Pancreatic Tumor Microvesicles Invade Immune Cells via CD36 and Distinctly Promote Tumor-associated Deep Vein Thrombosis

Tumor cell released microvesicles have been suggested to participate in the development of metastasis and cancer associated thrombosis but the mechanism and their in vivo relevance are largely unknown. We have characterized the microvesicles by proteomic analysis, nanoparticle tracking, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). We analyzed their immune cell interaction with superresolution microscopy in vitro and with immunohistochemistry in vivo. Moreover, intravital 2-photon microscopy was performed in a flow restriction model of the mouse vena cava to access cancer associated thrombosis. Nanoparticle tracking analysis, SEM and TEM confirmed that the diameter range of isolated tumor microvesicles was between 0.1 µm and 0.5 µm. Proteomic analyses showed that the intravesicular proteins were derived from various orgenelles of parent cell and included RNA-binding proteins. Through nanoscopic imaging of double-labelled tumor microvesicles, we observed that intravesicular RNA accumulated in the cytoplasm of macrophages while the vesicle membrane fused with the plasma membrane of the acceptor cell. This led to the insertion of microvesicle-derived coagulation initiator tissue factor into the plasma membrane. Lipid receptor CD36 was found to be a major mediator of the engulfment of tumor microvesicles by myeloid immune cells in vitro and by resident liver macrophages in vivo. In vitro, anti-CD36 antibody reduced the internalization of tumor microvesicles as compared to control antibody. Also, transfection with CD36 siRNA lowered uptake of tumor microvesicles by macrophages relative to control siRNA. CD36 triggered the colonization of perivascular Ly6C-macrophages for at least two wks. by blood derived microvesicles. This enhanced the formation of premetastatic niches and of macroscopic metastasis. In parallel, the circulating tumor microvesicles strongly promoted a deep vein thrombosis (DVT). This was mediated by vesicle surface exposure of phosphatidylethanolamine (PE). As shown by binding and neutralization of microvesicle procoagulant activity by the cyclic peptide duramycin. The prothrombotic effect of the microvesicles was independent of the recruitment of myeloid leukocytes or platelets. Intravital 2-photon microscopy analysis showed that tumor microvesicles were attached to the vessel wall and enhanced blood coagulation at the primary site of thrombus formation. In conclusion our results show that CD36 promotes internalization of tumor microvesicles by immune cells and their persistent accumulation in tissue macrophages which drives pancreatic cancer metastasis. Tumor associated thrombosis as promoted by tumor microvesicles is dependent on vesicle PE which may allow the selective therapeutic targeting of DVT during metastatic cancer.

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