Comprehensive immune landscape analysis of solid tumors using a 17-Plex immunofluorescence assay in clinical trials

Background: The tumor micro-environment is highly complex; accumulating evidence indicates that immune cell composition and spatial distribution can contribute to disease progression and inhibition of antitumor immunity. There is a growing effort to leverage new technologies for in-depth analysis of the complexities within the tumor immune microenvironment. We validated a 17-plex, subcellular resolution, pan-immune cell panel to interrogate the immune cell types and their spatial organization in whole tissue sections. Methods: We co-developed a 17-plex pan-immune cell immunofluorescence (IF) assay with RareCyte® utilizing the Orion™ platform to characterize immune cell populations and their functional states in formalin-fixed paraffin-embedded (FFPE) tissues from melanoma, breast, lung, prostate, and colorectal cancers. A single, 4-micron FFPE section was stained in one-step, using the intelliPATH™ FLX (Biocare Medical) with a cocktail of antibody-fluorophore conjugates targeting Granzyme B, Ki67, CD3e, CD20, CD4, CD163, CD8a, PDL1, NKp46, PD-1, LAG3, FOXP3, CD11b, CD138, SOX10, Cytokeratin, and Hoechst nuclear stain. Imaging was performed on the Orion™ platform, and data were analyzed using HALO® (Indica Labs). Results: The assay enabled comprehensive spatial profiling of T, B, NK, and myeloid cell distributions within tumor and stromal compartments. Functional states of T cells (cytotoxic, regulatory, exhausted) and macrophage/B-cell niches were distinguished based on immune checkpoint and activation marker expression. Proximity analyses identified immune synapses and organized lymphoid structures, facilitating robust microenvironment characterization. Conclusions: The Orion™ 17-plex pan-immune cell IF assay will enable comprehensive qualitative and quantitative analysis of the tumor microenvironment in clinical trial specimens. We anticipate that this approach will support the development of next-generation therapies by enabling detailed characterization of immune cell and tumor cell spatial biology across multiple cancer types.