MP06-11: Magnitude of STING Signaling Determines Antitumor Outcomes, and CD8+ T Cell Memory by BCG-STING in Urothelial Tumors

Alok Singh*, Monali Praharaj, Kara Lombardo, Liang Zhao, Joy Huang, Max Kates, David McConkey, William Bishai, Baltimore , MD, Trinity Bivalacqua, Philadelphia, PA

Introduction: Increased antitumor efficacy of BCG-STING (recombinant BCG overexpressing STING agonist, c-di-AMP) over BCG-WT is dependent on type I IFN-mediated myeloid and lymphoid cellular response. Tumor infiltrating macrophage and T cell phenotypes are not fully discussed following therapy with BCG-WT or BCG-STING in absence of functional STING signaling in host. We investigated direct role of STING signaling on tumor infiltrating macrophage polarization and CD8+T cell phenotypes (activation and memory) conferred by BCG-STING vs BCG-WT.

Methods: Syngeneic mouse MB49 flank tumors in female STING+/+ (wild-type C57BL/6J) and STING-/- (C57BL/6J-Tmem173gt/J) mice were intratumorally (IT) treated with BCG-WT and BCG-STING (5 x 106 CFU). Endpoint measurements included tumor volume, weights and multicolor flow-cytometry-based phenotyping of tumor-infiltrating macrophages and lymphocytes (TILs) as correlates of antitumor immunity. Data analyses was done using FACS Diva, FlowJoV.10, GSEA and Prism9.2 with ANNOVA for significance.

Results: STING-/- mice showed increased tumor growth following MB49 grafting as time taken to achieve maximum tumor growth and size was shorter when compared to STING+/+ mice. BCG-STING failed to achieve superior antitumor efficacy over BCG-WT in absence of functional STING signaling as no significant tumor attrition or weight reduction was observed in STING-/- mice. In the myeloid compartment, MB49 tumors in STING-/- had decreased recruitment of TNF-a+CD11b+F4/80+ macrophages in response to BCG-STING when compared with BCG-WT. Overall reduced TIL infiltration (CD3+ lymphocytes) was a hallmark of MB49 tumors in STING-/- mice in response to BCG therapy. The role of host STING in T cell immunity was evident since a decrease in both activation (CD69+) and effector (IFN-g+) marks on CD8+ T cells were observed in MB49 tumor bearing STING-/- mice following IT BCG-WT or BCG-STING. Increased induction of CD44+CD62L+ CD8+ T cells i.e., central memory T cells was observed in MB49 tumors specifically administered with BCG-STING in WT mice.

Conclusions: STING signaling is a prerequisite condition to achieve optimal or superior antitumor efficacies of BCG-WT and BCG-STING in urothelial cancer. Augmented antitumoral phenotypes on tumor infiltrating macrophages and T cells following BCG immunotherapy is lost in absence of STING. We for the first time provide supporting evidence of an undefined role of STING in conferring adaptive immune memory in CD8+ T cells specifically linked with BCG-STING. Detailed investigations could unveil underlying mechanisms of STING-dependent memory T cell responses in tumors.

Source of Funding: National Institute of Health, Maryland Tedco, Willowcroft Foundation, and Cigarette Restitution Fund