微乳头型肺腺癌类器官的构建及其靶向药物的筛选

doi:10.11958/20231326

天津医药 ›› 2024, Vol. 52 ›› Issue (1): 22-27.doi: 10.11958/20231326

• 专题研究·类器官与器官芯片（主编·陈怀永） • 上一篇下一篇

微乳头型肺腺癌类器官的构建及其靶向药物的筛选

姜忠敏¹^,²(), 张春艳²^,³^,⁴, 刘敏¹^,², 郑洁¹^,², 李艳霞²^,⁴, 仁青措⁴^,⁵, 孟纬⁴^,⁶, 刘晓智²^,⁴^,^△()

1.天津市第五中心医院病理科（邮编300450）
2.天津市早产儿器官发育表观遗传重点实验室
3.天津市第五中心医院普通外科
4.青海黄南藏族自治州人民医院高原特色医学研究所
5.青海黄南藏族自治州人民医院高原特色医学研究所传染科
6.青海黄南藏族自治州人民医院高原特色医学研究所骨科

收稿日期:2023-10-27 出版日期:2024-01-15 发布日期:2024-01-18
通讯作者: ^△E-mail：lxz7997@126.com
作者简介:姜忠敏（1979），女，副主任医师，主要从事临床肿瘤病理诊断方面研究。E-mail：jzm79913@163.com
基金资助:
天津市科技计划项目(22ZYQYSY00030);天津市自然科学基金资助项目(21JCZDJC01270);天津市卫生健康科技项目(TJWJ2022XK043);天津市卫生健康科技项目(TJWJ2023MS053);天津市卫生健康科技项目(TJWJ2023QN103);天津市卫生健康委(天津市中医药管理局)中医中西医结合科研课题(2021061);天津市医学重点学科（专科）建设项目(TJYXZDXK-062B);天津市医学重点学科（专科）建设项目(TJYXZDXK-079D);天津援建同仁市民生科技项目;天津市卫生健康委员会中医中西医结合科研课题(2023062)

Construction of micropapillary lung adenocarcinoma organoids and screening of targeted drugs

JIANG Zhongmin¹^,²(), ZHANG Chunyan²^,³^,⁴, LIU Min¹^,², ZHENG Jie¹^,², LI Yanxia²^,⁴, REN Qingcuo⁴^,⁵, MENG Wei⁴^,⁶, LIU Xiaozhi²^,⁴^,^△()

1. Department of Pathology, Tianjin Fifth Central Hospital, Tianjin 300450, China
2. Tianjin Key Laboratory of Epigenetics of Organ Development of Premature Infants
3. General Surgery, Tianjin Fifth Central Hospital
4. High Altitude Characteristic Medical Research Institute, Huangnan Tibetan Autonomous Prefecture People's Hospital
5. Department of Infectious Diseases, Huangnan Tibetan Autonomous Prefecture People's Hospital
6. Department of Orthopedics, Huangnan Tibetan Autonomous Prefecture People's Hospital

Received:2023-10-27 Published:2024-01-15 Online:2024-01-18
Contact: ^△E-mail：lxz7997@126.com

姜忠敏, 张春艳, 刘敏, 郑洁, 李艳霞, 仁青措, 孟纬, 刘晓智. 微乳头型肺腺癌类器官的构建及其靶向药物的筛选[J]. 天津医药, 2024, 52(1): 22-27.

JIANG Zhongmin, ZHANG Chunyan, LIU Min, ZHENG Jie, LI Yanxia, REN Qingcuo, MENG Wei, LIU Xiaozhi. Construction of micropapillary lung adenocarcinoma organoids and screening of targeted drugs[J]. Tianjin Medical Journal, 2024, 52(1): 22-27.

摘要/Abstract

摘要：

目的建立微乳头型肺腺癌类器官的培养方法，并开展靶向药物的筛选。方法自确诊为微乳头型肺腺癌患者手术组织样本中提取和培养原代肺癌类器官，动态观察和记录肺癌类器官生长情况；苏木精-伊红（HE）染色法及免疫组化染色法比较肺癌类器官与亲本组织间肿瘤细胞形态及蛋白表达特征；实时荧光定量聚核酶链反应检测肺癌亲本组织和类器官中基因突变情况；基于基因检测结果挑选靶向药物并验证其体外抑瘤效果。结果成功从微乳头型肺腺癌组织中培养出类球形肿瘤类器官，可传代至少3代。HE染色结果可见类器官中肿瘤细胞形态与亲本组织细胞基本一致；免疫组化染色结果显示肺癌类器官与亲本组织中各基因的蛋白表达水平大致相同；基因突变分析结果显示，肺癌亲本组织和类器官的突变基因结果一致，均体现为原癌基因酪氨酸蛋白激酶受体Ret（RET）融合突变。基于肺癌类器官的靶向药物筛选结果显示，凡德他尼的体外抑瘤效果最佳。结论基于微乳头型肺腺癌类器官的药筛实验可在短时间内筛选出高效靶向药物，可使微乳头型肺腺癌患者从中获益。

关键词: 肺腺癌, 类器官, 药物筛选试验, 抗肿瘤, 靶向制剂, 凡德他尼

Abstract:

Objective To establish a culture method for micropapillary lung adenocarcinoma organoids and conduct targeted drug screening. Methods Organoids were extracted and cultured from a surgical tissue sample of a patient diagnosed with micropapillary lung adenocarcinoma, and the growth of lung cancer organoids was observed and recorded dynamically. The morphological and gene expression characteristics of tumor cells between lung cancer organoids and parental tissue were compared using hematoxylin eosin (HE) staining and immunohistochemical methods. Real time fluorescence quantitative polynucleotide chain reaction (qRT-PCR) method was used to detect gene mutations in lung cancer parental tissue and organoids. Finally, based on results of genetic testing, targeted drugs were selected and their therapeutic effects were verified. Results We have successfully cultured spherical organoids from micropapillary lung adenocarcinoma tissue, which can be passaged for at least 3 generations. HE staining results showed that the morphology of tumor cells in organoids was roughly consistent with that of parental tissue. The immunohistochemical results showed that the protein expression levels of various genes in lung cancer organoids and parental tissue were roughly the same. Results of gene mutation analysis showed that the mutated genes in lung cancer parental tissue and organoids were consistent, both reflecting RET fusion. The screening results of targeted drugs based on lung cancer organoids showed that vandertinib had the best anti-tumor effect in vitro. Conclusion Drug screening experiments based on micropapillary lung adenocarcinoma organoids can screen highly efficient targeted drugs in a short period of time, which may benefit patients with micropapillary lung adenocarcinoma.

Key words: adenocarcinoma of lung, organoids, drug screening assays, antitumor, targeting preparation, vandetani

中图分类号:

R734.2

图/表 5

图1 微乳头型肺腺癌类器官的培养

Fig.1 Culture of micropapillary lung adenocarcinoid organoid

图2 HE染色及免疫组化实验鉴定微乳头型肺腺癌类器官的表型

Fig.2 HE staining and immunohistochemistry experiments identify the phenotype of micropapillary lung adenocarcinoma organoid

表1 免疫组化结果评分分析

Tab.1 Analysis of immunohistochemical result score

抗体	肿瘤亲本组织			类器官
抗体	阳性细胞占比评分	染色强度评分	总评分	阳性细胞占比评分	染色强度评分	总评分
TTF-1	3	3	9（高表达）	3	2	6（高表达）
CK7	4	3	12（高表达）	4	3	12（高表达）
Napsin A	4	2	8（高表达）	4	3	12（高表达）
Ki67	1	3	3（低表达）	2	3	6（高表达）

图3 肿瘤亲本组织和类器官RET融合基因检测结果

Fig.3 The detection results of RET fusion genes in parental tissue and its organoid

图4 受试靶向药物IC50检测结果

Fig.4 IC50 results of target drugs

参考文献 26

[1]	WANG W, HU Z, ZHAO J, et al. Both the presence of a micropapillary component and the micropapillary predominant subtype predict poor prognosis after lung adenocarcinoma resection:a meta-analysis[J]. J Cardiothorac Surg, 2020, 15(1):154. doi:10.1186/s13019-020-01199-8.
[2]	ZHAO Z R, TO K F, MOK T S, et al. Is there significance in identification of non-predominant micropapillary or solid components in early-stage lung adenocarcinoma?[J]. Interact Cardiovasc Thorac Surg, 2017, 24(1):121-125. doi:10.1093/icvts/ivw283.
[3]	YAN H H N, CHAN A S, LAI F P, et al. Organoid cultures for cancer modeling[J]. Cell Stem Cell, 2023, 30(7):917-937. doi:10.1016/j.stem.2023.05.012.
[4]	VENINGA V, VOEST E E. Tumor organoids:Opportunities and challenges to guide precision medicine[J]. Cancer Cell, 2021, 39(9):1190-1201. doi:10.1016/j.ccell.2021.07.020.
[5]	TRAVIS W D, BRAMBILLA E, RIELY G J. New pathologic classification of lung cancer:relevance for clinical practice and clinical trials[J]. J Clin Oncol, 2013, 31(8):992-1001. doi:10.1200/JCO.2012.46.9270.
[6]	BEASLEY M B, DEMBITZER F R, FLORES R M. Surgical pathology of early stage non-small cell lung carcinoma[J]. Ann Transl Med, 2016, 4(12):238. doi:10.21037/atm.2016.06.13.
[7]	CAO Y, ZHU L Z, JIANG M J, et al. Clinical impacts of a micropapillary pattern in lung adenocarcinoma:a review[J]. Onco Targets Ther, 2016, 9:149-158. doi:10.2147/OTT.S94747.
[8]	TRUINI A, SANTOS PEREIRA P, CAVAZZA A, et al. Classification of different patterns of pulmonary adenocarcinomas[J]. Expert Rev Respir Med, 2015, 9(5):571-586. doi:10.1586/17476348.2015.1083428.
[9]	MORALES-OYARVIDE V, MINO-KENUDSON M. High-grade lung adenocarcinomas with micropapillary and/or solid patterns:a review[J]. Curr Opin Pulm Med, 2014, 20(4):317-323. doi:10.1097/MCP.0000000000000070.
[10]	SUH J H. Current readings:pathology,prognosis,and lung cancer.[J]. Semin Thorac Cardiovasc Surg, 2013, 25(1):14-21. doi:10.1053/j.semtcvs.2013.01.003.
[11]	LESAVAGE B L, SUHAR R A, BROGUIERE N, et al. Next-generation cancer organoids[J]. Nat Mater, 2022, 21(2):143-159. doi:10.1038/s41563-021-01057-5.
[12]	KIM J, KOO B K, KNOBLICH J A. Human organoids:model systems for human biology and medicine[J]. Nat Rev Mol Cell Biol, 2020, 21(10):571-584. doi:10.1038/s41580-020-0259-3.
[13]	PORCEL J M. Biomarkers in the diagnosis of pleural diseases:a 2018 update[J]. Ther Adv Respir Dis, 2018, 12:1753466618808660. doi:10.1177/1753466618808660.
[14]	LIAN H, PAN X, HONG B, et al. Metastases to the kidney from primary lung cancer :clinicopathological analysis of six cases in a single center[J]. Diagn Pathol, 2023, 18(1):60. doi:10.1186/s13000-023-01344-6.
[15]	WANG M, CHEN P P, CAI G. GATA3 expression in primary lung carcinomas:correlation with histopathologic features and TTF-1,napsin A,and p40 expression[J]. Hum Pathol, 2023, 135:93-98. doi:10.1016/j.humpath.2023.01.007.
[16]	DUTU T, MICHIELS S, FOURET P, et al. Differential expression of biomarkers in lung adenocarcinoma:a comparative study between smokers and never-smokers[J]. Ann Oncol, 2005, 16(12):1906-1914. doi:10.1093/annonc/mdi408.
[17]	NAGASAKA M, BRAZEL D, BACA Y, et al. Pan-tumor survey of RET fusions as detected by next-generation RNA sequencing identified RET fusion positive colorectal carcinoma as a unique molecular subset[J]. Transl Oncol, 2023, 36:101744. doi:10.1016/j.tranon.2023.101744.
[18]	PECCIARINI L, BRUNETTO E, GRASSINI G, et al. Gene fusion detection in nsclc routine clinical practice:Targeted-NGS or FISH?[J]. Cells, 2023, 12(8):1135. doi:10.3390/cells12081135.
[19]	TAN A C, SEET AMAND A O L, LAI G G Y, et al. Molecular characterization and clinical outcomes in RET-rearranged NSCLC[J]. J Thorac Oncol, 2020, 15(12):1928-1934. doi:10.1016/j.jtho.2020.08.011.
[20]	潘放, 任庆兰. 酪氨酸激酶ALK、ROS1、RET融合基因与肺癌临床特征的关系分析[J]. 实用癌症杂志, 2015, 30(7):958-960,966.
	PAN F, REN Q L. Relation between tyrosine kinase ALK,ROS1,RET fusion gene and clinical features of lung cancer[J]. J Pract Cancer, 2015, 30(7):958-960,966. doi:10.3969/j.issn.1001-5930.2015.07.003.
[21]	李小雪, 史可鉴, 姚舒洋, 等. RET基因融合非小细胞肺癌的治疗进展[J]. 肿瘤研究与临床, 2022, 34(7):547-550.
	LI X X, SHI K J, YAO S Y, et al. Treatment progress of non-small cell lung cancer with RET gene fusion[J]. Cancer Res Clinic, 2022, 34(7):547-550. doi:10.3760/cma.j.cn115355-20211210-00544.
[22]	DUKE E S, BRADFORD D, MARCOVITZ M, et al. FDA approval summary:Selpercatinib for the treatment of advanced RET fusion-positive solid tumors[J]. Clin Cancer Res, 2023, 29(18):3573-3578. doi:10.1158/1078-0432.CCR-23-0459.
[23]	NOKIHARA H, NISHIO M, YAMAMOTO N, et al. Phase 1 study of cabozantinib in Japanese patients with expansion cohorts in non-small-cell lung cancer[J]. Clin Lung Cancer, 2019, 20(3):e317-e328. doi:10.1016/j.cllc.2018.12.018.
[24]	WANG Y, XU Y, WANG X, et al. RET fusion in advanced non-small-cell lung cancer and response to cabozantinib:a case report[J]. Medicine(Baltimore), 2019, 98(3):e14120. doi:10.1097/MD.0000000000014120.
[25]	YOH K, SETO T, SATOUCHI M, et al. Vandetanib in patients with previously treated RET-rearranged advanced non-small-cell lung cancer(LURET):an open-label,multicentre phase 2 trial[J]. Lancet Respir Med, 2017, 5(1):42-50. doi:10.1016/S2213-2600(16)30322-8.
[26]	CHEN J Y, XU C W, LV J W, et al. Clinical characteristics and targeted therapy of different gene fusions in non-small cell lung cancer:a narrative review[J]. Transl Lung Cancer Res, 2023, 12(4):895-908. doi:10.21037/tlcr-22-566.

微乳头型肺腺癌类器官的构建及其靶向药物的筛选

Construction of micropapillary lung adenocarcinoma organoids and screening of targeted drugs

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