Preliminary Insights Into the Combined Antitumor Efficacy of Onco‐VV‐TT and Natural Killer Cells in Glioblastoma

This study investigates the therapeutic efficacy and safety of Onco-VV-TT, a genetically engineered oncolytic vaccinia virus, in the treatment of glioblastoma (GBM), with and without the combination of natural killer (NK) cell immunotherapy. A comprehensive set of in vitro and in vivo experiments was conducted to assess viral cytotoxicity, replication, tumor penetration, and immunomodulatory effects. Onco-VV-TT selectively reduced the viability of GBM cell lines (U251, U87, and C6) in a time- and dose-dependent manner while sparing normal fibroblasts. The virus significantly impaired clonogenicity, migration, and stemness-related gene expression in U251 cells. Confocal imaging confirmed viral replication and deep penetration into 3D spheroids. NK cells demonstrated strong cytotoxicity against U251 cells in both 2D and 3D models. In a xenograft mouse model, intratumoral administration of Onco-VV-TT suppressed tumor growth and significantly prolonged survival; NK cell monotherapy showed limited efficacy, while the addition of NK cells further enhanced therapeutic outcomes. Immunological analyses revealed that combination therapy elevated pro-inflammatory cytokines (TNF-α, IFN-γ, and IL-6) and increased WBC count, reflecting an expected transient systemic inflammatory response to vaccinia virus exposure rather than a direct enhancement of antitumor immunity. Histopathological and immunohistochemical analysis showed reduced mitosis, increased apoptosis, decreased cancer stem cell marker CD133, and lower VEGF expression in tumors treated with Onco-VV-TT and NK cells. Safety evaluations in immunocompetent mice demonstrated no significant systemic toxicity or organ damage following intravenous injection of Onco-VV-TT. Transient inflammatory marker elevation resolved by day 21, and all hematological and biochemical parameters remained within normal range. These findings suggest that Onco-VV-TT, particularly in combination with NK cell therapy, is a promising and well-tolerated approach for GBM treatment, meriting further preclinical and clinical development.