Abstract 6483: Powerful end-to-end spatial analyses using precise spatial proteomics

Abstract Spatial biology supports our understanding of the progression and treatment of cancer by distilling complex cellular interactions into actionable spatial signatures. The most actionable targets to study with spatial methods are protein biomarkers, but non-destructive, high-plex spatial proteomics has been challenging to achieve due to technical limitations posted by available technologies. With the recent advent of enhanced photobleaching in cyclic immunofluorescence (EpicIF™) technology, multiplex immunofluorescence (mIF) can now be achieved more easily and inexpensively. EpicIF technology quickly and safely removes signal from even the brightest organic fluorophores, enabling the use of standard fluorescent antibodies in high-plex spatial proteomic protocols. We leveraged EpicIF technology to design modular mIF assay panels targeting key tumor immune microenvironment (TiME) biomarkers, tissue structural features, and markers enabling accurate cell segmentation. These assay panels were validated for performance (including sensitivity, specificity, and reproducibility) across a range of carcinoma tissues using the CellScape™ Precise Spatial Proteomics platform. Our validated panels enabled robust segmentation of immune, tumor and stromal cell types, ultimately permitting robust and comprehensive end-to-end spatial analysis of the TiME. We identified complex spatial neighborhoods composed of cellular interactions between immune infiltrates containing B-cells and T-cells, with mosaic expression of developmental and activation status markers in the TiME. Together, the use of these modular mIF panels, coined Next-Generation VistaPlex™ Assays, provided a fast foundation for accurate spatial analyses. The pairing of EpicIF technology with ready-to-use mIF assay panels provides the first simple, achievable access to spatial proteomics, including those without previous expertise in spatial biology. Citation Format: Charles E. Jackson, Matthew H. Ingalls, Kayla E. Cashion, J Spencer Schwarz, Adam Northcutt, Arne Christians, Oliver Braubach. Powerful end-to-end spatial analyses using precise spatial proteomics [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 6483.