Tumor cell-derived extracellular vesicles (EV) convert normal myeloid cells into myeloid-derived suppressor cells (MDSC), inhibiting antitumor immune responses. Here, we show that EV from Ret mouse melanoma cells upregulate the expression of programmed cell death ligand 1 (PD-L1) on mouse immature myeloid cells (IMC), leading to suppression of T-cell activation. PD-L1 expression and the immunosuppressive potential of EV-generated MDSC were dependent on the expression of Toll-like receptors (TLR). IMC from Tlr4 À/À mice failed to increase T-cell PD-L1 expression and immunosuppression with Ret-EV treatment, and this effect was dependent on heat-shock protein 86 (HSP86) as HSP86-deficient Ret cells could not stimulate PD-L1 expression on normal IMC; IMC from Tlr2 À/À and Tlr7 À/À mice demonstrated similar results, although to a lesser extent. HSP86-deficient Ret cells slowed tumor progression in vivo associated with decreased frequency of tumor-infiltrating PD-L1 þ CD11b þ Gr1 þ MDSC. EV from human melanoma cells upregulated PD-L1 and immunosuppression of normal monocytes dependent on HSP86. These findings highlight a novel EV-mediated mechanism of MDSC generation from normal myeloid cells, suggesting the importance of EV targeting for tumor therapy. Significance: These findings validate the importance of TLR4 signaling in reprogramming normal myeloid cells into functional myeloid-derived suppressor cells.

Despite substantial advances in the treatment of solid cancers, resistance to therapy remains a major obstacle to prolonged progression-free survival. Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, with a high level of liver metastasis. Primary PDAC is highly hypoxic, and metastases are resistant to first-line treatment, including gemcitabine. Recent studies have indicated that endothelial cell (EC) focal adhesion kinase (FAK) regulates DNA damaging therapy-induced angiocrine factors and chemosensitivity in primary tumor models. Here, we show that inducible loss of EC-FAK in both orthotopic and spontaneous mouse models of PDAC is not sufficient to affect primary tumor growth but reduces liver and lung metastasis load and improves survival rates in gemcitabine-treated, but not untreated, mice. EC-FAK loss did not affect primary tumor angiogenesis, tumor blood vessel leakage, or early events in metastasis, including the numbers of circulating tumor cells, tumor cell homing or metastatic seeding. Phosphoproteomics analysis showed a downregulation of the MAPK, RAF, and PAK signaling pathways in gemcitabine-treated FAK-depleted ECs compared to gemcitabine-treated wild-type ECs. Moreover, low levels of EC-FAK correlated with increased survival and reduced relapse in gemcitabine-treated PDAC patients, supporting the clinical relevance of these findings. Altogether, we have identified a new role of EC-FAK in regulating PDAC metastasis upon gemcitabine treatment that impacts outcome.