LAUSR

Background: ~75% of diagnosed bladder tumors are non-muscle-invasive, requiring instillation of M. bovis Bacillus Calmette-Guérin (BCG) with recurrence rates observed in ~50% of patients. Treatments for BCG-resistant bladder tumors have lagged because few studies have tried to understand the relationship between timing of tumor recurrence and the state of immune system at the time of recurrence. Natural Killer (NK) cells are very early responders to tumor cells. HLA-E, like PD-L1, is sensitive to IFN-γ and strongly inhibits NKG2A+ NK and CD8 T cells and is commonly upregulated on tumors. We observed that BCG exposure results in chronic activation of NK and CD8 T cells and their acquisition of NKG2A and PD-1 phenotypes. Upon tumor recurrence in all patients sampled, a subset of the tumor cells aggregated in nests, were activated in response to NK/CD8 T cell-derived IFN-γ, and upregulate HLA-E and PD-L1. Spatial sequencing revealed HLA-EBright tumor nests with high levels of CXCL9/10/11 and in significantly closer proximity to NK/CD8 T cells (and Tregs) compared with HLA-EDim tumor nests that lacked CXCL9/10/11. Here, we apply single cell spatial phenotyping to study major subsets of NK cells in the TME of bladder tumors obtained from patient matched tumor sections before and after BCG therapy. Purpose of the study: The goal of this study is to understand the underlying cell-to-cell interactions that promote BCG resistance in non-muscle-invasive bladder cancer (NMIBC). Methods: The PhenoCycler technology centers around an oligo-based barcoding strategy, in which oligonucleotide-labeled antibodies are imaged in iterative cycles of hybridized/dehybridized complementary oligonucleotide fluorescent reporters. The platform enables (1) whole-slide imaging, (2) single-cell and subcellular resolution, (3) compatibility with a wide range of FFPE tissues, (4) customized content and plexing capability and (5) sophisticated analysis of cell phenotypes and cellular neighborhoods. Results and conclusions: We developed a comprehensive antibody panel for in-depth analysis of the TME of bladder tumors. Our panel centers around a core of 67 markers for cell lineage, immune checkpoints, immune activation markers, tissue structure and vasculature. This core was supplemented with a module of additional antibodies that define complex NK cell phenotypes, including markers of maturation and differentiation, metabolism, activation and inhibition, trafficking, cytolytic killing, as well as cytokines/chemokines that shape the TME. The power of single cell spatial biology is the ability to visualize all major subsets of NK cells and their associated microenvironment simultaneously in single tissue sections. In doing so, we are defining novel functional interactions as new correlates of BCG resistance and identifying pathways that can be exploited for next-gen immunotherapies. Citation Format: Dmytro Klymyshyn, Niyati Jhaveri, Nadine Nelson, Michael Prater, Zhenqin Wu, Steven Hamel, Matthew D. Galsky, Nina Bhardwaj, John P. Sfakianos, Subham Basu, Oliver Braubach, Amir Horowitz. Single cell spatial phenotyping of bladder tumors with a novel mechanism of NKG2A and HLA-E mediated resistance to BCG immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5648.