Abstract 4649: Design and validation of ultrahigh-plex discovery panels for immuno-oncology and oncology

Abstract Introduction Ultrahigh-plex single-cell spatial phenotyping is revolutionizing cancer research through deeper interrogation of cellular and protein-level co-expression, localization, and arrangements within cancer tissues. To enable in-depth characterization of the tumor microenvironment (TME), we have developed and validated pre-optimized antibody panels on cancer tissues to reveal key cell types and cellular architecture using the PhenoCycler™-Fusion system. Here, we present our methodology for the development and validation of multiplex spatial phenotyping panels for oncology and immuno-oncology. Methods We designed panel modules such that each module answers specific biological questions about the TME and can be multiplexed together for a more comprehensive view of the structure and function of the tumor and TME. The resulting PhenoCode™ Discovery panel modules include markers that identify key immune cell types, immune activations and checkpoints, mediators of proliferation, and other hallmarks of cancer. Each antibody in the panel module was conjugated to an oligo barcode and stained on human formalin-fixed paraffin-embedded (FFPE) tissue. The slide was imaged on a PhenoCycler™-Fusion platform, where dye-labeled oligo reporters (complementary to the barcodes) are hybridized to the barcode to visualize the antibody. The results were compared serial sections run with a 3,3’-Diaminobenzidine (DAB) chromogenic immunohistochemistry assay to ensure specificity of the antibody was retained. Antibody concentration, exposure time, and corresponding dye were further optimized, verified, and validated as a whole module using tonsil and cancer tissues based on image analysis and intensity analysis, to achieve an ultrahigh-plex detection. Results and Conclusions In this study, we showcased the rigorous process used for development and validation of PhenoCode™ Discovery panel modules and demonstrated how they can seamlessly work together to collect more data out of one tissue sample. The introduction of these pre-optimized antibody panel modules mark a pivotal point in the evolution of ultrahigh-plex spatial phenotyping. They not only simplify experimental design and assay set up, with panels that make it easier for researchers to answer important questions with this technology, but also save precious time and resources, while reducing overall costs. Citation Format: Yan He, Shannon Berry, Olive Shang, Michael McLane, Nicholas Ihley, Yi Zheng. Design and validation of ultrahigh-plex discovery panels for immuno-oncology and oncology. [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 4649.