UPAR-expressing melanoma exosomes promote angiogenesis by VE-Cadherin, EGFR and uPAR overexpression and rise of ERK1,2 signaling in endothelial cells

Alessio Biagioni,Beatrice Menicacci,Elena Andreucci,Mario Del Rosso,Daniele Guasti,Francesca Bianchini,Simona Serratì,Lido Calorini,Anna Laurenzana,Anastasia Chillà,Francesca Margheri,Paolo Paoli,Gabriella Fibbi,Silvia Peppicelli,Alessandra Mocali

doi:10.1007/s00018-020-03707-4

Abstract

Exosomes (Exos) have been reported to promote pre-metastatic niche formation, proliferation, angiogenesis and metastasis. We have investigated the role of uPAR in melanoma cell lines-derived Exos and their pro-angiogenic effects on human microvascular endothelial cells (HMVECs) and endothelial colony-forming cells (ECFCs). Melanoma Exos were isolated from conditioned media of A375 and M6 cells by differential centrifugation and filtration. Tunable Resistive Pulse Sensing (TRPS) and Nanoparticle tracking analysis were performed to analyze dimension and concentration of Exos. The CRISPR–Cas 9 technology was exploited to obtain a robust uPAR knockout. uPAR is expressed in melanoma Exos that are internalized by HMVECs and ECFCs, enhancing VE-Cadherin, EGFR and uPAR expression in endothelial cells that undergo a complete angiogenic program, including proliferation, migration and tube formation. uPAR loss reduced the pro-angiogenic effects of melanoma Exos in vitro and in vivo by inhibition of VE-Cadherin, EGFR and uPAR expression and of ERK1,2 signaling in endothelial cells. A similar effect was obtained with a peptide that inhibits uPAR–EGFR interaction and with the EGFR inhibitor Gefitinib, which also inhibited melanoma Exos-dependent EGFR phosphorylation. This study suggests that uPAR is required for the pro-angiogenic activity of melanoma Exos. We propose the identification of uPAR-expressing Exos as a potentially useful biomarker for assessing pro-angiogenic propensity and eventually monitoring the response to treatment in metastatic melanoma patients.

Highlights

Melanoma is the deadliest skin cancer, with stage IV melanoma patients having a 5-year survival rate of less than 15% [1]
We have previously shown that uPAR is strongly up-regulated in A375 and in metastasis-prone M6 melanoma cells with respect to normal melanocytes [16]. uPAR overexpression in melanoma cells controls an invasive and glycolytic phenotype depending on alpha 5-beta1 integrin -mediated uPAR connection with EGFR [17]
Melanoma-derived Exos were isolated from culture media (CM) of A375 and M6, the metastatic clone of A375, after

Summary

Introduction

Melanoma is the deadliest skin cancer, with stage IV melanoma patients having a 5-year survival rate of less than 15% [1]. Emerging evidence suggests that cancer-derived extracellular vesicles (EVs) play a major role in conditioning the tumor microenvironment and in preparing the “soil” of the pre-metastatic niche for metastasis. Exos are nano-sized (40–150 nm) membrane-bound vesicles that originate from the late endosomal trafficking, are gathered intracellularly into multivesicular bodies (MVBs) and released by fusion with the plasma membrane [7]. They are critical mediators of intercellular communication between tumor and stromal cells via their biologically active payload, including proteins, lipids and metabolites, RNA and DNA [8, 9]. Exos are reported to promote proliferation, invasion, and chemoresistance of cancer cells, to stimulate reprogramming of stromal cells to Cancer-Associated Fibroblasts (CAFs) and to promote angiogenesis which is critical for tumor cells release in the circulation and their spread to distant sites [10,11,12,13]

Methods

Results

Conclusion