Development of an ultra-high-plex immunofluorescence assay to investigate the tumor-immune microenvironment on the CellScape platform with EpicIF 2309991

Abstract Introduction Spatial biology has transformed cancer immunology, but single-cell in-situ proteomic data remain challenging to acquire. To streamline spatial proteomic assay development we developed EpicIF™, a proprietary signal removal chemistry for the CellScape platform for precise spatial phenotyping (PSP). We previously demonstrated that EpicIF efficiently eliminates fluorophores between imaging cycles while preserving tissue morphology and antigenicity. Here we demonstrate how EpicIF, with key enhancements to the CellScape platform, enable ultra-high-plex PSP to study the tumor immune microenvironment (TIME). Methods To test the feasibility of an ultra-high-plex spatial assay on CellScape, we subjected tissue to 50 EpicIF signal removal cycles and then applied a small 30-plex antibody panel to image protein markers and compared their staining to known expression patterns. We then developed a 100-plex panel to profile the TIME with commercially sourced fluorophore-conjugated antibodies targeting various immune phenotypes, signaling pathways, and tissue architecture. Downstream analysis was performed to highlight spatial phenotypes within the TIME. Results Following 50 rounds of EpicIF-based signal removal we found signal reduction in only 2 out of 30 markers from our representative panel, suggesting a marginal effect on antigenicity and high likelihood of success for an ultra-high-plex assay. We then successfully applied our 100-plex panel to additional tumor tissues, enabling deep phenotyping of the TIME. Conclusion Key enhancements in imaging throughput and stability across long assays on the CellScape, along with EpicIF technology, establish a new benchmark for rapid, reliable, and ultra-high-plex spatial proteomic studies of the TIME. Funding Source N/A Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)