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| 携带TP53和ERBB2基因突变的肝细胞癌类器官的培养及药物敏感性鉴定 |
| 刘二冬1,侯明慧2,王向3,宗雅丽1,王红阳1,4*,付静4* |
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(1. 复旦大学生命科学学院和代谢与整合生物学研究院, 上海 200438;
2. 郑州大学第一附属医院转化医学中心类器官实验室, 郑州 450052;
3. 海军军医大学（第二军医大学）第三附属医院胆道二科, 上海 200438;
4. 海军军医大学（第二军医大学）国家肝癌科学中心, 上海 201805
*通信作者) |
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| 摘要: |
| 目的 培养肝细胞癌类器官用于药物筛选，为肝细胞癌患者的个体化治疗提供参考。方法 将1例肝细胞癌患者的肿瘤组织消化为单细胞后接种于基质胶中培养。通过H-E染色观察类器官与原代肿瘤组织的细胞形态。采用免疫组织化学染色观察类器官与原代肿瘤组织中细胞角蛋白7（CK7）、细胞角蛋白8（CK8）、p53、磷脂酰肌醇蛋白聚糖3（GPC-3）等分子的表达情况。通过靶向测序检测类器官中肿瘤相关基因的突变情况。根据靶向测序结果进行药物筛选，采用CellTiter-Glo细胞活性检测试剂盒检测varlitinib［表皮生长因子受体/erb-b2受体酪氨酸激酶2（ERBB2）特异性抑制剂］、索拉非尼（激酶多靶点抑制剂）、顺铂和nutlin3（p53激活剂）对类器官活性的影响。结果 成功建立了携带肿瘤蛋白p53（TP53）和ERBB2基因突变的肝细胞癌类器官。H-E染色结果显示，类器官与原代肿瘤组织具有极为相似的结构和细胞学特征。免疫组织化学染色结果显示，类器官与原代肿瘤组织中CK7均为阴性，CK8、p53和GPC-3均为阳性。靶向测序结果显示，类器官中的突变基因有TP53（E271K，100%）、ERBB2（A1232V，62.29%）、RUNX转录因子1（RUNX1）（E395A，20.21%）、雄激素受体（AR）（Q91dup，70%）。细胞活性检测结果显示，varlitinib和索拉非尼对类器官的IC50分别为2.81 μmol/L和1.327 μmol/L，顺铂对类器官的IC50为40.98 μmol/L，nutlin3对类器官生长无明显影响。结论 成功建立了1例携带TP53和ERBB2基因突变的肝细胞癌类器官，该类器官保留了原代肿瘤组织的结构和分子特征，药物筛选结果可能为肝细胞癌患者的个体化治疗提供参考。 |
| 关键词: 肿瘤蛋白p53 erb-b2受体酪氨酸激酶2 肝细胞癌 类器官 药物筛选 |
| DOI：10.16781/j.CN31-2187/R.20211266 |
| 投稿时间：2021-12-15修订日期：2022-03-28 |
| 基金项目:国家自然科学基金（82073411）. |
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| Culture and drug sensitivity identification of a hepatocellular carcinoma organoid with TP53 and ERBB2 mutations |
| LIU Er-dong1,HOU Ming-hui2,WANG Xiang3,ZONG Ya-li1,WANG Hong-yang1,4*,FU Jing4* |
(1. School of Life Sciences and Institute of Metabolism and Integrative Biology, Fudan University, Shanghai 200438, China;
2. Laboratory of Organoids, Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China;
3. Department of Biliary Surgery(Ⅱ), The Third Affiliated Hospital of Naval Medical University (Second Military Medical University), Shanghai 200438, China;
4. National Center for Liver Cancer, Naval Medical University (Second Military Medical University), Shanghai 201805, China
*Corresponding authors) |
| Abstract: |
| Objective To explore the culture of a hepatocellular carcinoma organoid for drug screening, providing clinical reference for individualized treatment. Methods The tumor tissue from a hepatocellular carcinoma patient was digested into single cells and inoculated into Matrigel for culture. The cell morphology of the organoid and primary tumor tissue was observed by hematoxylin-eosin (H-E) staining. Immunohistochemistry staining (IHC) was used to verify the expression of cytokeratin 7 (CK7), cytokeratin 8 (CK8), p53, and glypican 3 (GPC-3) in the organoid and primary tumor tissue; the mutations of tumor-related genes in the organoid were detected by targeted sequencing. Drug screening was carried out according to the targeted sequencing results. The effects of varlitinib (a specific inhibitor of epidermal growth factor receptor/erb-b2 receptor tyrosine kinase 2[ERBB2]), sorafenib (a multi-targeted inhibitor of kinase), cisplatin and nutlin3 (a p53 activator) on the organoid viability were detected by CellTiter-Glo luminescent cell viability assay. Results The hepatocellular carcinoma organoid with tumor protein p53 (TP53) and ERBB2 gene mutations was successfully established.H-E staining showed that the structural and cytological characteristics were very similar between the organoid and primary tumor tissue. IHC staining showed that CK7 was negative in the organoid and primary tumor tissue, while CK8, p53, and GPC-3 were positive. Targeted sequencing results showed that the mutation genes in the organoid were TP53 (E271K, 100%), ERBB2 (A1232V, 62.29%), RUNX family transcription factor 1 (RUNX1) (E395A, 20.21%), and androgen receptor (AR) (Q91dup, 70%). The results of the cell viability assay showed that the half inhibitory concentration (IC50) values of varlitinib and sorafenib on the organoid were 2.81 μmol/L and 1.327 μmol/L, respectively; the IC50 of cisplatin was 40.98 μmol/L; while nutlin3 had no significant effect on the organoid. Conclusion We have successfully established a hepatocellular carcinoma organoid with TP53 and ERBB2 mutations. This organoid retains the structural and molecular characteristics of the primary tumor tissue. The results of drug screening may provide a reference for the individualized treatment. |
| Key words: tumor protein p53 erb-b2 receptor tyrosine kinase 2 hepatocellular carcinoma organoid drug screening |
