Background The next breakthroughs in immuno-oncology will be driven by high-plex tools that decipher the spatial arrangement of different cell types within the tumor microenvironment (TME).

Materials and Methods Imaging Mass Cytometry™ (IMC™) is a proven tool for the study of complex cellular interactions in the TME. It utilizes CyTOF®technology for simultaneous assessment of 40-plus protein markers at subcellular resolution without spectral overlap or background autofluorescence, thus providing unprecedented insight into the organization and function of the TME. Despite this, some protein targets are challenging to include in IMC as they have very few or no commercial antibodies available. Moreover, although cellular identity can easily be deciphered through detection of protein targets, knowledge of the cell’s transcriptome improves understanding of cellular function and activation state.

Results Here,we present a robust and reliable workflow that combines the highly sensitive and specific RNAscope™ technology for RNA detection with the multiplexing capability of IMC to visualize key RNA and protein markers in the same tumor samples.

Conclusions The RNAscopeHiPlexv2 assay was combined with protein detection using IMC to evaluate expression of both RNA and protein targets in formalin-fixed, paraffin-embedded (FFPE) tumor tissue microarray (TMA).

J. Pemberton: A. Employment (full or part-time); Significant; Standard BioTools. S. Kala: A. Employment (full or part-time); Significant; Standard BioTools. A. Dikshit: A. Employment (full or part-time); Significant; Standard BioTools. C. Hupple: A. Employment (full or part-time); Significant; Standard BioTools.