The αvβ6 integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis

ABSTRACT

Prostate cancer (PrCa) cells crosstalk with the tumour microenvironment by releasing small extracellular vesicles (sEVs). sEVs, as well as large extracellular vesicles (LEVs), isolated via iodixanol density gradients from PrCa cell culture media, express the epithelial-specific αvβ6 integrin, which is known to be induced in cancer. In this study, we show sEV-mediated protein transfer of αvβ6 integrin to microvascular endothelial cells (human microvascular endothelial cells 1 – HMEC1); we demonstrate that de novo αvβ6 integrin expression is not caused by increased mRNA levels. Incubation of HMEC1 with sEVs isolated from PrCa PC3 cells that express the αvβ6 integrin results in a highly significant increase in the number of nodes, junctions and tubules. In contrast, incubation of HMEC1 with sEVs isolated from β6 negative PC3 cells, generated by shRNA against β6, results in a reduction in the number of nodes, junctions and tubules, a decrease in survivin levels and an increase in a negative regulator of angiogenesis, pSTAT1. Furthermore, treatment of HMEC1 with sEVs generated by CRISPR/Cas9-mediated down-regulation of β6, causes up-regulation of pSTAT1. Overall, our findings suggest that αvβ6 integrin in cancer sEVs regulates angiogenesis during PrCa progression.

KEYWORDS:

• Angiogenesis
• endothelial cell
• extracellular vesicle
• integrin
• prostate cancer
• survivin

Acknowledgments

We would like to thank Dr. Eric B. Kmiec and Dr. Pawel A. Bialk from the Gene Editing Institute, Christiana Health Care System, for generating the cell lines with CRISPR/Cas9-mediated β6 integrin subunit down-regulation; Dr. Joseph A. Madri, Department of Pathology, Yale University School of Medicine, New Haven, CT, USA for BAEC; Dr. Michael Root at Thomas Jefferson University for refractometer; Dr. James Keen and Yolanda Covarrubias, Sidney Kimmel Cancer Center (SKCC) Bio-imaging facility at Thomas Jefferson University for support with confocal imaging; Dr. Lei Yu and Amir Yarmahmoodi, SKCC Flow Cytometry facility at Thomas Jefferson University for technical support with NTA and FACS experiments; Dr. Zhijiu Zhong, Translational Research/Pathology facility at Thomas Jefferson University for technical support with immunohistochemistry experiments; Cancer Genomics & Bioinformatics Core facility, led by Dr. Paolo Fortina at Thomas Jefferson University for technical support with q-PCR experiments; Dr. Mark Fortini and Jennifer Wilson at Thomas Jefferson University for editing the manuscript; Veronica Robles for administrative assistance with the preparation of the manuscript.

Author contributions

SRK and LRL conceptualised the study and designed experiments. SRK performed the experiments. IS generated Figure 1b and reviewed the manuscript. FQ assisted in performing an experiment and reviewed the manuscript. NN generated Figure 1a and reviewed the manuscript. EA assisted in performing an experiment and reviewed the manuscript. QL performed statistical analysis on data. SS and JK assisted in performing an experiment and reviewed the manuscript. PAM provided PrCa tissue sections, reviewed immunohistochemical staining and the manuscript. PHW and SV provided anti-αvβ6 integrin Abs (6.2A1 and 6.4B4) and reviewed the manuscript. SRK, DCA and LRL analysed results and wrote the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s). Paul H. Weinreb is an employee and shareholder of Biogen. Shelia M. Violette is currently an employee of Admirx and, during this study, was an employee and shareholder of Biogen.

Correction Statement

This article has been republished with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

This study was supported by NCI-P01-140043, NCI-R01-224769. This project is also funded, in part, under a Commonwealth University Research Enhancement Program grant with the Pennsylvania Department of Health (H.R.): SAP 4100072566; the Department specifically disclaims responsibility for any analyses, interpretations or conclusions. The research reported in this publication utilised the shared Flow Cytometry and Bio-imaging facilities at the SKCC and was supported by the National Cancer Institute of the National Institutes of Health under award number P30CA056036. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH; National Cancer Institute (US) [140043]; National Cancer Institute (US) [P30CA056036]; National Cancer Institute (US) [224769]; Pennsylvania Department of Health [SAP 4100072566].