Mesenchymal stem cell mediated delivery of Extracellular Vesicle (EV)-encapsulated microRNAs for the treatment of breast cancer

Abstract

Mesenchymal Stem Cells (MSCs) hold the ability to home to the site of the tumour and bypass the host immune system. Extracellular Vesicles (EVs) are thought to be a fingerprint of the parent cell and therefore MSC-EVs may retain these cancer targeting properties and immune privilege. The development of a novel MSC-EV therapeutic for breast cancer in parallel with a diagnostic approach using circulating EV-miRNAs shows strong potential. However limited research has focused on the impact of EV administration on the host immune system. Therefore, this study aimed to determine whether MSC-EV administration in immunocompetent animals would initiate a host immune response, and to analyse secreted EV-microRNAs in vitro and in vivo. Secreted EVs were isolated from the serum of tumour-bearing animals, and the conditioned media of breast cancer cells or MSCs by differential centrifugation, microfiltration and ultracentrifugation. EVs were characterised by western blot, transmission electron microscopy and nanoparticle tracking analysis. EV-miRNA expression was analysed using next generation sequencing (NGS) and RQ-PCR. Immune competent Balb/c mice bearing 4T1 breast tumours, or healthy control animals, received an IV injection of either murine or human MSC-EVs. Following sacrifice, tumours, draining lymph nodes and spleens were digested into a single cell suspension and flow cytometry analysis performed targeting a range of immune cell markers for T cells, B cells, Myeloid Derived Suppressor Cells (MDSC) and natural killer (NK) cells. Small EVs (30-150nm) were successfully isolated with the appropriate lipid bilayer and expression of CD63 and CD81 confirmed. There was no significant correlation between the number of circulating EVs (6.34 x 109 – 1.30 x 1010) and protein yield (181.2 – 1914.5μg/mL; R2 =0.26, p= 0.3). However, a moderate correlation between tumour burden and the number of circulating EVs was observed (R2 =0.386, p=0.047). Although miR-16 was detected in all serum EVs, wide variation suggested it would not make a stable endogenous control. NGS analysis of 4T1 breast cancer cells and secreted EVs revealed the presence of 242 miRNAs. 72 miRNAs were expressed at significantly higher levels in the EVs, with a number of these validated by RQ-PCR. When immune response to tumour growth was analysed in vivo, increased T cells and MDSCs and decreased NK cells were observed. More importantly however the proportion of T cells, B cells, MDSCs, Dendritic cells and NK cells remained stable in the tumour, draining lymph node and spleen of all tumour-bearing/healthy control animals that received either human or murine MSC-EVs, with no significant change detected. EVs contain a range of miRNA that could potentially help monitor tumour burden and therapeutic efficacy. The data presented supports the hypothesis that MSC-EVs retain the immune privilege of the secretory cell, with human cell-derived EVs eliciting no immune response in mice. This is a promising step forward for MSC-EV therapy and reinforces the potential for the use of EVs in the cancer setting.