Cancer is one of the most common causes of death around the world. The high rates of disease and mortality require the development of novel treatments for cancer. Another significant issue is the toxicity typically linked to conventional therapeutic approaches, such as chemotherapy and radiotherapy. Oncolytic viruses are still one of the most promising and rapidly developing fields of research among the many potential antitumoral therapies. Among all oncolytic viruses, the Vaccinia virus is arguably one of the safest, with an extremely long and prominent history of use, since it was the one and only vaccine used in the Smallpox Eradication Program in the 1970s. Interestingly enough, it was the first oncolytic virus proven to have tumor tropism in vitro and in vivo in laboratory settings. While being highly immunogenic, Vaccinia virus DNA replication takes place in the cytoplasm of the infected cell, and virus genes never integrate into the host genome. Challenges in VV therapy remain, particularly in delivering the virus efficiently to tumor sites. Intravenously administered VV must navigate multiple host immune barriers, including complement proteins, coagulation factors, blood cell components, and pre-existing neutralizing antibodies (nAbs), to target effectively the tumor mass. To enhance therapeutic efficacy, VV is often combined with other anticancer treatments or delivered via cellular carriers, which can both circumvent host immune defenses and boost antitumor immunity. In this study, we investigated an innovative approach to enhance targeted cancer therapy by using immune cells (T cells, CAR-T cells, and Microglia cells) as carriers for the oncolytic Vaccinia virus (strain LIVP 1.1.1). The main goal was to assess the susceptibility of these immune cells to VV infection and to investigate the combined effects of VV-loaded (VV-infected) cells on human Neuroblastoma (SH-SY5Y, SK-N-AS) and Glioblastoma (U87) cancer cell lines in both two-dimensional (2D) monolayer and three-dimensional (3D) spheroid culture systems. In the first project, we focused on using VV-loaded (VV-infected) T cells and CAR-T cells against Neuroblastoma. A comparison among six donors demonstrated that CAR.GD2 cells exhibited significantly higher susceptibility to Vaccinia virus infection and produce significantly greater viral titers in contrast to T cells. Despite VV loading decreasing overall cell yield in a dose- and time-dependent manner, CAR.GD2 cells showed heightened resistance to VV-induced cytotoxicity and maintained viability for an extended duration compared to T cells. In co-culture experiments, VV-loaded CAR.GD2 cells nearly completely eliminated Neuroblastoma cells in 2D models at all ratios within 72 hours, showing that they are more effective than non-loaded cells. In 3D models, VV-loaded CAR.GD2 cells efficiently delivered the virus, leading to viral transfer, infection, and subsequent destruction of the tumor spheroids. In the second project, we investigated using Microglia cells (BV2) as carriers to against Neuroblastoma and Glioblastoma cancer cell lines. BV2 cells effectively replicated and released the Vaccinia virus, with higher MOIs leading to an approximate 50% loss of BV2 cells within 48 hours. In co-culture studies, non-loaded BV2 cells exhibited no impact; conversely, VV-loaded BV2 cells successfully eradicated Neuroblastoma (SH-SY5Y) cells after 72 hours and Glioblastoma (U87) cells after 48 hours. The co-culture system facilitated increased viral replication and release in comparison to direct infection of cancer cells. This strategy was further confirmed in 3D spheroid models, where VV-loaded BV2 cells effectively delivered the virus, resulting in effective infection, transmission, and considerable damage to both SH-SY5Y and U87 spheroids. In conclusion, we have tested different immune cells, and the results demonstrate a novel therapeutic model in which these immune cells function as targeted delivery systems for Oncolytic Vaccinia Virus. This approach, especially employing VV-loaded CAR.GD2 cells against Neuroblastoma and VV-loaded Microglia cells against both Neuroblastoma and Glioblastoma, demonstrated enhanced cytotoxic effects compared to individual therapies, particularly in 3D models that replicate the tumour microenvironment.
Elena Ekrami (2026) studied this question.