CAV1-DOT1L axis in TAM-derived EVs orchestrates VM and sensitises PDAC to combined VM and VEGF targeting.

Vasculogenic mimicry (VM) is a non-endothelial vascularisation programme sustaining pancreatic ductal adenocarcinoma (PDAC) perfusion and metastasis, yet its regulators and therapeutic vulnerabilities remain unclear. To elucidate the immune and epigenetic mechanisms regulating VM and identify strategies to overcome VM-driven PDAC progression. Histopathology, three-dimensional tissue clearing, spatial transcriptomics and single-cell RNA sequencing were combined to map VM distribution and its immune contexture. Tissue microarrays, co-culture assays and xenograft models were used to assess tumour-associated macrophage (TAM) contributions. Extracellular vesicle (EV) proteomics and mechanistic studies identified cargo molecules and signalling pathways. DOT1L (disruptor of telomeric silencing 1-like) inhibitor EPZ-5676 and vascular endothelial growth factor receptor (VEGFR) inhibitor axitinib were used for therapeutic validation. VM was abundant in PDAC, increased with tumour stage and was preferentially surrounded by TAMs. M2-like TAMs promoted tube formation, invasion and tumour growth, while blockade of TAM-derived EVs abolished these effects. EV proteomics identified caveolin-1 (CAV1) as a key cargo correlating with VM density and TAM infiltration. Mechanistically, EV-delivered CAV1 interacted with DOT1L, promoted DOT1L EV loading and drove H3K79 methylation-dependent autophagy-related 5 (ATG5) transcription, sustaining VM and invasive phenotypes. Notably, while DOT1L inhibition suppressed VM and tumour progression, it paradoxically induced compensatory endothelial angiogenesis. Combined DOT1L and VEGFR blockade overcame this compensatory feedback, achieving superior tumour control without toxicity. TAM-derived EVs drive VM through a CAV1-DOT1L-ATG5 axis. We identify a compensatory link between VM and angiogenesis and demonstrate that dual targeting of these two vascular modalities offers a promising therapeutic strategy for PDAC.