Abstract 2589: Folate receptor α confers colonization advantage of cholangiocarcinoma in peritoneal microenvironment with therapeutic vulnerability

Abstract Purpose: Peritoneal metastasis/recurrence results in poor prognosis and failure of radical resection in cholangiocarcinoma (CCA) patients, but the molecular mechanisms and potential therapy strategies remain an enigma. Methods: Freshly resected primary lesions and peritoneal metastases from CCA patients were collected for single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics to identify the crucial target during peritoneal colonization of CCA cells. Tumor-adipocytes in vitro co-culture model and tumor peritoneal implantation mouse model were utilized to investigate the role and therapeutic promises of the identified target in peritoneal metastasis of CCA. Metabolomics, metabolic flux, and flow cytometry assays were employed to delve into the underlying molecular mechanisms. Results: The scRNA-seq analysis of CCA tumors (N=5 pairs) revealed an up-regulated expression of FOLR1, encoding folate receptor alpha (FRα), in CCA cells after colonization in the peritoneal microenvironment, which was validated by spatial transcriptomics (N=2 pairs). The specifical overexpression of FRα in CCA cells in the peritoneal metastases was validated by immunohistochemistry and multiplex immunofluorescence staining, and it was associated with a poorer prognosis, larger metastases size, and increased ascites volume in CCA patients (N=68). Despite no significant impact on the proliferative and invasive capacities of CCA cells in conventional culturing, overexpression of FRα enhanced their proliferation in the tumor-adipocytes co-culture model and tumor peritoneal implantation mouse model. Mechanistically, utilization of enriched fatty acids from adipocytes for fatty acid oxidation fuels CCA cells outgrowth and colonization in peritoneal metastases, but it also leads to excessive oxidative stress. FRα-dependent folate metabolism and one-carbon metabolism contribute to production of NADPH and GSH, the essential substrates for cellular anti-oxidative stress, thus leading to growth advantage of CCA cells in the peritoneal microenvironment. Herein, the utilization of Mirvetuximab Soravtansine, the antibody-drug conjugate targeting FRα, demonstrated an encouraging therapeutic efficacy in CCA peritoneal implantation mouse models, including reduced tumor size, decreased number of disseminated lesions, lowered ascites volume and ascites CTCs, prolonged mouse survival, and delayed recurrence of peritoneal metastases after peritoneal metastasectomy. Conclusions: Hence, FRα plays a pivotal role in facilitating the colonization of CCA in the peritoneal microenvironment, rendering it a promising therapeutic target for alleviating peritoneal metastases burden in advanced CCA patients. The potential therapeutic advantages of Mirvetuximab Soravtansine warrant preliminary investigation in our planned clinical trial. Citation Format: Honghua Zhang, Linwei Guo, Shusheng Lin, Xiuxian Li, Zhixiao Song, Leibo Xu, Chao Liu. Folate receptor α confers colonization advantage of cholangiocarcinoma in peritoneal microenvironment with therapeutic vulnerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2589.