[Establishment and preliminary application of organoids in ovarian cancer].

Objective: To explore the establishment and application of ovarian cancer organoids. Methods: Fresh ovarian tumor tissues, obtaining from patients underwent surgery in the First Affiliated Hospital of Nanjing Medical University between October 2021 and March 2022, were collected, enzymatic degraded, digested, and embedded into matrigel to establish organoids. A total of 32 ovarian cancer samples were collected. Hematoxylin eosin (HE) staining and immunofluorescence (IF) procedure were used to verify the morphological structure of organoids and their expression of molecular markers. 3D cyto-live or dead assay was used to detecte the live or dead cells in organoids. Carboplatin with a concentration ranging from 5 to 80 μmol/L (5, 10, 20, 40, 80 μmol/L) was added to organoids to calculate the 50% inhibitory concentration (IC50) in different organoids. Results: (1) Organoids from a total of 32 patients were established, of which 18 cases could be passaged stably in the long term in vitro, while 14 could be passaged in the short time. The average amplification time of long-term passage in vitro was over 3 months, and the longest reached 9 months. (2) In HE staining, significant nuclei atypia and local micropapillary structures were observed in organoids. IF staining revealed that ovarian cancer organoids expressed molecular markers similar to primary tumor tissues, such as Pan cytokeratin (Pan-CK), p53, paired box gene 8 (PAX8), and Wilms tumor gene 1 (WT1). (3) In 3D cyto-live or dead assay, a large number of apoptotic cells were observed inside and around the organoids after added carboplatin. The sensitivity to carboplatin varied in 18 organoids could amplify in the long term, with an average IC50 of (29.5±15.8) μmol/L. Moreover, IC50 values of 4 organoids derived from patients received neoadjuvant chemotherapy were much higher than the 14 organoids which did not received neoadjuvant chemotherapy [(48.7±11.3) μmol/L vs (24.0±12.1) μmol/L; t=3.429, P=0.022]. Conclusions: Organoids recapitulate ovarian cancers in vitro and could be stably passaged. Organoids derived from patients received neoadjuvant chemotherapy have higher resistance to carboplatin.目的： 探索卵巢上皮性癌（卵巢癌）类器官的建立方法及初步应用。 方法： 收集2021年10月至2022年3月于南京医科大学第一附属医院行手术治疗的32例卵巢癌患者的新鲜癌组织标本，通过组织酶解、消化、基质凝胶包埋等流程建立卵巢癌类器官。采用HE染色与免疫荧光染色验证卵巢癌类器官的形态结构与分子标志物的表达，3D类器官细胞活性染色法检测卵巢癌类器官细胞的活性状态。在卵巢癌类器官培养液中分别加入不同浓度（5、10、20、40、80 μmol/L）的卡铂，计算卵巢癌类器官对卡铂的50%抑制浓度（IC50）。 结果： （1）32份卵巢癌组织标本中，18份在体外产生可稳定传代的类器官，体外稳定扩增时间均>3个月，最长者达9个月；14份可短期传代的类器官，连续扩增时间均<2个月。（2）HE染色显示，卵巢癌类器官的细胞核呈明显的异型性，且局部可形成微乳头状结构。免疫荧光染色显示，卵巢癌类器官表达与原代卵巢癌相似的分子标志物，如广谱细胞角蛋白（Pan-CK）、p53、配对盒基因8（PAX8）、Wilms瘤基因1（WT1）。（3）3D类器官细胞活性染色法检测显示，卵巢癌类器官内部及类器官的周围出现大量凋亡细胞。在体外可稳定扩增的18份类器官对卡铂的IC50为（29.5±15.8）μmol/L；其中，接受了新辅助化疗后生成的4份类器官对卡铂的IC50为（48.7±11.3）μmol/L，高于14份未接收新辅助化疗者［（24.0±12.1）μmol/L］，两者比较，差异有统计学意义（t=3.429，P=0.022）。 结论： 卵巢癌类器官能够在体外模拟卵巢癌的特征，并且能够稳定传代，新辅助化疗后卵巢癌组织产生的类器官对卡铂具有更高的耐药性。.