Small extracellular vesicles modulated by αVβ3 integrin induce neuroendocrine differentiation in recipient cancer cells

ABSTRACT

The ability of small extracellular vesicles (sEVs) to reprogram cancer cells is well established. However, the specific sEV components able to mediate aberrant effects in cancer cells have not been characterized. Integrins are major players in mediating sEV functions. We have previously reported that the αVβ3 integrin is detected in sEVs of prostate cancer (PrCa) cells and transferred into recipient cells. Here, we investigate whether sEVs from αVβ3-expressing cells affect tumour growth differently than sEVs from control cells that do not express αVβ3. We compared the ability of sEVs to stimulate tumour growth, using sEVs isolated from PrCa C4-2B cells by iodixanol density gradient and characterized with immunoblotting, nanoparticle tracking analysis, immunocapturing and single vesicle analysis. We incubated PrCa cells with sEVs and injected them subcutaneously into nude mice to measure in vivo tumour growth or analysed in vitro their anchorage-independent growth. Our results demonstrate that a single treatment with sEVs shed from C4-2B cells that express αVβ3, but not from control cells, stimulates tumour growth and induces differentiation of PrCa cells towards a neuroendocrine phenotype, as quantified by increased levels of neuroendocrine markers. In conclusion, the expression of αVβ3 integrin generates sEVs capable of reprogramming cells towards an aggressive phenotype.

KEYWORDS:

• Prostate cancer
• tumour growth
• synaptophysin
• aurora kinase A
• iodixanol density gradient

Acknowledgments

The authors would like to thank Dr Michael Root for access to the refractometer, Dr Lorraine Iacovitti and Dr Eric Kostuk for the use of the Keyence BZ-x710 microscope. Dr Beatrice Knudsen for her expert advice on the xenograft histology and Dr Matthew Schiewer for the useful discussion on AR.

We would also like to thank Dr James Keen and Yolanda Covarrubias of the Sidney Kimmel Cancer Center (SKCC) Bioimaging Facility at Thomas Jefferson University for support with confocal imaging; Dr Jianke Zhang and Amir Yarmahmoodi of SKCC Flow Cytometry Facility at Thomas Jefferson University for technical support with NTA; Dr Mark Fortini, Pamela Walter and Jennifer Wilson at Thomas Jefferson University for editing comments; Veronica Robles for administrative assistance with the preparation of the manuscript. We would like to thank Raymond O’Neill for the IHC staining for SYP. We would also like to thank Dr Haifeng Yang and Danielle Wentworth of the Thomas Jefferson University – Sidney Kimmel Cancer Center Biorepository, a College of American Pathologists (CAP)-accredited biorepository for providing the human specimens used in this study as well as Wei Jiang and Zhijiu Zhong, Translational Research/Pathology Facility at Thomas Jefferson University for technical support with immunohistochemistry experiments.

Author contributions

FQ and LRL conceptualized the study and designed experiments. FQ performed the experiments. SRK assisted in performing an experiment and reviewed the manuscript. SS assisted in performing experiments. GD performed the SP-IRIS experiments shown in Figure 2 and analysed the results. QL performed the statistical analysis. FQ and LRL analysed all the results. RP, GK and JDD and PF performed the gene amplification analysis (Figure 6(a, b)). PMC reviewed the pathological features of the specimens used, analysed the immunohistochemical data and provided insights regarding the results and conclusions of the study. WKK performed the clinical evaluation of the human specimens used in this study and provided insights regarding the results and conclusions of the study. HB provided insights with respect to the NE gene analysis and on the results and conclusions of the paper. FQ and LRL wrote the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s). George Daaboul is CSO and co-founder of NanoView Bioscience.

Additional information

Funding

This study was supported by NCI-P01-140043, NCI-R01-224769 to LRL. This project was 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 utilized the shared Flow Cytometry, Histopathology, and Bioimaging facilities at the SKCC that are 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. RP is funded by the Prostate Cancer Foundation 19Youn18;National Cancer Institute (US) [140043];National Cancer Institute (US) [P30CA056036];National Cancer Institute (US) [224769];Pennsylvania Department of Health [SAP 4100072566];