WO2023004888A1 - 一种用于口腔癌类器官培养的培养基、及其培养方法和应用 - Google Patents

一种用于口腔癌类器官培养的培养基、及其培养方法和应用 Download PDF

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WO2023004888A1
WO2023004888A1 PCT/CN2021/112175 CN2021112175W WO2023004888A1 WO 2023004888 A1 WO2023004888 A1 WO 2023004888A1 CN 2021112175 W CN2021112175 W CN 2021112175W WO 2023004888 A1 WO2023004888 A1 WO 2023004888A1
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oral cancer
alkyl
medium
culture
organoids
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刘青松
汪文亮
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 oral cancer organoids, a method for cultivating oral cancer organoids using the medium, and its application in drug efficacy evaluation and screening.
  • Oral cancer is a general term for malignant tumors that occur in the oral cavity.
  • Oral cancer includes cancers that originate in the tongue (anterior 2/3 of the tongue), buccal mucosa, gums, floor of the mouth, and hard palate. It is one of the more common malignant tumors in the sixth most malignant tumor-prone area (head and neck) in the world , second only to nasopharyngeal carcinoma, ranking second in head and neck malignancies.
  • Oral cancer can occur at any age, with the peak incidence at the age of 40 to 60, and the male to female ratio is about 2:1.
  • oral cancer has shown the following new characteristics: first, the incidence rate of tongue cancer is increasing rapidly, which is close to half of all oral cancers; Patients are not uncommon; the third is that female patients are increasing year by year.
  • traditional treatment methods including surgery, radiotherapy, and chemotherapy have developed to varying degrees, the prognosis of oral cancer is still unsatisfactory.
  • the 5-year survival rate is only about 50% to 60%, about 1/3 of Patients will relapse, and the swallowing, language function and facial appearance of patients who have undergone surgical treatment are seriously affected, and there are obvious and serious appearance injuries, eating and language disorders.
  • the main reasons affecting the survival rate are local recurrence and lymphatic metastasis.
  • 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 rapid expansion of oral cancer organoids in vitro.
  • One aspect of the present invention is to provide a culture medium for oral cancer organoids, said culture medium comprising an MST1/2 kinase inhibitor, at least one cell culture additive selected from N2 and B27, fibroblast growth factor, CHIR99021, Epidermal Growth Factor, Insulin, ITS Cell Culture Supplement, SB202190, Y27632, Dexamethasone, GlutaMAX, and Non-Essential Amino Acids.
  • 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, or solvate thereof,
  • 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
  • the concentration of fibroblast growth factor is preferably 3 ⁇ 30ng/mL
  • the concentration of CHIR99021 is preferably 1.25-5 ⁇ M
  • the concentration of epidermal growth factor is preferably 2-18 ⁇ M
  • the concentration of insulin is preferably 1-10 ⁇ g/mL
  • the volume ratio of the ITS cell culture additive to the medium is preferably 1:30 to 1:300;
  • the concentration of SB202190 is preferably 100-400nM
  • the concentration of Y27632 is preferably 3-30 ⁇ M
  • the concentration of dexamethasone is preferably 0.02-0.5 ⁇ M
  • the volume ratio of GlutaMAX to the medium is preferably 1:30 to 1:300;
  • the non-essential amino acid is one or more selected from glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine, and the concentration of the non-essential amino acid is preferably 50- 200 ⁇ M.
  • 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 oral cancer organoids.
  • oral cancer organoids are cultured using the oral cancer organoid culture medium of the present invention.
  • the oral cancer organoid culture method of the present invention includes the following steps.
  • tissue samples Separate oral cancer tissue samples, add 1:3 ratio of basal medium and tissue digestion solution (the amount of tissue digestion solution is about 10 mL of tissue digestion solution per 1 g of tumor tissue) and place in a constant temperature shaker for digestion , the digestion temperature is 4-37°C, the shaker speed is 200rpm-350rpm, and 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 oral cancer primary cells obtained in the above step 1 were resuspended with the oral cancer organoid medium of the present invention and counted, and the cell density was diluted to 5-10 ⁇ 106 cells/mL, and the diluted cell suspension was taken out and added to Mix in an equal volume of Matrigel matrigel, then inoculate the mixture on a multi-well plate, put the inoculated multi-well plate in the incubator for 30-60 minutes, wait until the Matrigel is completely solidified, and then add the oral cancer organoid medium to expand the culture.
  • the present invention also provides a method for evaluating or screening drugs for the treatment of oral cancer, which comprises the following steps:
  • the culture cost is controllable, and the medium does not need to add expensive Wnt agonists, R-spondin family proteins, Noggin proteins, BMP inhibitors, fibroblast growth factor 10 (FGF10) and other factors;
  • 1A-1L are graphs showing the effects of different concentrations of factors added to the oral cancer organoid medium of the present invention on the proliferation of oral cancer organoids.
  • Figures 2A-2D are photographs of oral cancer organoids cultured using the oral cancer organoid medium of the present invention under a microscope, wherein Figure 2A shows photos of organoids cultured from sample OZ-018 after 8 days; Figure 2B shows Photos of organoids cultured from sample OZ-019 after 10 days; Figure 2C shows photos of organoids cultured from sample OZ-020 after 7 days; Figure 2D shows photos of organoids cultured from sample OZ-021 after 8 days .
  • Figure 3A is the results of pathological and immunohistochemical identification of oral cancer organoids cultured in the oral cancer organoid medium of the present invention for sample OZ-020;
  • Figure 3B is the pathological and immunohistochemical identification of sample OZ-020 tissue The result of the identification;
  • Figure 3C is the result of pathological and immunohistochemical identification of the oral cancer organoid cultured in the oral cancer organoid medium of the present invention for the sample OZ-021;
  • Figure 3D is the pathological identification of the sample OZ-021 tissue and the results of immunohistochemical identification.
  • Fig. 4 is the comparison result of using the oral cancer organoid culture medium of the present invention and the existing medium to culture oral cancer organoids, wherein Fig. 4A shows the photos after culturing with the OC-3 medium of the present invention for 10 days; Fig. 4B The photos after 10 days of culture in ED medium are shown; FIG. 4C shows a bar graph comparing the relative sizes of organoids cultured in OC-3 medium and ED medium.
  • Figure 5 shows the results of oral cancer organoids cultured using the oral cancer organoid medium of the present invention for different drug sensitivity tests, wherein Figure 5A shows the photos of the growth of the organoids without drug treatment and the organoids after 3 days of drug treatment Photographs of growth; Figure 5B shows a histogram of inhibition rates of oral cancer organoid growth inhibition by different concentrations of test drugs.
  • 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 may 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 ).
  • Oral 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 0.25 cm 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 was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; BSA was purchased from Biofroxx.
  • “+” 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 oral cancer organoids isolated from oral cancer tissue; “-” means that the medium with this additive added
  • the culture medium showed an effect of inhibiting the proliferation of at least one of the oral cancer organoids isolated from oral cancer tissues; Proliferation was not significantly affected in both cases.
  • B27 fibroblast growth factor bFGF, CHIR99021, epidermal growth factor EGF, insulin, ITS cell culture supplement, SB202190, Y27632, dexamethasone, GlutaMAX, compound 1, non-essential amino acids and other factors were selected for further investigation.
  • Oral cancer primary cells were obtained from the intraoperative tissue samples (numbered OZ013, OZ014) according to the method of (2) of Example 1, and the organoids in Table 2 below were used for organoid culture.
  • bFGF prepared on the basis of formula 2 is added to the 96-well plate inoculated with organoids, and the final concentrations of bFGF are 3 ng/mL, 10 ng/mL, and 30 ng respectively. /mL; and use the medium of formula 2 to set up control wells (BC).
  • CHIR99021 prepared on the basis of formula 3 is added to the 96-well plate inoculated with organoids, and the final concentrations of CHIR99021 are 1.25 ⁇ M, 2.5 ⁇ M, and 5 ⁇ M respectively; and use The media of formulation 3 set up control wells (BC).
  • Figures 1A-1L the ratios are the diameters of organoids cultured for 7 days using each medium compared to the diameters of organoids cultured for 7 days in the corresponding BC control wells.
  • 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 1:25-1:100; the content of fibroblast growth factor bFGF is preferably 3-30 ng/mL; the content of CHIR99021 is preferably 1.25-5 ⁇ M; epidermal cell growth
  • the content of factor EGF is preferably 2-18ng/mL; the content of insulin is preferably 1-10 ⁇ g/mL; the volume concentration of ITS cell culture additive is preferably 1:30-1:300; the content of SB202190 is preferably 100-400nM; Y27632
  • the content of dexamethasone is preferably 3-30 ⁇ M; the content of dexamethasone is preferably 0.02-0.5 ⁇ M; the volume concentration of GlutaMAX is preferably 1:30-1:300; the content of MST1/2 kinase inhibitor is preferably 2.5-10 ⁇ M; optional
  • the amino acid content is preferably 50 to 200 ⁇ M.
  • the oral cancer primary cells obtained according to the method described in (2) of Example 1 were resuspended with the oral cancer organoid medium OC-3 of the present invention and counted, and the cell density was diluted to 5-10 ⁇ 10 6 cells/ mL, 400 ⁇ L of the diluted cell suspension was taken out and added to an equal volume of Matrigel (Corning) to mix gently, and then the mixture was seeded in a 24-well plate at 50 ⁇ L/well. Put the inoculated culture plate into the incubator for 30 minutes, wait until the Matrigel is completely solidified, then add the oral cancer organoid medium OC-3 that has been returned to room temperature in advance, 500 ⁇ L per well, and expand the culture by replacing the medium every three days .
  • Figures 2A-2D are photographs of oral cancer organoids obtained after culture of samples OZ-018, OZ-019, OZ-020, and OZ-021 taken under a 10x objective lens. Under the microscope, oral cancer organoids are spherical in shape with a smooth surface.
  • the pathological and immunohistochemical identification of the cultured oral cancer organoids are carried out, and the corresponding tissue samples are sent for pathological and immunohistochemical identification to compare the consistency of the organoid and tissue results.
  • Figures 3A and 3C are the results of pathological and immunohistochemical identification of oral cancer organoids obtained from samples OZ-020 and OZ-021 cultured in vitro, respectively, and are pictures taken under a 20x objective lens.
  • HE results showed that the structural morphology of organoids was cancerous; the expression of CK, 34 ⁇ E12, P63, CK5/6, and Ki67 suggested that the sample was oral cancer.
  • Figure 3B and Figure 3D show the pathological and immunohistochemical results of the corresponding tissues of OZ-020 and OZ-021, and the results show that the oral cancer organoids cultured with the medium OC-3 of the present invention are consistent with the diagnostic results of oral cancer tissues.
  • Oral cancer primary cells were obtained from the intraoperative tissue sample OZ-022 according to the method of (2) of Example 1, and organoid culture was carried out according to the method of Example 3 using OC-3 medium and ED medium respectively.
  • Figures 4A and 4B are photographs of organoids cultured in OC-3 medium and ED medium respectively under a 10x objective lens
  • Figure 4C is a histogram comparing the relative sizes of organoids cultured in the two media.
  • OC-3 medium can significantly promote the expansion and culture of oral cancer organoids.
  • Example 5 Oral cancer organoids amplified using the medium of the present invention are used for drug screening
  • Oral cancer primary cells were isolated from the obtained oral cancer intraoperative sample (OZ-016) according to the method of (2) of Example 1, and the organoid culture was carried out using OC-3 medium, and the diameter of the oral cancer organoid was Drug screening was carried out above 50 ⁇ m.
  • doxorubicin Three drugs (daunorubicin, ararubicin, and doxorubicin; all purchased from MCE Company) with two concentration gradients were prepared according to the table below, and stored for later use.
  • the drug inhibition rate (%) 100%-(the chemiluminescence value drug treatment group of the culture well on the third 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 third 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. 5A and 5B .
  • Figure 5A is a photo of organoid growth without drug treatment and a photo of organoid growth after 3 days of drug treatment taken under a microscope (Invitrogen EVOS M500) with a 4x objective lens
  • Figure 5B is a test drug at different concentrations inhibiting oral cancer Histogram of organ growth inhibition rate.
  • FIG. 5A It can be confirmed from FIG. 5A that the organoids cultured with the oral cancer organoid medium of the present invention grow well, and the growth of the organoids is significantly inhibited after being treated with daunorubicin and arubicin.
  • Fig. 5B is a histogram of the inhibition rate of three test drugs at different concentrations to inhibit the growth of oral cancer organoids. It can be seen from Figure 5B that the data error value of the drug treatment group is very small, indicating that using this system for drug screening, the data between the repeated wells of the same drug are basically consistent.
  • arubicin has a strong inhibitory effect on the growth of organoids at two concentrations, and the inhibitory effects of different concentrations of daunorubicin are significantly different.
  • the effect of doxorubicin on the growth of organoids The inhibitory effect was weaker, suggesting that organoids from the same patient have different sensitivities to different drugs. According to the results, the effectiveness and effective dosage of the drug can be judged when the oral cancer patient is used clinically.
  • the invention provides a culture medium and a culture method for culturing oral cancer organoids in vitro, 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

一种用于口腔癌类器官培养的培养基，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、CHIR99021、表皮细胞生长因子、胰岛素、ITS细胞培养添加剂、SB202190、Y27632、地塞米松、GlutaMAX、和非必需氨基酸。本发明还涉及口腔癌类器官的培养方法及其用途。通过使用本发明的口腔癌类器官培养基，能够实现口腔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，提高了口腔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

一种用于口腔癌类器官培养的培养基、及其培养方法和应用 技术领域
本发明属于生物技术领域，具体涉及一种用于口腔癌类器官培养的培养基、使用该培养基培养口腔癌类器官的方法、及其在药物的疗效评估和筛选中的应用。
背景技术
口腔癌是发生在口腔的恶性肿瘤的总称。口腔癌包括原发于口腔舌(舌前2/3)、颊黏膜、牙龈、口底、硬腭的癌症，是全球第六大恶性肿瘤好发区(头颈部)的较常见恶性肿瘤之一，仅次于鼻咽癌，居头颈部恶性肿瘤的第二位。口腔癌可发生于任何年龄，以40～60岁为发病高峰，男女构成比约为2:1。近年来，口腔癌呈现出以下新特点：一是舌癌发病率的增长速度较快，已接近所有口腔癌症的一半；二是口腔癌的患病年龄有年轻化的趋势，20～30岁青年患者并不少见；三是女性患者逐年增多。过去20年，虽然传统治疗方法包括手术、放疗、化疗都有不同程度的发展，但是口腔癌的预后仍然难以让人满意，其5年生存率只有50％～60％左右，大约1/3的患者会复发，而且经过手术治疗的患者吞咽、语言功能及面部容貌等受到严重的影响，存在明显和严重的容貌伤害、进食和语言障碍。影响生存率的主要原因是局部复发和淋巴道转移。因此，新的治疗方法如靶向肿瘤治疗等生物治疗手段正受到越来越多的关注。同时，如何提高药物治疗的抗癌效能，降低药物的毒、副作用，亦即抗癌治疗的靶向性，是抗肿瘤基础和临床研究所关心的问题。
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，口腔癌类器官培养方法多采用基础培养基(DMEM或是DMEM/F12)、R-spondin-1、Noggin以及一些昂贵的蛋白因子，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口腔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口腔癌类器官的培养基，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、CHIR99021、表皮细胞生长因子、胰岛素、ITS细胞培养添加剂、SB202190、Y27632、地塞米松、GlutaMAX、和非必需氨基酸。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 PCTCN2021112175-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 PCTCN2021112175-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 PCTCN2021112175-appb-000003
Figure PCTCN2021112175-appb-000004
Figure PCTCN2021112175-appb-000005
Figure PCTCN2021112175-appb-000006
Figure PCTCN2021112175-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；
(2)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；
(3)成纤维细胞生长因子的浓度优选为3～30ng/mL；
(4)CHIR99021的浓度优选为1.25～5μM；
(5)表皮细胞生长因子的浓度优选为2～18μM；
(6)胰岛素的浓度优选为1～10μg/mL；
(7)ITS细胞培养添加剂相对于培养基的体积比优选为1:30～1:300；
(8)SB202190的浓度优选为100～400nM；
(9)Y27632的浓度优选为3～30μM；
(10)地塞米松的浓度优选为0.02～0.5μM；
(11)GlutaMAX相对于培养基的体积比优选为1:30～1:300；
(12)非必需氨基酸为选自甘氨酸、丙氨酸、天冬酰胺、天冬氨酸、谷氨酸、脯氨酸和丝氨酸中的一种或多种，非必需氨基酸的浓度优选为50～200μM。
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。
本发明还提供一种口腔癌类器官的培养方法。在本发明的口腔癌类器官的培养方法中，使用本发明的口腔癌类器官培养基对口腔癌类器官进行培养。
本发明的口腔癌类器官培养方法包括以下步骤。
1.从口腔癌实体瘤组织分离样本，获得口腔癌原代细胞。该处理过程包括以下步骤：
(1)分离口腔癌组织样本，加入1:3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～350rpm，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1200～1600rpm，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.配制本发明的口腔癌类器官培养基，并对上述步骤获得的口腔癌原代细胞进行培养。
将上述步骤1中获得的口腔癌原代细胞用本发明的口腔癌类器官培养基重悬并计数，将细胞密度稀释为5～10×10 6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，然后加入口腔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口腔癌疾病的药物的方法，其包括以下步骤：
(1)使用本发明的口腔癌类器官的培养方法培养口腔癌类器官；
(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)提高口腔癌类器官培养的成功率，成功率达到90％以上；
(2)保证体外原代培养的口腔癌类器官能够保持病人的病理特性；
(3)扩增效率高，能快速培养出口腔癌类器官，扩增出的口腔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、Noggin蛋白、BMP抑制剂、成纤维细胞生长因子10(FGF10)等因子；
(5)所述技术培养获得的口腔癌类器官数量大，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-1L为显示本发明口腔癌类器官培养基所添加因子的不同浓度对口腔癌类器官增殖的影响的图。
图2A-2D为利用显微镜观察使用本发明的口腔癌类器官培养基培养得到的口腔癌类器官的照片，其中图2A显示8天后由样本OZ-018培养获得的类器官的照片；图2B显示10天后由样本OZ-019培养获得的类器官的照片；图2C显示7天后由样本OZ-020培养获得的类器官的照片；图2D显示8天后由样本OZ-021培养获得的类器官的照片。
图3A为对样本OZ-020使用本发明的口腔癌类器官培养基培养得到的口腔癌类器官进行病理和免疫组化鉴定的结果；图3B为对样本OZ-020组织进行病理和免疫组化鉴定的结果；图3C为对样本OZ-021使用本发明的口腔癌类器官培养基培养得到的口腔癌类器官进行病理和免疫组化鉴定的结果；图3D为对样本OZ-021组织进行病理和免疫组化鉴定的结果。
图4为使用本发明的口腔癌类器官培养基与现有培养基对口腔癌类器官进行培养的比较结果，其中图4A显示用本发明的OC-3培养基培养10天后的照片；图4B显示用ED培养基培养10天后的照片；图4C显示OC-3培养基和ED培养基培养得到的类器官相对大小的对比柱状图。
图5显示使用本发明的口腔癌类器官培养基培养得到口腔癌类器官进行不同药物敏感性测试的结果，其中图5A显示未加药处理时类器官生长的照片和加药处理3天后类器官生长的照片；图5B显示不同浓度的测试药物抑制口腔癌类器官生长的抑制率柱状图。
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 PCTCN2021112175-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 PCTCN2021112175-appb-000009
Figure PCTCN2021112175-appb-000010
Figure PCTCN2021112175-appb-000011
Figure PCTCN2021112175-appb-000012
Figure PCTCN2021112175-appb-000013
实施例1 口腔癌类器官培养基中各添加因子对口腔癌类器官增殖的影响
(1)口腔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口腔癌类器官培养基。
(2)口腔癌原代细胞的分离和处理
1样品选择
口腔癌实体瘤组织样品(术中)由专业医疗机构的专业医务人员从患者获取，患者均签署了知情同意书。术中样本0.25cm 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DMEM/F12(Corning)，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；氯化钙购自生工生物工程(上海)股份有限公司；BSA购自Biofroxx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*1*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4分钟；
3.3弃上清，加入1:3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100μm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，于25℃下1500rpm离心4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，25℃下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)口腔癌类器官的培养
将上述步骤中获得的口腔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 6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5μL/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，按照每三天更换一次培养基进行扩大培养。7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，比较各因子对口腔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 EGF R&D 5ng/mL +
3 R-spondin1 R&D 20ng/mL +
4 前列腺素E2 Tocris 0.5μM ○
5 胰岛素 Peprotech 1.5μg/mL +
6 B27 Gibco 1:50 +
7 A8301 MCE 100nM -
8 SB202190 MCE 200nM +
9 bFGF R&D 10ng/mL +
10 氢化可的松 Sigma 10ng/mL ○
11 Noggin R&D 30ng/mL ○
12 FBS Excell 5％ +
13 IGF-1 R&D 45ng/mL ○
14 KGF R&D 5ng/mL ○
15 GlutaMAX Gibco 1:100 +
16 非必需氨基酸 Corning 100μM +
17 地塞米松 MCE 0.1μM +
18 NRG1 sino biological 5ng/mL ○
19 Y27632 MCE 10μM +
20 ITS Gibco 1:100 +
21 化合物1 制备例 5μM +
22 CHIR99021 MCE 2.5μM +
其中，“+”表示与基础培养基相比，加入该添加剂的培养基对从口腔癌组织分离出的口腔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口腔癌组织分离出的口腔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口腔癌组织分离出的口腔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B27、成纤维细胞生长因子bFGF、CHIR99021、表皮细胞生长因子EGF、胰岛素、ITS细胞培养添加剂、SB202190、Y27632、地塞米松、GlutaMAX、化合物1、非必需氨基酸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口腔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OZ013、 OZ014)获得口腔癌原代细胞，并使用下表2中的进行类器官培养。
表2 培养基组分(浓度为终浓度)
Figure PCTCN2021112175-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OC-3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bFGF每孔200μL，bFGF的终浓度分别为3ng/mL、10ng/mL、30ng/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CHIR99021每孔200μL，CHIR99021终浓度分别为1.25μM、2.5μM、5μM；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EGF每孔200μL，EGF的终浓度分别为2ng/mL、6ng/mL、18ng/mL；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胰岛素每孔200μL，胰岛素的终浓度分别为1μg/mL、3μg/mL、10μg/mL；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ITS每孔200μL，ITS终浓度分别为1:300、1:100、1:30；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SB202190每孔200μL，SB202190的终浓度分别为100nM、200nM、400nM；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Y27632每孔200μL，Y27632的终浓度分别为3μM、10μM、30μM；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地塞米松每孔200μL，地塞米松的终浓度分别为0.02μM、0.1μM、0.5μM；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GlutaMAX每孔200μL，GlutaMAX的终浓度分别为1:300、1:100、1:30；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11的培养基设置对照孔(BC)。
在使用配方12的培养基时，在接种有类器官的96孔板中在配方12的基础上分别添加配制好的非必需氨基酸每孔200μL，非必需氨基酸的终浓度分别为50μM、100μM、200μM；并使用配方12的培养基设置对照孔(BC)。
7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，比较各因子浓度对口腔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～1L。图1A～1L中，比值为使用各培养基培养7天得到的类器官直径与对应的BC对照孔培养7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～1L的结果，B27的体积浓度优选为1:25～1:100；成纤维细胞生长因子bFGF的含量优选为3～30ng/mL；CHIR99021的含量优选为1.25～5μM；表皮细胞生长因子EGF的含量优选为2～18ng/mL；胰岛素的含量优选为1～10μg/mL；ITS细胞培养添加剂的体积浓度优选为1:30～1:300；SB202190的含量优选为100～400nM；Y27632的含量优选为3～30μM；地塞米松的含量优选为0.02～0.5μM；GlutaMAX的体积浓度优选为1:30～1:300；MST1/2激酶抑制剂的含量优选为2.5～10μM；非必需氨基酸的含量优选为50～200μM。
实施例3 口腔癌类器官培养及鉴定
将按照实施例1之(2)所述方法获得的口腔癌原代细胞用本发明的口腔癌类器官培养基OC-3重悬并计数，将细胞密度稀释为5～10×10 6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50μL/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口腔癌类器官培养基OC-3，每孔500μL，按照每三天更换一次培养基进行扩大培养。
在第7-10天，使用显微镜(Invitrogen公司EVOS M500)观察培养得到的口腔癌类器官。图2A-2D的是10倍物镜下拍摄样本OZ-018、OZ-019、OZ-020、OZ-021培养后得到的口腔癌类器官的照片。口腔癌类器官在镜下呈规则球状，表面较光滑。
对培养得到的口腔癌类器官进行病理和免疫组化鉴定，同时将对应的组织样本送去进行病理和免疫组化鉴定，比较类器官和组织结果的一致性。
图3A和3C分别为由样本OZ-020、OZ-021体外培养后得到的口腔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3A和3C所示，HE结果显示类器官的结构形态为癌组织形态；CK、34βE12、P63、CK5/6、Ki67表达，提示该样本为口腔癌。图3B和图3D为OZ-020、OZ-021对应组织的病理和免疫组化结果，结果表明使用本发明的培养基OC-3培养的口腔癌类器官与口腔癌组织诊断结果一致。
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(Else Driehuis等，Cancer Discov.2019,9:852-871)中使用的培养基，其配方为DMEM/F12培养基(购自Corning公司)+1:50B27(购自Gibco公司)+1.25mmol/L N-乙酰半胱氨酸(购自MCE公司)+10mmol/L烟酰胺(购自MCE公司)+250ng/ml两性霉素B(购自Selleck公司)+10μg/ml庆大霉素(购自MCE公司)+500nM A8301(购自MCE公司)+50ng/ml表皮细胞生长因子(购自R&D公司)+10ng/mL成纤维细胞生长因子10(购自sino biological公司)+5ng/mL成纤维细胞生长因子2(购自R&D公司)+1μmol/L前列腺素E2(购自Tocris公司)+0.3μmol/L CHIR99021(购自MCE公司)+1μmol/L Forskolin(购自MCE公司)+10ng/mL R-spondin(购自R&D公司)+10ng/mL Noggin(购自R&D公司)。以下简称为ED培养基。
(2)口腔癌类器官培养
按照实施例1之(2)的方法从术中组织样本OZ-022获得口腔癌原代细胞，并分别用OC-3培养基和ED培养基按照实施例3的方法进行类器官培养。
在培养第10天，使用显微镜(Invitrogen公司EVOS M500)观察培养得到的口腔癌类器官。图4A和4B是10倍物镜下拍摄分别由OC-3培养基和ED培养基培养得到的类器官的照片，图4C是两种培养基培养得到的类器官相对大小的对比柱状图。
根据图4A-4C的结果可知，与ED培养基相比，OC-3培养基能显著促进口腔癌类器官的扩增和培养。
实施例5 使用本发明的培养基扩增得到的口腔癌类器官用于药物筛选
(1)口腔癌类器官培养
从得到的口腔癌术中样本(OZ-016)按照与实施例1之(2)的方法分离得到口腔癌原代细胞，并使用OC-3培养基进行类器官培养，待口腔癌类器官直径超过50μm时进行药物筛选。
(2)筛选药物配制
按照下表配制2个浓度梯度的3种药物(柔红霉素、阿柔比星、多柔比星；均购自MCE公司)，保存待用。
表3 柔红霉素、阿柔比星、多柔比星药物添加液的配制
柔红霉素 最终浓度(μM) 1 0.1
阿柔比星 最终浓度(μM) 1 0.1
多柔比星 最终浓度(μM) 0.1 0.01
(3)加药
取出配制好的药物，置于室温，将药物用OC-3培养基稀释1000倍后备用。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沿着孔壁慢慢将入到培养孔。加药结束后，96孔板表面消毒后移至培养箱中继续培养，72小时后测定类器官活力。
(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20μL CellTiter-Glo发光试剂，静置10分钟后混匀，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％-(第三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)/(第三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5A和5B。图5A为4倍物镜下显微镜(Invitrogen公司EVOS M500)拍摄的未加药处理时类器官生长的照 片和加药处理3天后类器官生长的照片，图5B为不同浓度的测试药物抑制口腔癌类器官生长的抑制率柱状图。
由图5A可以确认，使用本发明的口腔癌类器官培养基培养得到的类器官生长状态良好，用柔红霉素和阿柔比星处理之后类器官生长明显受到抑制。图5B是不同浓度的三种测试药物抑制口腔癌类器官生长的抑制率柱状图。由图5B可以看出，药物处理组的数据误差值很小，表明利用此套体系进行药物筛选，相同药物重复孔之间数据基本保持一致。三个抗肿瘤药物中，阿柔比星在两个浓度下都具有较强的抑制类器官生长的效果，不同浓度的柔红霉素的抑制效果差异显著，多柔比星对类器官生长的抑制作用较弱，这表明同一病人的类器官对不同药物的敏感性不同。根据结果可以判断口腔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在体外培养口腔癌类器官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 (10)

  1. 一种用于口腔癌类器官的培养基，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、CHIR99021、表皮细胞生长因子、胰岛素、ITS细胞培养添加剂、SB202190、Y27632、地塞米松、GlutaMAX、和非必需氨基酸，
    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 PCTCN2021112175-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 PCTCN2021112175-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 PCTCN2021112175-appb-100003
    Figure PCTCN2021112175-appb-100004
    Figure PCTCN2021112175-appb-100005
    Figure PCTCN2021112175-appb-100006
    Figure PCTCN2021112175-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的浓度为3～30ng/mL；
    所述CHIR99021的浓度为1.25～5μM；
    所述表皮细胞生长因子的浓度为2～18μM；
    所述胰岛素的浓度为1～10μg/mL；
    所述ITS细胞培养添加剂相对于培养基的体积比为1:30～1:300；
    所述SB202190的浓度为100～400nM；
    所述Y27632的浓度为3～30μM；
    所述地塞米松的浓度为0.02～0.5μM；
    所述GlutaMAX相对于培养基的体积比为1:30～1:300；
    所述非必需氨基酸为选自甘氨酸、丙氨酸、天冬酰胺、天冬氨酸、谷氨酸、脯氨酸和丝氨酸中的一种或多种，其浓度为50～200μM。
  7. 如权利要求1～5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。
  8. 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不含Wnt激动剂、R-spondin家族蛋白、Noggin蛋白、BMP抑制剂、成纤维细胞生长因子10。
  9. 一种口腔癌类器官的培养方法，其特征在于包括以下步骤：
    (1)从口腔癌实体瘤组织分离样本，获得口腔癌原代细胞；
    (2)配制根据权利要求1-8中任一项所述的口腔癌类器官的培养基，并对步骤(1)获得的口腔癌原代细胞进行类器官培养。
  10. 一种用于评估或筛选治疗口腔癌疾病的药物的方法，其特征在于，包括以下步骤：
    (1)使用权利要求9所述的口腔癌类器官的培养方法培养口腔癌类器官；
    (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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