Abstract 2568: Cancer-associated fibroblasts-derived periostin promotes lymph node metastasis of cholangiocarcinoma through lymphatic endothelial reprogramming

Abstract Purpose: Cholangiocarcinoma (CCA) is a bile duct malignancy with poor outcomes, especially in patients with lymph node metastasis (LNM). However, the mechanism of LNM in CCA as well as its therapeutic options remain undefined. Understanding crosstalk between the cellular components in the complex tumor microenvironment (TME) of CCA, and how it influence LNM, may help identifying novel therapeutic targets in LNM of CCA. Method: Freshly resected tumors from 10 CCA patients were collected for single-cell RNA sequencing (scRNA-seq), aiming to identify the differences between CCA with or without LNM. Immunohistochemistry (IHC) and enzyme-linked immune sorbent assay (ELISA) were applied to validate the identified targets. In vivo study was conducted using foot pad-popliteal LNM mouse model to find out main factors affect LNM. Co-culture system was established to interrogate cellular crosstalk between human lymphatic endothelial cells (HLECs) and patient-derived CAFs in TME of CCA. Other phenotypic and mechanistic experiments include tube formation assay, trans-endothelial migration assay, cell adhesion assay, pull down assay, etc. Results: Our scRNA-seq revealed the enrichment of a distinguish subtype of CAFs that express periostin (POSTN) in CCA tumors with LNM. IHC and immunofluorescence staining in CCA sections validated that the CAF-specific expressing protein, POSTN, expresses significantly higher in CCA with LNM, which is also associated with higher circulating tumor cells (CTCs), and worse prognosis. In vivo study shows that CAFs highly expressing POSTN (POSTN+CAFs) lead to higher incidence of LNM. In vitro study indicates that either with co-culture of POSTN+CAFs or treatment of recombinant human POSTN (rhPOSTN), HLECs developed increased capacity of tube formation and migration, while CCA cells also show increased capacity of migration in trans-endothelial assay but not traditional migration assay. Meanwhile, POSTN also impairs integrity of lymphatic vessels, providing routes for CCA cells to disseminate. Pull down assay identifies the binding of POSTN to its receptor, αVβ3 integrin, on HLECs. We demonstrate that the effects of POSTN aforementioned can be abrogated by knockdown of ITGB3 in HLECs, suggesting that POSTN exerts its effects in an αVβ3-dependent manner. We further show that POSTN’s effects are attributed to the activation of integrin-FAK/Src pathway. Moreover, in vivo application of Cilengitide, an αVβ3/5 inhibitor, decreases the incidence of LNM in CCA, indicating its therapeutic efficacy in mitigating LNM burden in CCA patients with higher expression of POSTN. Conclusion: This study uncovers the novel role of CAF-derived POSTN in promoting LNM of CCA, presenting the potential therapeutic promise of the FDA-approved αVβ3/5 inhibitor, Cilengitide, in inhibiting LNM in CCA. Citation Format: Zhixiao Song, Xiuxian Li, Honghua Zhang, Chao Liu. Cancer-associated fibroblasts-derived periostin promotes lymph node metastasis of cholangiocarcinoma through lymphatic endothelial reprogramming [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 2568.