PO.TB04.01 · 肿瘤生物学
建立腹水来源的类器官模型用于卵巢癌的转录组学和治疗学研究
Developing an ascitic fluid-derived organoid model for transcriptomic and therapeutic investigations in ovarian cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言：上皮性卵巢癌（EOC）在腹膜腔内自由扩散，通过淋巴管阻塞引起肿瘤播散、腹腔器官粘连和腹水形成。由于减瘤手术后实体瘤样本稀缺，腹水为疾病演变提供了关键窗口。其丰富的细胞和非细胞成分助长肿瘤存活、免疫逃逸和化疗耐药，但在临床前模型中仍未得到充分利用。因此，我们旨在建立并表征一个腹水来源的类器官（AsO）平台，该平台能够捕捉这种异质性肿瘤微环境（TME）并改善药物反应分析。
方法：在获批的IRB方案下，从知情同意的西班牙裔患者收集恶性腹水和肿瘤组织。腹水经离心并反复裂解以去除红细胞。全腹水细胞（WhAs）被分为类器官培养和生物样本库两部分。部分WhAs被包埋于细胞外基质中，并覆盖优化的类器官培养基。类器官每8-13天传代一次，并在冷冻培养基中冷冻保存。我们用明场成像记录形态学，用H&E染色FFPE切片，并对整装类器官进行免疫染色。对匹配的原发肿瘤（WhTu）、WhAs和AsO进行了bulk RNA-seq，我们使用标准生物信息学流程量化表达，随后进行相关性和通路分析。类器官中的药物敏感性通过活/死细胞检测（CC50）和发光检测（IC50）测量。
结果：类器官培养基优化显著改善了类器官形成、传代效率和长期扩增。一项重大成就是成功冷冻保存了WhAs和AsO，二者在复苏后几乎在所有情况下均恢复生长。H&E染色揭示了核异型性，这是EOC的标志，免疫荧光证实了包括PAX8、p53、CK8和Ki67在内的关键生物标志物的表达。Pearson相关系数表明AsO与WhAs之间存在强烈的整体转录相似性（R2 = 0.87）。为鉴定卵巢癌（OC）TME内腹水特异性基因，我们还考察了AsO与WhTu之间的转录差异（DEG）。将来自AsO的前50个高表达基因与来自Gene Expression Omnibus（GEO）的OC肿瘤来源类器官数据集进行比较时，我们鉴定出22个重叠基因。已观察到患者对卡铂（Carboplatin）、紫杉醇（Paclitaxel）、仑伐替尼（Lenvatinib）和Saruparib的特异性药物反应。
结论：在组织学、蛋白标志物和转录组学水平上对腹水来源类器官的全面表征，以及跨多个实验平台的系统性药物敏感性比较，将显著增强其临床和转化相关性。
查看英文原文 English abstract
Introduction: Epithelial ovarian cancer (EOC) spreads freely within the intraperitoneal cavity, enabling tumor dissemination, abdominal organ adhesion, and ascites formation through lymphatic obstruction. Because solid-tumor samples are scarce after debulking surgery, ascites provide a crucial window into disease evolution. Its rich cellular and acellular components fuel tumor survival, immune evasion, and chemoresistance but remain underused in preclinical models. We therefore aim to establish and characterize an ascites-derived organoid (AsO) platform that captures this heterogeneous tumor microenvironment (TME) and improves drug-response profiling.
Methods: Malignant ascites and tumor tissues were collected from consenting Hispanic patients under an approved IRB protocol. Ascitic fluid was centrifuged and repeatedly lysed to remove red blood cells. The whole ascitic cells (WhAs) were divided for organoid culture and biobanking. Part of WhAs was embedded in extracellular matrix and overlaid with optimized organoid culture media. Organoids were passaged every 8-13 days and cryopreserved in freezing media. We documented morphology with brightfield imaging, stained FFPE sections with H&E, and immuno-stained whole-mount organoids. Bulk RNA-seq was performed on matched primary tumors (WhTu), WhAs and AsO, and we quantified expression using standard bioinformatics pipelines followed by correlation and pathway analyses. Drug sensitivity in organoids was measured by live/dead assay (CC50) and luminescence assay (IC50).
Results: Organoid culture media optimization markedly improved organoid formation, passaging efficiency, and long-term expansion. A major achievement was the successful cryopreservation of both WhAs and AsO, which resumed growth after resuscitation in nearly all cases. H&E staining revealed nuclear atypia, a hallmark of EOC, and immunofluorescence confirmed expression of key biomarkers including PAX8, p53, CK8, and Ki67. Pearson correlation coefficients demonstrated strong global transcriptional similarity (R 2 = 0.87) between AsO and WhAs. To identify ascites-specific genes within the ovarian cancer (OC) TME, we also examined transcriptional differences (DEGs) between AsO and WhTu. We identified 22 overlapping genes when comparing between the top 50 highly expressed genes from AsO and OC tumor-derived organoid datasets from Gene Expression Omnibus (GEO). Patient-specific drug responses to Carboplatin, Paclitaxel, Lenvatinib, and Saruparib have been observed.
Conclusion: The comprehensive characterization of ascites-derived organoids at histological, protein marker, and transcriptomic levels, along with systematic drug sensitivity comparisons across multiple experimental platforms, will significantly enhance their clinical and translational relevance.
利益披露 Disclosure
M. Hassan, None..
Z. Azam, None..
T. Al-Hilal, None..
S. Roy, None.