Abstract 2179: Multiomic co-detection of proteins, mRNA and protein-protein interactions reveal PD1-PDL1 interactions in the tumor microenvironment of bladder cancer patients treated with anti-PD-L1

Abstract Protein-protein interaction (PPI) is one of the mechanisms where individual cells communicate with nearby cells or the extracellular matrix through autocrine, juxtacrine and paracrine mechanisms. The network of interactions and the underlying stoichiometry both within a tissue and with the immune repertoire is adversely impacted in many diseases, including cancer. While screening technologies to predict and map the protein interactome are advancing our understanding of intercellular/intracellular protein-protein interactions (PPIs), no orthogonal technologies exist that can validate inferred PPIs in the context of known mRNA/protein markers. To address this need, we have developed a technology that uses modifications to RNAscopeTM - a validation technology known for its high sensitivity and specificity - to visualize PPIs, proteins, and mRNA in situ on a single tissue section. Oligonucleotides that bind RNAscope signal generation complexes were conjugated to antibodies and protein targets were visualized in situ along with PPIs/mRNAs using a fully automated, protease-free RNAscope Multiomic LS workflow on a BOND RX instrument followed by whole slide scanning and analysis. The assay, which interrogate one PPI and any combination of up to 5 mRNA and protein targets, was deployed on samples from patients with localized, muscle-invasive bladder cancer who received neoadjuvant anti-PD-L1 checkpoint inhibitor therapy prior to planned surgical cystectomy on a clinical trial (NCT02451423). To characterize tumor immune microenvironment of these patients, both responders and non-responders to the treatment, PD-1/PD-L1 interactions were assessed using a panel of targets to include cell phenotyping protein markers for both immune and tumor cells (CD3, CD4, CD8, FoxP3, and PanCK) and mRNA markers for effector molecules (IFNG, GZMB, GZMK). We visualized PD-1/PD-L1 interactions, and changes in cellular interactions between cytotoxic CD8+ T cells or regulatory CD4+ T cells and CK+ tumor cells before and after immunotherapy treatment. We will present data to illustrate the novelty of the assay as well as secondary analysis to characterize the importance of PPIs for more granular dissection of immune modulation in the tumor microenvironment. Citation Format: Ge-Ah Kim, Sonali A. Deshpande, Li-Chong Wang, Julia Yu, Hamidreza Sepasizangabadi, Ji Zhang, Jason Au, Kathryn Allaire, Junyan He, Maithreyan Srinivasan, David Y. Oh, Lawrence Fong. Multiomic co-detection of proteins, mRNA and protein-protein interactions reveal PD1-PDL1 interactions in the tumor microenvironment of bladder cancer patients treated with anti-PD-L1 [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 2179.