Netrin-1/UNC5B-TFF3 axis modulates cancer stem cells self-renewal and chemoresistance in metastatic colorectal cancer

Purpose: Metastatic colorectal cancer (mCRC) exhibits poor outcomes due to recurrence and resistance to chemotherapy driven by cancer stem cells (CSCs). We investigated the role of the dependence receptor ligand netrin-1 and its receptor UNC5B in CSC self-renewal and evaluated therapeutic inhibition of netrin-1 using the anti-netrin-1 antibody NP137. Experimental Procedures: Patient-derived organoids (PDOs) from colorectal liver metastases were treated with recombinant netrin-1, NP137, or controls. Self-renewal was quantified by extreme limiting dilution assays. UNC5B was silenced by CRISPR/Cas9 to determine receptor the implication of this Netrin-1 receptor. Single-cell RNA-sequencing elucidated signaling pathways affected by NP137. In vivo efficacy of NP137 alone or combined with FOLFOX chemotherapy was tested in PDO-xenografted mice, and paired tumor biopsies from an mCRC patient enrolled in an NP137 clinical trial were analyzed by RNA-seq. Results: Netrin-1 enhanced CSC self-renewal and survival, effects abolished by NP137 or UNC5B knockout. NP137 treatment triggered CSC apoptosis, an effect reversed by caspase inhibition. Single-cell transcriptomics revealed that UNC5B-positive cells secreted trefoil factor 3 (TFF3), which acted paracrinally to maintain stemness gene expression (LGR5, SOX4, SMOC2, PROM1). NP137 suppressed TFF3 and stemness transcripts in both PDOs and a treated patient's tumor. Blocking TFF3 dimerization phenocopied NP137 activity, confirming TFF3 as a critical downstream effector. FOLFOX exposure upregulated netrin-1 and UNC5B, and combination therapy (FOLFOX + NP137) significantly reduced self-renewal and tumor growth in mice while decreasing intratumoral TFF3. Conclusions: Netrin-1 sustains mCRC CSC self-renewal through an UNC5B-dependent, TFF3-mediated paracrine survival mechanism. Pharmacologic inhibition of netrin-1 with NP137 induces CSC apoptosis and enhances chemotherapy efficacy, identifying the netrin-1/UNC5B/TFF3 axis as a promising therapeutic target to overcome stemness-driven resistance in metastatic colorectal cancer.