Abstract 799: Integrated spatial multiomics pipeline for visualizing protein interactions in the tumor microenvironment.

Abstract Protein-protein interaction is one of the many mechanisms where individual cells communicate with nearby cells or extracellular matrix to modulate the tissue environment. Visualizing these interactions using a spatial platform can validate known interactions implicated in disease pathology within relevant spatial domains, especially in cancer. We developed the ProximityScope™ assay that can be used with the RNAscope™ Multiomic LS assay to detect protein interactions and their impact on molecular pathways by simultaneously visualizing proteins and RNA on a single tissue section. This workflow is fully automated on Leica Biosystems’ BOND RX staining platform. The assay detects one protein-protein interaction and can be combined with five RNA/protein targets simultaneously on the same section. Here, we demonstrate a complete end-to-end workflow by staining FFPE human tonsil tissue samples to detect PD1-PDL1 interactions along with immune cell markers such as PDCD1 RNA, CD68 RNA, CD4 protein, CD8 protein, and PanCK protein. Images were acquired using the Aperio FL 120 by Leica Biosystems.[AD1] Using the HALO® image analysis software from Indica Labs, we performed RNA and protein quantification as well as cell phenotyping. PD-1/PD-L1 interactions were successfully visualized between immune cells and tumor cells. T cells were identified by either CD8 or CD4 protein detection, macrophages were identified with CD68 RNA, while tumor cells were identified by PanCK protein staining. Interactions appear as punctate dots or dot clusters between two cells or on the cell surface. HALO software identified the PD-1/PD-L1 interaction between adjacent cells. This study demonstrates the importance of having an end-to-end spatial solution for staining, imaging, and quantification of target protein interactions to evaluate protein function. The ProximityScope assay has the potential to study a broad range of protein interactions to evaluate signaling pathways and gain biological insights, assess therapeutic success, or detect PD-1/PD-L1 and related interactions that can serve as biomarkers for patient stratification.For Research Use Only. Not for use in diagnostic procedures. Citation Format: Anushka Dikshit, Josh Brownlee, Ge-Ah Kim, Sonali Deshpande, Diane Robirds, Anne Hellebust. Integrated spatial multiomics pipeline for visualizing protein interactions in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 799.