Abstract 3070: Cytomegalovirus infection enhances spheroid formation and drives aggressive phenotype in glioblastoma via upregulation of SOX2 pRB pAKT and STAT3/IL-6 signaling

Abstract Background: Cytomegalovirus (CMV) infection has been implicated in glioblastoma (GBM) progression, with effects on stemness, immune evasion, and angiogenesis. Our work investigates the impact of CMV infection on spheroid formation, oncogenic pathways including SOX2, retinoblastoma protein (pRB), phospho-AKT (pAKT), and STAT3/IL-6 signaling, as well as cytokine modulation. Additionally, we evaluate the therapeutic potential of ganciclovir in reversing CMV-induced effects. Methods: GBM cell lines LN229, U251, U87, U138, and T98G were infected with the GFP-expressing CMV TB40 strain at a multiplicity of infection (MOI) of 0.5. Infection was confirmed via fluorescence microscopy and Western blotting for CMV pp65 protein. The stable cell line with infection was confirmed after multiple passage and presence of CMV protein. Spheroid formation assays assessed morphological changes and aggressiveness in vitro. Western blotting analyzed the expression of SOX2, pRB, pAKT, and phospho-STAT3 (pSTAT3), while cytokine profiles, including PD-L1, VEGF, angiopoietin-2, and IL-6, were measured in non-infected, infected, and ganciclovir-treated cells using Luminex panel. Results: CMV infection was confirmed in all GBM cell lines, with infection rates ranging from 60% to 90%. CMV-infected cells exhibited accelerated and more aggressive spheroid formation compared to non-infected cells. Infection led to upregulation of SOX2, pRB, pAKT, and pSTAT3, accompanied by elevated IL-6, indicating enhanced stemness, proliferation, and pro-inflammatory signaling. Cytokine profiling revealed significant increases in PD-L1and angiopoietin-2 with decrease in soluble VEGF levels following infection and treatment with Gancylovir, reflecting immune suppression and angiogenesis. Ganciclovir treatment modulate pro-tumorigenic cytokines, creating a more favorable tumor microenvironment. Conclusions: CMV infection accelerates spheroid formation and enhances the aggressive phenotype in GBM via upregulation of SOX2, pRB, pAKT, and STAT3/IL-6 signaling, alongside immune evasion and angiogenic cytokine shifts. Our data suggest the therapeutic potential of targeting CMV and its downstream pathways in GBM. These findings highlight the critical role of CMV in GBM pathogenesis and its promise as a therapeutic target. Citation Format: Vida Tajiknia, Noe Mercado, Jacquelin Real, Eain Murphy, Sean Lawler. Cytomegalovirus infection enhances spheroid formation and drives aggressive phenotype in glioblastoma via upregulation of SOX2 pRB pAKT and STAT3/IL-6 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3070.