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.
