BF/PC-13

Nucleic acids, lipids, and other cell components can be found within different types of extracellular vesicles (EVs), which include apoptotic bodies (ABs), large extracellular vesicles (LEVs), and small extracellular vesicles (SEVs). Release of LEVs from cells can be reduced by genetic or pharmacological inhibition of the enzyme acid sphinogomyelinase (aSMase), and indeed several studies have demonstrated a role for the clinically approved aSMase inhibitor imipramine in blocking LEV release, including in response to UVB exposure. Given that exposure of keratinocytes to UVB radiation results in the generation of UVR photoproducts in DNA that can subsequently be found in association with ABs and SEVs, we examined how imipramine impacts the release of extracellular DNA containing UVR photoproducts at an early time point after UVR exposure. Using several different model systems, including cultured keratinocytes in vitro, discarded human surgical skin ex vivo, and skin biopsies obtained from treated human subjects, these pilot studies suggest that imipramine treatment stimulates the release of CPD‐containing, SEV‐associated DNA. These surprising findings indicate that LEV and SEV generation pathways could be linked in UVB‐irradiated cells and that imipramine may exacerbate the systemic effects of extracellular UVR‐damaged DNA throughout the body.

Small extracellular vesicles (SEV) have attracted much attention both as mediators of intercellular communication and as drug delivery systems. In addition, recent studies have shown that SEV containing virus components and virus particles are released from virus-infected cells. Oncolytic viruses, which efficiently kill tumor cells by tumor cell-specific replication, have been actively studied as novel anticancer agents in clinical and preclinical studies. However, it remains to be fully elucidated whether SEV released from oncolytic virus-infected cells are involved in the antitumor effects of oncolytic viruses. In this study, we examined the tumor cell killing efficiencies and innate immune responses following treatment with SEV released from oncolytic reovirus-infected tumor cells in vitro and in vivo. Reovirus-infected B16 cells secreted SEV associated with or containing reovirus particles (Reo-SEV) with a diameter of approximately 130 nm and a zeta potential of -17 mV, although death of reovirus-infected B16 cells was not observed. The secreted Reo-SEV also contained interferon (IFN)-β, tumor antigens, and damage-associated molecular patterns (DAMPs), including heat shock proteins (HSPs). Reo-SEV were secreted from the tumor tissues of reovirus-injected mice. Inhibition of the SEV secretion pathway using GW4869, which is a neutral sphingomyelinase inhibitor, resulted in significant reduction in the infectious titers of reovirus in the culture supernatants, suggesting that the cells released progeny virus via the SEV secretion pathway. Reo-SEV more efficiently killed mouse tumor cells and induced innate immune responses in mouse bone marrow-derived dendritic cells than reovirus. Reovirus and Reo-SEV mediated efficient and comparable levels of growth suppression of B16 subcutaneous tumors and induction of tumor infiltration of CD8+ T cells following intravenous administration. These results indicate that Reo-SEV are a promising oncolytic agent and that SEV are an effective delivery vehicle for oncolytic virus.