Exosome-Powered Oncolytic Viruses Change the Game in Breast Cancer Treatment

Oncolytic viruses (OVs) are viruses engineered to selectively infect and destroy cancer cells while sparing normal tissues. However, the clinical application of OVs, including coxsackievirus B3 (CVB3), has been limited by rapid clearance of the virus due to pre-existing neutralizing antibodies. To over this challenge, new study published in the journal ACS NANO and conducted by Dr. Amirhossein Bahreyni, Dr. Yasir Mohamud, Sanaz Ashraf Nouhegar, Dr. Jingchun Zhang, and led by Professor Honglin Luo from the University of British Columbia developed a novel therapeutic strategy that integrates oncolytic virotherapy with chemotherapy and immunotherapy. The researchers developed bioengineered exosomes as delivery vehicles to protect oncolytic virus from immune clearance and to enhance its targeting and therapeutic efficacy. They engineered a modified version of CVB3, known as miR-CVB3 which was designed to selectively target breast cancer cells. To do this, they inserted specific microRNA target sequences into the viral genome which are downregulated in tumor cells but present in normal tissues thereby enhances the virus’s selectivity. To test the effectiveness of miR-CVB3, the authors infected breast cancer cell lines and observed a reduction in immune checkpoint proteins such as PD-L1 and B7-H3, as well as the “do not eat me” signal CD47, while there was an increase in “eat me” signals of calreticulin on the surface of cancer cells. These results demonstrated to the authors that miR-CVB3 successfully remodeled the tumor microenvironment from an immunosuppressive to an immunostimulatory state. Additionally, the researchers evaluated the exosomes released by miR-CVB3-infected cancer cells and found that exosomes from infected cells, which they named ExomiR-CVB3 carried the virus and exhibited an enriched cargo of immunostimulatory molecules, including HSP70 and HMGB1 which are known to activate immune responses. They performed cryogenic transmission electron microscopy and confirmed the presence of miR-CVB3 within these exosomes, and conducted dynamic light scattering analysis which indicated that ExomiR-CVB3 had the favorable nano size and charge for efficient cellular uptake. They also demonstrated that these exosomes could infect breast cancer cells more effectively than free miR-CVB3, specially in cells with low levels of viral entry receptors.

To assess the therapeutic efficacy of ExomiR-CVB3 in vivo, the researchers treated tumor-bearing mice with either free miR-CVB3 or ExomiR-CVB3 and showed that ExomiR-CVB3 significantly outperformed free miR-CVB3 in terms of tumor growth inhibition and survival rates. Moreover, tumor sizes were notably smaller in mice treated with ExomiR-CVB3, and these mice also exhibited higher levels of immune cell infiltration, including CD8+ T cells and macrophages into the tumor microenvironment as well as increased levels of pro-inflammatory cytokines and co-stimulatory molecules. According to the authors, the engineered exosome-based delivery system improved the targeting and retention of miR-CVB3 within tumors and amplified the immune response against the cancer cells. The researchers also investigated the ability of ExomiR-CVB3 to protect miR-CVB3 from immune clearance which is a common challenge in the systemic administration of oncolytic viruses. To do this, they incubated ExomiR-CVB3 and free miR-CVB3 with serum containing neutralizing antibodies against CVB3 and then exposed to breast cancer cells and found that ExomiR-CVB3 retained its infectivity and cytotoxicity in the presence of neutralizing antibodies whereas free miR-CVB3 was significantly neutralized. In mouse models pre-immunized with CVB3 to simulate pre-existing immunity, ExomiR-CVB3 treatment led to reduced tumor sizes and increased survival rates compared to free miR-CVB3, demonstrating the protective effect of exosome encapsulation in shielding the virus from immune clearance. The authors increased the therapeutic potential of ExomiR-CVB3 by incorporating the AS1411 aptamer which is a DNA aptamer that specifically targets nucleolin as well as incorporating doxorubicin to form a new complex termed ExomiR-CVB3/DoxApt. They showed ExomiR-CVB3/DoxApt to have increased cellular uptake and cytotoxicity against breast cancer cells compared to ExomiR-CVB3 alone which indicates a synergistic effect between the anti-cancer components. Moreover, when they treated mice with the ExomiR-CVB3/DoxApt complex, they found enhanced antitumor efficacy compared to other treatment groups. Further validation using histological analysis of tumor tissues revealed increased apoptosis and reduced proliferation in tumors treated with ExomiR-CVB3/DoxApt and increased immune cell infiltration with elevated levels of CD8+ T cells and macrophages. In conclusion, Professor Honglin Luo and colleagues provided compelling evidence that integrating miR-CVB3 with engineered exosomes combined with targeted aptamers and chemotherapy is a highly effective for treating breast cancer. Additionally, their use of bioengineered exosomes as delivery vehicles for the miR-CVB3 enhanced the virus’s selectivity and efficacy against breast cancer cells and also mitigated rapid immune clearance. Furthermore, the detailed mechanism studies that revealed ability of ExomiR-CVB3 to remodel the tumor microenvironment into a more immunostimulatory state suggests that their approach could enhance the effectiveness of existing immunotherapies and potentially leads to more durable responses in patients. On the other hand, the ExomiR-CVB3/DoxApt complex demonstrated the potential for synergistic therapeutic effects which could reduce the required dosages of chemotherapeutic agents and ultimately minimize risk of systemic toxicity. It is worthy to mention the approaches used by Professor Honglin Luo and team may be applied to other types of cancer beyond breast cancer. Finally, while CVB3 or modified versions such as miR-CVB3 has not yet been tested in clinical trials for breast cancer, the promising results from preclinical studies suggest that it could eventually make its way into early-phase clinical trials provided that safety studies are adequately addressed. Indeed, the success of other oncolytic viruses in clinical trials such as talimogene laherparepvec (T-VEC) for melanoma provides a hopeful outlook for the future of CVB3 and similar oncolytic viruses in cancer therapy.