Wang Zhiwei, Yu Yuanquan, Wu Peiyao, Ye Qinghuang, Guo Yinghao, Zhang Xiaoxiao, Xi Longfu, Li Qi, Jin Yun, Zhou Donger, Luo Yan, Peng Shuyou, Li Jiangtao
Department of Surgery, the Second Affiliated Hospital, Zhejiang University School of Medicine, 310009, Hangzhou, Zhejiang Province, China.
Gastroenterology Endoscopy Center, Jiangsu Province Hospital of Chinese Medicine, Affiliated Hospital of Nanjing University of Chinese Medicine, 210029, Nanjing, Jiangsu Province, China.
Cell Death Discov. 2022 Apr 20;8(1):214. doi: 10.1038/s41420-022-01014-4.
The long culture duration of patient-derived organoids (PDOs) have severely limited their clinical applications. The aim of this study was to determine the effect of lactate supplementation on the growth, genetic profiles and drug sensitivities of PDOs from hepatopancreatobiliary tumors. LM3, Huh7, Panc02, and RBE cell lines were cultured as organoids in the presence or absence of lactate, and total protein was extracted to measure the expression of α-enolase (ENO1), hypoxia-inducible factor-1α (HIF1α), AKT, and PI3 kinase (PI3K). Thirteen hepatopancreatobiliary tumor specimens were collected during surgical resection and cultured as PDOs with or without L-lactate. Hematoxylin and eosin (H&E) staining and immunohistochemical staining were performed on the original tissues and PDOs to compare their pathological structures, and their genetic profiles were analyzed by whole-exome sequencing (WES). The sensitivity of the PDOs to gemcitabine, 5-fluorouracil, cisplatin, paclitaxel, ivosidenib, infigratinib, and lenvatinib were evaluated in terms of cell viability. Peripheral blood mononuclear cells (PBMCs) were isolated and co-cultured with PDOs to test the sensitivity of PDOs to tislelizumab. The addition of 20 mM lactate significantly promoted the growth of LM3 and Huh 7 organoids by 217% and 36%, respectively, compared to the control group, and the inhibition of lactate transporter decreased their growth. The HIF1α/ENO1/AKT/PI3K pathway was also activated by lactate. The inhibition of enolase also partly decreased the growth of organoids treated with lactate. Furthermore, 20 mM lactate increased the viability of 9 PDOs from 135% to 317% without affecting their pathological features. The genetic similarity, in terms of single nucleotide variations, insertions, and deletions, between original tissues and lactate-treated PDOs ranged from 83.2% to 94.1%, and that between the untreated and lactate-treated PDOs was at least 93.2%. Furthermore, the addition of lactate did not significantly change the dose-response curves of the PDOs to chemotherapeutic drugs, targeted drugs, and immune checkpoint inhibitor, especially for the drugs to which the cells were sensitive. Thus, lactate can be added to the culture medium of PDOs to promote their growth without altering their genetic profiles and drug sensitivities.
患者来源的类器官（PDO）培养时间长，严重限制了其临床应用。本研究旨在确定补充乳酸对肝胰胆肿瘤来源的PDO的生长、基因图谱和药物敏感性的影响。将LM3、Huh7、Panc02和RBE细胞系在有或无乳酸的情况下培养成类器官，提取总蛋白以检测α-烯醇化酶（ENO1）、缺氧诱导因子-1α（HIF1α）、AKT和PI3激酶（PI3K）的表达。在手术切除过程中收集13例肝胰胆肿瘤标本，将其培养成添加或不添加L-乳酸的PDO。对原始组织和PDO进行苏木精和伊红（H&E）染色及免疫组化染色，以比较其病理结构，并通过全外显子测序（WES）分析其基因图谱。从细胞活力方面评估PDO对吉西他滨、5-氟尿嘧啶、顺铂、紫杉醇、艾伏尼布、英菲格拉替尼和仑伐替尼的敏感性。分离外周血单个核细胞（PBMC）并与PDO共培养，以测试PDO对替雷利珠单抗的敏感性。与对照组相比，添加20 mM乳酸分别显著促进了LM3和Huh 7类器官生长217%和36%，抑制乳酸转运蛋白则降低了它们的生长。乳酸还激活了HIF1α/ENO1/AKT/PI3K通路。抑制烯醇化酶也部分降低了用乳酸处理的类器官的生长。此外，20 mM乳酸使13个PDO中的9个的活力从135%提高到317%，而不影响其病理特征。原始组织与乳酸处理的PDO之间在单核苷酸变异、插入和缺失方面的基因相似性在83.2%至94.1%之间，未处理与乳酸处理的PDO之间的基因相似性至少为93.2%。此外，添加乳酸并未显著改变PDO对化疗药物、靶向药物和免疫检查点抑制剂的剂量反应曲线，尤其是对细胞敏感的药物。因此，可以在PDO的培养基中添加乳酸以促进其生长，而不改变其基因图谱和药物敏感性。
