Ultra-high-plex immunofluorescence analysis of the tumor-immune microenvironment with EpicIF technology on the CellScape platform

Spatial biology continues to transform cancer research by revealing the intricate molecular and cellular architecture of tissues. Yet, our understanding of the complex processes in the tumor microenvironment is still incomplete, partly because single-cell in situ proteomic data remain challenging to achieve. Most high-plex imaging approaches rely on complex and indirect detection schemes that restrict accessibility and decouple detection from direct visualization. To overcome these limitations, we developed EpicIF™ technology for the CellScape platform for precise spatial phenotyping. EpicIF™ technology is a novel multiplex immunofluorescence (mIF) method that employs a proprietary signal-removal chemistry to efficiently eliminate fluorophores between imaging cycles while preserving tissue morphology and antigenicity. This allows repeated rounds of staining and imaging with fluorophore-labeled primary antibodies, thereby eliminating the need for barcoding, oligo labeling, or sequencing-based decoding. Using this chemistry, we first determined biomarker and tissue integrity after 50 EpicIF-based signal removal cycles to ensure feasibility of an ultra-high plex assay. Of 30 representative markers tested, only 2 showed reduction in signal intensities, while the majority of biomarkers showed little or no significant reduction at all. We then performed direct cyclic immunofluorescence imaging of more than 200 protein targets within a single FFPE tissue section. Our antibody panel was entirely sourced from commercially available fluorophore-conjugated antibodies, and it targets a broad spectrum of biomarkers to profile tumor cells, immune cell subsets, the tissue architecture, and key signaling pathways. This broad immune-oncology focused antibody panel was used to stain more than 200 tumor tissues, including breast cancer, lung cancer, melanoma, glioma, colorectal cancer, and other cancer types. The practical realization of this 200-plex immunofluorescence assay was made possible through key enhancements to the CellScape platform, enabling higher imaging throughput, and sustained stability across extended acquisition runs. This platform establishes a new benchmark for spatial proteomics by combining assay innovation with high-throughput instrumentation to deliver rapid, reliable, and ultra-high-plex imaging at single-cell resolution. This approach provides a powerful framework for dissecting the complex cellular and molecular landscape of the TME and opens new avenues for biomarker discovery, therapeutic stratification, and mechanistic insights into tumor-immune dynamics.