Small extracellular vesicles carrying reovirus, tumor antigens, interferon-ß, and damage-associated molecular patterns for efficient tumor treatment.

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)-ß, 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.

Pre-treatment of oncolytic reovirus improves tumor accumulation and intratumoral distribution of PEG-liposomes.

PEGylated liposomes (PEG-liposomes) are a promising drug delivery vehicle for tumor targeting because of their efficient tumor disposition profiles via the enhanced permeability and retention (EPR) effect. However, tumor targeting of PEG-liposomes, particularly their delivery inside the tumors, is often disturbed by physical barriers in the tumor, including tumor cells themselves, extracellular matrices, and interstitial pressures. In this study, B16 melanoma tumor-bearing mice were injected intravenously with oncolytic reovirus before administration of PEG-liposomes to enhance PEG-liposomes' tumor disposition. Three days after reovirus administration, significant expression of reovirus sigma 3 protein, elevation of apoptosis-related gene expression, and activation of caspase 3 in the tumors were found. Apoptotic cells were found inside the tumors. These data indicated that reovirus efficiently replicated in the tumors and induced apoptosis of tumor cells. The tumor disposition levels of PEG-liposomes were approximately doubled by reovirus pre-administration, compared with a PBS-pretreated group. PEG-liposomes were widely distributed in the tumors of reovirus-pretreated mice, whereas in the PBS-pretreated group, PEG-liposomes were found mainly around or inside the blood vessels in the tumors. Pre-treatment with reovirus also improved the tumor accumulation of PEG-liposomes in human pancreatic BxPC-3 tumors. 3D imaging analysis of whole BxPC-3 tumors demonstrated that pretreatment with reovirus led to the enhancement of PEG-liposome accumulation inside the tumors. Combination treatment with reovirus and paclitaxel-loaded PEG-liposomes (PTX-PEG-liposomes) significantly suppressed B16 tumor growth. These results provide important information for clinical use of combination therapy of reovirus and nanoparticle-based drug delivery system (DDS).

Oncolytic reovirus-mediated killing of mouse cancer-associated fibroblasts.

Oncolytic viruses, which mediate tumor cell-specific infection, resulting in efficient tumor cell killing, have attracted much attention as a novel class of anti-cancer biopharmaceutical agents. Cancer-associated fibroblasts (CAFs) are an important component of the tumor microenvironment that strongly supports the growth, survival, and metastasis of tumor cells, suggesting that CAFs would have influence to the antitumor effects of oncolytic viruses; however, it remains to be fully evaluated whether oncolytic viruses affect the viabilities and properties of CAFs following treatment. Oncolytic reovirus, which is a non-enveloped virus that contains 10-segmented double-stranded RNA genome, shows efficient tumor cell lysis without apparent cytotoxicity to normal cells and has been tested worldwide in clinical trials against various types of tumors. In this study, we demonstrated that reovirus exhibited cytotoxicity against mouse primary CAFs isolated from subcutaneous tumors, but not against tail-tip fibroblasts. Infection with reovirus resulted in activation of caspase 3 and up-regulation of apoptosis-related gene expression, indicating that reovirus induced apoptosis of mouse primary CAFs. Intratumoral administration of reovirus induced apoptosis of mouse CAFs in the tumor. Taken together, these results indicate that reovirus has the potential to mediate antitumor effects by killing not only cancer cells but also CAFs.

A TGFß Signaling Inhibitor, SB431542, Inhibits Reovirus-mediated Lysis of Human Hepatocellular Carcinoma Cells in a TGFß-independent Manner.

BACKGROUND/AIM: Oncolytic reovirus, which is a non-enveloped virus possessing a 10-segmented double-stranded RNA genome, has been anticipated as a novel class of antitumor agent. Hepatocellular carcinoma (HCC) is considered to be a target suitable for reovirus-mediated virotherapy. Transforming growth factor (TGF)-ß plays an important role in the pathogenesis of HCC. TGF-ß-signaling inhibitors have proceeded to clinical trials as potential antitumor agents for HCC. On the other hand, TGF-ß is involved in induction of expression of cathepsins B and L, which are important for reovirus infection. It remains to be examined whether TGF-ß signaling inhibitors affect reovirus-mediated lysis of HCC cells. The aim of this study was to evaluate the effects of TGF-ß-signaling inhibitors on tumor cell lysis efficiency of reovirus in human HCC cells. MATERIALS AND METHODS: Reovirus was added to four types of human HCC cell lines pretreated with one of three TGF-ß type I receptor inhibitors: SB431542, A-83-01, or galunisertib (LY2157299). Cell viability, virus genome copy numbers, and virus protein expression were evaluated following reovirus infection. RESULTS: SB431542 significantly inhibited reovirus-mediated killing of human HCC cell lines, while A-83-01 and galunisertib did not inhibit. CONCLUSION: These data indicate that SB431542 inhibited reovirus-mediated lysis of human HCC cells in a TGF-ß signaling-independent manner.