EGFR-dependent delivery of phosphorylated IGFBP3 by cancer stem cells drives chemotherapy resistance in colorectal cancer.

The primary systemic treatment for advanced colorectal cancer (CRC) is chemotherapy, but inevitable drug resistance leads to poor prognosis. Cancer stem cells (CSCs) are recognized as significant contributors to chemotherapy resistance, yet the mechanisms underlying their ability to induce widespread resistance across tumor cell populations remain poorly understood. In this study, we demonstrate a novel mechanism by which colorectal CSC-like spheroid cells (CCSCs) drive chemotherapy resistance through the release of small extracellular vesicles (sEVs) enriched with highly phosphorylated insulin-like growth factor binding protein-3 (IGFBP3) into the tumor microenvironment (TME). While cell-free IGFBP3 or endogenous IGFBP3 directly induces apoptosis in tumor cells, IGFBP3 originating from quiescent CCSCs functions through an epidermal growth factor receptor (EGFR)-dependent pathway to promote chemotherapy resistance. Mechanistically, the protein kinase VRK2, which is highly enriched in CCSCs, leads to the phosphorylation of IGFBP3 at the S201 site. This phosphorylation enables IGFBP3 to bind to EGFR, facilitating its sorting and subsequent recruitment into sEVs. Both EGFR and phosphorylated IGFBP3 are co-delivered via sEVs to recipient tumor cells, where they inhibit chemotherapy-induced DNA damage and promote resistance. Importantly, siRNA targeting VRK2 or the inhibitor D4476 significantly reduced IGFBP3 phosphorylation and its incorporation into sEVs, thereby reversing chemotherapy resistance. These findings highlight the dual role of IGFBP3 in CRC and provide critical insights into the core mechanism by which CCSCs drive chemotherapy resistance through EGFR and sEVs-mediated signaling, offering a novel therapeutic approach for personalized treatment of CRC.