肺癌类器官培养
从肺癌术中样本(OB6)按照实施例1之(2)的方法分离得到肺癌原代细胞，并使用LOM培养基进行类器官培养，待肺癌类器官直径超过50μm时进行药物筛选。
(2)筛选药物配制
按照下表配制10个浓度梯度的2种药物(硼替佐米和阿法替尼；均购自MCE公司)，保存待用。
不同浓度硼替佐米和阿法替尼药物添加液的配制：将硼替佐米和阿法替尼配制成10个不同浓度的储存液，最高浓度为20000μM，然后2倍稀释比例进行稀释，得到10000μM、5000μM、2500μM、1250μM、625μM、312.5μM、156.25μM、78.125μM和39.0625μM不同浓度的储存液。
(3)加药
取出配制好的药物储存液，置于室温，将药物用LOM培养基稀释1000倍后得到20000nM、10000nM、5000nM、2500nM、1250nM、625nM、312.5nM、156.25nM、78.13nM和39.06nM。从孵箱取出按照步骤(1)培养获得的类器官，去除培养孔中的培养基，将含有药物的培养基按照每孔100μL沿着孔壁慢慢将入到96孔透明培养板中。加药结束后，96孔板表面消毒后移至培养箱中继续培养，5天后测定类器官活力。
(4)类器官活力测试
4℃冰箱取出CellTiter-Glo发光试剂(购自Promega公司)，取10毫升试剂于加样槽中，培养箱中取出待检测96孔板，每孔加入50μL CellTiter-Glo发光试剂，静置30分钟后观察96孔板中细胞状态，若细胞大部分已经裂解，则轻轻震荡混匀，吸取100μL至另外一块白色96孔板中，使用多功能酶标仪(Perkin Elmer公司Envision)检测。
(5)数据处理
按照公式药物抑制率(％)＝100％-(第五天培养孔化学发光数值 药物处理组/第零天培养孔化学发光数值 药物处理组)/(第五天培养孔化学发光数值 DMSO/第零天培养孔化学发光数值 DMSO)*100％，计算得到不同药物的抑制率，将结果示于图6A和6B。图6A和6B为不同浓度的测试药物抑制肺癌类器官生长的抑制率曲线。两个抗肿瘤药物中，硼 替佐米在10个浓度下都具有较强的抑制类器官生长的效果，不同浓度的阿法替尼的抑制效果有一定的差异，呈剂量依赖性，这表明同一病人的类器官对不同药物的有效性和敏感性不同。根据结果可以判断肺癌病人在临床使用该种药物时的有效性及有效用量。
工业应用性
本发明提供一种用于肺癌类器官培养的培养基及培养方法，可将培养得到的类器官应用于药物的疗效评估和筛选。因而，本发明适于工业应用。
尽管本文对本发明作了详细说明，但本发明不限于此，本技术领域的技术人员可以根据本发明的原理进行修改，因此，凡按照本发明的原理进行的各种修改都应当理解为落入本发明的保护范围。

Claims (11)

  1. 一种用于肺癌类器官的培养基，其特征在于包括MST1/2激酶抑制剂、选自N2和B27的至少一种细胞培养添加剂、成纤维细胞生长因子10、SB202190、Y27632、A83-01、神经调节蛋白1、胰岛素样生长因子-1、角化细胞生长因子、GlutaMAX、和烟酰胺，
    其中，所述MST1/2激酶抑制剂包括式(I)的化合物或其药学可接受的盐、或溶剂化物，
    Figure PCTCN2021130659-appb-100001
    其中，
    R 1选自C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C2-C6螺环烷基、以及任选地被1-2个独立地R 6取代的芳基、芳基C1-C6烷基和杂芳基；
    R 2和R 3各自独立地选自C1-C6烷基；
    R 4和R 5各自独立地选自氢、C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C1-C6烷基羟基、C1-C6卤代烷基、C1-C6烷基氨基C1-C6烷基、C1-C6烷氧基C1-C6烷基、和C3-C6杂环基C1-C6烷基；
    R 6选自卤素、C1-C6烷基、C1-C6烷氧基、和C1-C6卤代烷基。
  2. 如权利要求1所述的培养基，其中
    R 1选自C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C2-C6螺环烷基、以及任选地被1-2个独立地R 6取代的苯基、萘基、苯甲基和噻吩基；
    R 2和R 3各自独立地选自C1-C3烷基；
    R 4和R 5各自独立地选自氢、C1-C6烷基、C3-C6环烷基、C4-C8 环烷基烷基、C1-C6烷基羟基、C1-C6卤代烷基、C1-C6烷基氨基C1-C6烷基、C1-C6烷氧基C1-C6烷基、哌啶基C1-C6烷基、和四氢吡喃基C1-C6烷基；
    R 6选自卤素、C1-C6烷基、C1-C6烷氧基、和C1-C6卤代烷基。
  3. 如权利要求1所述的培养基，其中所述MST1/2激酶抑制剂包括式(Ia)的化合物或其药学可接受的盐、或溶剂化物，
    Figure PCTCN2021130659-appb-100002
    其中，
    R 1选自C1-C6烷基、任选地被1-2个独立地R 6取代的苯基、任选地被1-2个独立地R 6取代的噻吩基、和任选地被1-2个独立地R 6取代的苯甲基；
    R 5选自氢、C1-C6烷基、和C3-C6环烷基；
    R 6各自独立地选自卤素、C1-C6烷基、和C1-C6卤代烷基。
  4. 如权利要求3所述的培养基，其中
    R 1为任选地被1-2个独立地R 6取代的苯基；
    R 5为氢；
    R 6优选为氟、甲基或三氟甲基。
  5. 如权利要求1所述的培养基，其中所述MST1/2激酶抑制剂选自以下化合物或其药学可接受的盐中的至少一种：
    Figure PCTCN2021130659-appb-100003
    Figure PCTCN2021130659-appb-100004
    Figure PCTCN2021130659-appb-100005
    Figure PCTCN2021130659-appb-100006
    Figure PCTCN2021130659-appb-100007
  6. 如权利要求1～5中任一项所述的培养基，其特征在于所述培养基中各成分的含量满足以下任意一项或多项或全部满足：
    所述MST1/2激酶抑制剂的浓度为2.5～10μM；
    所述B27或N2细胞培养添加剂相对于培养基的体积比为1:25～1:100；
    所述成纤维细胞生长因子10的浓度为25～200ng/mL；
    所述SB202190的浓度为200～1000nM；
    所述Y27632的浓度为2.5～10μM；
    所述A83-01的浓度为200～1000nM；
    所述神经调节蛋白1的浓度为1～40ng/mL；
    所述胰岛素样生长因子-1的浓度为2～40ng/mL；
    所述角化细胞生长因子的浓度为2～40ng/mL；
    所述GlutaMAX相对于培养基的体积比为1:50～1:200；
    所述烟酰胺的浓度为1～10mM。
  7. 如权利要求1～6中任一项所述的培养基，其特征在于所述培养基中各成分的含量满足以下任意一项或多项或全部满足：
    所述MST1/2激酶抑制剂的浓度为5～10μM；
    所述B27或N2细胞培养添加剂相对于培养基的体积比为1:50～1:100；
    所述成纤维细胞生长因子10的浓度为25～100ng/mL；
    所述SB202190的浓度为200～1000nM；
    所述Y27632的浓度为2.5～10μM；
    所述A83-01的浓度为200～1000nM；
    所述神经调节蛋白1的浓度为5～40ng/mL；
    所述胰岛素样生长因子-1的浓度为10～40ng/mL；
    所述角化细胞生长因子的浓度为10～40ng/mL；
    所述GlutaMAX相对于培养基的体积比为1:50～1:200；
    所述烟酰胺的浓度为2.5～10mM。
  8. 如权利要求1～7中任一项所述的培养基，其特征在于还包括：
    选自DMEM/F12、DMEM、F12或RPMI-1640的初始培养基；和
    选自链霉素/青霉素、两性霉素B和Primocin中的一种或多种的抗生素。
  9. 如权利要求1～8中任一项所述的培养基，其特征在于所述培养基不含Wnt激动剂、R-spondin家族蛋白、Noggin蛋白、BMP抑制剂。
  10. 一种肺癌类器官的培养方法，其特征在于包括以下步骤：
    (1)从肺癌实体瘤组织分离样本，获得肺癌原代细胞；
    (2)配制根据权利要求1～9中任一项所述的肺癌类器官的培养基，并对步骤(1)获得的肺癌原代细胞进行类器官培养。
  11. 一种用于评估或筛选治疗肺癌的药物的方法，其特征在于，包括以下步骤：
    (1)使用根据权利要求10所述的肺癌类器官的培养方法培养肺癌类器官；
    (2)选定需要检测的药物并按照所需浓度梯度进行稀释；
    (3)对(1)中培养得到的类器官添加稀释后的所述药物；
    (4)进行类器官大小或类器官活力检测。
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