Precise spatial multiplexing of immune cell diversity in clinical FFPE tumor samples with ChipCytometry™

Abstract Highly multiplexed spatial biomarker analysis has demonstrated the potential to advance our current understanding of the immune system and its role in cancer – from tumor initiation to metastatic progression. Previously, a trade-off between plex and spatial context meant that our understanding of immune cell involvement in cancer was limited by either single-plex technologies with spatial context (i.e., immunohistochemistry) or highly multiplex technologies without spatial context (i.e., single-cell RNA sequencing). ChipCytometryTM is a novel, highly multiplexed technology that preserves both plex and spatial context to deeply profile immune cell diversity at single-cell resolution. ChipCytometry uses commercially available antibodies and combines iterative immuno-fluorescent staining with high-dynamic range imaging to profile dozens of protein biomarkers in a single tissue specimen. Cellular phenotypes are identified with via flow cytometry-like hierarchical gating from standard multichannel OME-TIFF images, compatible with a variety of computational tools being developed for multiplexed analysis and visualization. Here, we use ChipCytometry to identify and quantify key immune cell subtypes in both normal (i.e., appendix) and tumor tissues (i.e., melanoma and non-small cell lung cancer) FFPE samples. The results show precise expression levels for each biomarker in the assay in each individual cell in the sample, while maintaining spatial positioning of each cell. Spatial analysis reveals quantifiable heterogeneity of immune cell infiltration within the tumor samples, demonstrating the utility of the ChipCytometry platform for the in-depth immune profiling in clinical FFPE samples.