Sacc Ev-Col1 Homotrimer Promote Tregs Infiltration Via Fibroblast Activation

The poor prognosis of salivary adenoid cystic carcinoma (SACC) is strongly associated with its propensity for lung metastasis. This study explores extracellular vesicle (EV)-driven pre-metastatic niche formation and immunosuppressive remodeling to identify therapeutic targets for early intervention and immunotherapy in SACC lung metastasis. EVs from high-metastasis SACC-LM and parental SACC-83 cells were isolated via ultracentrifugation. Collagen I (Col1) homotrimer expression were detected by western blotting and LC-MS. Pre-metastatic niche formation and metastatic models was assessed in 6-week-old female C57BL/6J mice. Fibroblast activation and regulatory T cell (Treg) infiltration was analyzed using collagen gel contraction assays, spatial proteomics, and multiplex immunofluorescence, respectively. SACC-LM EVs significantly enhanced lung metastasis by establishing a pre-metastatic niche. These EVs carried unique Col1 homotrimer (α1/α1/α1), distinct from fibroblast-derived Col1 heterotrimer (α1/α2/α1). EV-transferred Col1 homotrimers drove lung fibroblast differentiation into metastasis-associated fibroblasts (MAFs) expressing α-SMA and FAP. Activated MAFs secreted interleukin-6, interleukin-8, and MMP-2, promoting Treg infiltration and immunosuppressive microenvironment formation. Spatial proteomics revealed EV-induced α-SMA+FAP+MAFs-Tregs crosstalk during pre-metastatic niche development. We identify an EV-fibroblast-Treg axis driving SACC lung metastasis, where tumor-derived Col1 homotrimer activates MAFs to recruit Tregs and establish immunosuppression. Targeting MAF activation and Treg infiltration disrupts metastatic progression, enhancing immunotherapy efficacy. This mechanism guides niche-focused diagnostics and immunotherapy to prevent SACC lung metastasis.