The role of tertiary lymphoid structures in brain tumor therapy with oncolytic adenoviruses - Brain tumors pose formidable challenges to immunotherapy. The unique immune environment of the brain, combined with clinical and biological barriers, severely limits therapeutic efficacy. Glioblastoma and diffuse midline glioma represent the most lethal primary brain tumors, with median overall survival of approximately 12 months or less. Concurrently, advances in cancer therapies have extended the lives of patients with extracranial solid tumors, leading to a rising incidence of central nervous system (CNS) metastases. Metastatic brain tumors affect an estimated 300,000 individuals annually in the United States. Given their profound impact on neurological function and quality of life, there is an urgent need for safer and more effective therapeutic strategies. Oncolytic viruses (OVs) offer unique advantages in overcoming these challenges. By selectively lysing tumor cells and activating both innate and adaptive immune responses, OVs can convert immunologically “cold” tumors into “hot” ones. Our sustained high-impact research on oncolytic adenoviruses (OAs) led to phase I clinical trials demonstrating that Delta-24-RGD is safe and can induce durable responses and long-term survival. Building on this success, we developed Delta-24-RGDOX, an OA expressing the immune co-stimulatory molecule OX40 ligand, which enhanced both local and systemic antitumor immunity in preclinical models. While preclinical and clinical studies show that host immune responses clear OVs within weeks, antitumor immunity often persists for months. The mechanisms sustaining this prolonged immune activity remain poorly understood. In our studies using immunocompetent mouse tumor models and clinical samples, we observed immune cell aggregates, some identified as tertiary lymphoid structures (TLS), within the brains of OA-treated subjects. The presence of TLS correlated with extended median survival. TLS has been associated with improved responses to immunotherapy across cancer types. Unlike secondary lymphoid organs, TLSs arise within the tumor microenvironment, maintaining continuous exposure to tumor antigens and inflammatory cues. This positions them as potent on-site hubs for tumor-specific T cell activation, particularly advantageous within the “immune-privileged” CNS. We also observed TLSs adjacent to tumor stromal cells exhibiting upregulated podoplanin (PDPN) expression. While PDPN+ cancer-associated fibroblasts remodel the extracellular matrix to promote tumor progression, they can also facilitate TLS formation. Based on these findings, we hypothesize that OAs promote TLS formation, in part through stromal remodeling, to sustain antitumor immunity in brain tumors. To test this hypothesis, we propose two specific aims: Aim 1, Define the role of OA-induced TLSs in antitumor immunity in brain; Aim 2, Investigate the role of tumor stroma remodeling in TLS formation during OA therapy. The study requires immunocompetent orthotopic mouse models because OA-induced TLS formation depends on complex interactions between the tumor, immune system, and brain microenvironment that cannot be recapitulated in vitro. It will be the first to define the role of TLSs in OA therapy for brain tumors. Our findings will clarify whether TLSs are essential for effective OA therapy and establish a foundation for developing novel OA-based strategies that harness TLS biology to improve therapeutic efficacy.
