COMBINING BCG IMMUNOTHERAPY WITH STING PATHWAY ACTIVATION TO INDUCE TERTIARY LYMPHOID STRUCTURES IN THE TREATMENT OF NON-MUSCLE-INVASIVE BLADDER CANCER
Bacillus Calmette Guerin (BCG) , Stimulator of Interferon Genes (STING) , Tertiary Lymphoid Structures (TLS) , Non-Muscle-Invasive Bladder Cancer (NMIBC) , Combination Immunotherapy
Following surgical resection, high-risk non-muscle invasive bladder cancer (NMIBC) is best treated with intravesical Bacillus Calmette-Guérin (BCG) immunotherapy. BCG is the live-attenuated form of Mycobacterium bovis and is utilized in an adjuvant setting to recruit immune cells and potentiate anti-tumor immunity. Like other mucosal sites, the bladder has the potential to harbor immune outposts known as tertiary lymphoid structures (TLSs). These ectopic structures resemble the germinal centers of secondary lymphoid organs and are highly organized aggregates of various immune cell populations, including T cells, B cells, and follicular dendritic cells. TLS induction is a type 1 interferon (IFN1)- dependent response to chronic inflammation and has been associated with positive prognosis in multiple cancer types. However, the attenuation of Mycobacterium bovis for clinical use as BCG removes the ability of the mycobacteria to trigger the Stimulator of Interferon Genes (STING) pathway. This pathway plays a crucial role in producing a potent IFN-1 response, and its removal hinders the induction of TLS formation. Therefore, we hypothesized that combining BCG with exogenous STING pathway activation has the potential to induce TLS formation in the bladder mucosa, which could potentially improve outcomes for patients with NMIBC. We have previously demonstrated the potential of this combination to synergistically upregulate IFN1-associated genes and promote the release of chemokines in vitro. Therefore, our current objective is to determine if simultaneous administration of STING pathway agonist (SA) and BCG immunotherapy will induce TLS formation in the bladder mucosa. Here we show that the combination of BCG and SA induces MB49 murine bladder cancer cell death in vitro. Furthermore, we demonstrate that this combination leads to the upregulation of TLS-associated genes, recruitment of immune cells, and production of pro-inflammatory cytokines in the MB49 orthotopic murine model of bladder cancer. Finally, we characterize the organization and developmental stages of pre-existing, tumor-associated lymphoid aggregates present in tumors from patients with NMIBC. Overall, this work has the potential to improve the treatment of NMIBC by providing rationale for the utilization of synergistic immunotherapy consisting of BCG and STING pathway activation.