Cancer-associated fibroblasts expressing FSTL3 promote vasculogenic mimicry formation and drive colon cancer malignancy.

Anti-angiogenic therapies are commonly employed in colon cancer management, yet many patients eventually develop resistance and experience disease progression. Vasculogenic mimicry (VM)-the formation of tumor-derived vessel-like networks-has been recognized as one mechanism contributing to this resistance, although the underlying details remain incompletely understood. Here, by integrating bioinformatic analyses of publicly available datasets and validating the results in patient samples (n = 157), we identified follistatin-like 3 (FSTL3) as a critical factor predominantly expressed in colon cancer-associated fibroblasts (CCAFs), with its expression strongly correlating with increased VM formation, intratumoral blood vessels, and poor prognosis. Single-cell RNA sequencing of tumors from VM and non-VM patients revealed that hypoxia drives FSTL3 expression in CCAFs, leading to extracellular matrix remodeling and enhancing cancer cell endothelial-like plasticity. Mechanistically, FSTL3 binds to transferrin receptor (TfR1), an iron-uptake receptor on cancer cells, thereby activating the TfR1/AKT/mTOR pathway and elevating VE-Cadherin to support endothelial-like transformation, VM, and metastatic progression. Notably, FSTL3-targeting antibodies (aFSTL3) effectively inhibited VM and angiogenesis in both in vitro and in vivo models, while the combination of aFSTL3 with bevacizumab produced synergistic suppression of neovascular-like structures and distant metastases. These findings demonstrate a pivotal role for FSTL3+ CCAFs in facilitating VM through TfR1-mediated signaling and offer a promising dual-target approach to overcome anti-angiogenic therapy resistance in colon cancer.