Lysosomal Pathways and Autophagy Distinctively Control Endothelial Cell Behavior to Affect Tumor Vasculature

Abstract

Cancer cell-stromal cell crosstalk is orchestrated by a plethora of ligand-receptor interactions generating a tumor microenvironment (TME) which favors tumor growth. The high pro-angiogenic nature of the TME perpetuates the chaotic network of structurally immature, low pericyte-covered vessels characteristic of the tumor vasculature. We previously demonstrated that chloroquine (CQ) -a lysosomotropic agent used as first-generation autophagy blocker in clinical trials- induced tumor vessel normalization and reduced tumor hypoxia. CQ improved both vessel structure and maturation, whereas the conditional knockout of the crucial autophagy gene <i>Atg5</i> in endothelial cells (ECs) did not, thus highlighting a potential differential role for EC-associated autophagy and the lysosomes in pathological tumor angiogenesis. However, how CQ or ATG5-deficiency in ECs affect angiogenic signals regulating EC-pericyte interface and therefore vessel maturation, remains unknown. Here, we show that in ECs CQ constrained VEGF-A-mediated VEGF receptor (VEGFR)2 phosphorylation, a driver of angiogenic signaling. In the presence of CQ we observed increased expression of the decoy receptor VEGFR1 and of a lower molecular weight form of VEGFR2, suggesting receptor cleavage. Consequently, VEGF-A-driven EC spheroid sprouting was reduced by CQ treatment. Furthermore, CQ significantly affected the transcription and secretion of platelet-derived growth factor (PDGF)-AB/BB (upregulated) and Endothelin-1 (EDN1, downregulated), both modulators of perivascular cell (PC) behavior. In contrast, silencing of ATG5 in ECs had no effect on <i>VEGFR2</i> to <i>VEGFR1</i> ratio nor on <i>PDGFB</i> and <i>EDN1</i> expression. Accordingly, mice harboring B16F10 melanoma tumors treated with CQ, displayed both an increased number of αSMA⁺ PCs covering tumor vessels and co-expressed PDGF receptor-β, enabling PDGF ligand dependent recruitment. Moreover, upon CQ treatment the tumoral expression of angiopoietin-1 (<i>Angpt1)</i>, which retains mural cells, and induces vessel stabilization by binding to the EC-localized cognate receptor (TIE2), was increased thus supporting the vessel normalization function of CQ. These features associated with improved tumor vasculature were not phenocopied by the specific deletion of <i>Atg5</i> in ECs. In conclusion, this study further unravels endothelial cell autonomous and non-autonomous mechanisms by which CQ "normalizes" the intercellular communication in the tumor vasculature independent of autophagy.