Ficolin‐3 Activates Complement and Triggers Necroptosis in Cholangiocarcinoma Cells via the <scp>RIPK1</scp> / <scp>RIPK3</scp> / <scp>MLKL</scp> Signaling Pathway

Ficolin 3 (FCN3) is a pattern recognition molecule that activates the complement system via the lectin pathway. While its immunological roles are known, the specific mechanisms by which FCN3 affects cholangiocarcinoma (CCA) pathogenesis remain unclear. In this study, we investigated FCN3 expression in CCA and benign cells, as well as tumor versus non-tumor tissues, using RT-qPCR and Western blotting analyses. The effects of FCN3 on CCA cell proliferation, migration, and invasion were analyzed through CCK-8, EdU, transwell, and wound-healing assays, with in vivo studies supporting these findings. The complement-mediated cytotoxicity of CCA cells was assessed using human serum with or without heat inactivation and an anti-C6 blocking antibody. Immunocytochemical staining was used to examine membrane attack complex (MAC) deposition, and an immunoprecipitation assay was adopted to evaluate the interaction between FCN3 and MASP family members. The role of FCN3 in inducing necroptosis was explored through transmission electron microscopy (TEM) and Western blotting analysis, focusing on the RIPK1/RIPK3/MLKL pathway. The results of the study demonstrate that FCN3 expression was significantly lower in CCA cells and tissues. Overexpressing FCN3 suppressed cell proliferation and migration, enhanced complement-mediated cytotoxicity via MASP2 binding, and increased MAC deposition. FCN3 also induced necroptosis through activating the RIPK1/RIPK3/MLKL pathway. These results highlight FCN3 as a tumor suppressor in CCA and suggest its potential as a therapeutic target for this malignancy.