Breast cancer extracellular vesicles transfer viral cargo following oncolytic virotherapy

Abstract

Background: Breast cancer is the major kind of cancer among women in the United Kingdom. To treat radio-/chemo-resistant cancer as well as advanced illness, novel medicines are necessary. Oncolytic viruses (OV) are naturally cytotoxic and infect and kill tumour cells whilst sparing healthy tissues. The full mechanism by which this occurs remains to be elucidated, but it may in part be mediated by extracellular vesicles (EVs). EVs are nanosized, membrane-enclosed vesicles that contain molecular cargo. EVs can be taken up by cells, for instance immune cells, at local or distant sites, causing phenotypic changes in the recipient cells. We hypothesise that infection of breast cancer cells with an oncolytic virus causes release of EVs carrying viral and immunogenic cargo that leads to activation of anti-tumour immunity. Methods: To determine this, we infected breast cancer cells lines MCF-7 and MDA-MB-231 with the herpes simplex virus (HSV1716). EVs were isolated from the OV conditioned medium of infected cells and control cells by differential ultra-centrifugation (dUC). Characterization of these EVs' physical properties in order to determine the size ranges and rates of EV generation in breast cell lines and investigation on the oncolytic potential of EV-OV was carried out using Nanoparticle tracking analysis (NTA), Transmission electron microscopy (TEM) and mass spectroscopy. The antitumour efficacy of purified EVs was tested in an immunocompetent mouse model of mammary cancer using the luciferase labelled triple negative breast cancer cell line E0771. Results: EVs were isolated from MCF-7 and MDA-MB-231 cells after infection with OV, these are typically exosome-like with a diameter of ~ 150 nm. The purified EVs expressed exosome markers including CD9, CD63, TSG101 and carried viral and immune cargo. These EVs were internalised by breast cancer cells and inhibited tumour cell migration in vitro. Furthermore, systemic delivery of EVs derived from OV (EV-OV) infected breast cancer cells was able to inhibit primary tumour growth and pulmonary metastasis in vivo, more effectively that OV given alone. Conclusion: This indicates that EVs generated from cells that have been infected with OV may have antitumour characteristics. The use of EV-OV as a treatment with other cancer medications, such as immune checkpoint inhibitors, which may attract and activate T cells, changing a "cold" tumour into a "hot," tumor might lead to the creation of a new therapeutic approach for treating breast cancer.