Oncolytic Virus VV-GMCSF-Lact and Human GM-CSF Against GL261 Glioma in Immunocompetent Mice

<b>Background/Objectives:</b> Oncolytic viruses are an immunotherapeutic approach that can modulate the tumor microenvironment (TME), transforming immunologically 'cold' tumors into 'hot' ones. Insertion of genes encoding immunomodulatory proteins can further enhance antitumor immune responses. In this study, we compared the antitumor and immunomodulatory effects of the double recombinant vaccinia virus VV-GMCSF-Lact, which carries the human GM-CSF gene, with those of recombinant human GM-CSF (rhGM-CSF) in an immunocompetent murine GL261 glioma model. <b>Methods:</b> The study was conducted using a subcutaneous GL261 glioma model in immunocompetent C57BL/6 mice, comparing intratumoral VV-GMCSF-Lact and rhGM-CSF treatments with evaluation of immune cell populations by flow cytometry, tumor morphology by H&E staining, and tumor transcriptome profiles by RNA sequencing. <b>Results:</b> Flow cytometry showed that VV-GMCSF-Lact reduced the number of immunosuppressive cells in the TME of subcutaneously transplanted gliomas, targeting different components of the TME depending on animal sex. The immunotherapeutic effects of rhGM-CSF were less pronounced and primarily affected peripheral immune cells. Histological analysis revealed a decrease in mitotic figures in tumors from female mice after viral therapy. Transcriptome profiling of GL261 tumors demonstrated divergent gene expression patterns and cellular compositions between treatment groups. VV-GMCSF-Lact treatment was associated with a decreased proportion of malignant GL261 cells and CD8⁺ T lymphocytes, while rhGM-CSF treatment increased proportions of MDSCs, macrophages, NK cells, and tumor-associated neutrophils. <b>Conclusions:</b> Taken together, our data demonstrate that VV-GMCSF-Lact induces antitumor immune responses in murine GL261 glioma in vivo and modulates the tumor microenvironment more effectively than rhGM-CSF alone, supporting its potential for developing new strategies for glioma treatment.