Abstract 4636: High-plex co-detection of RNA and protein to explore tumor-immune interactions utilizing RNAscope with Imaging Mass Cytometry
Authors: Pemberton James , Kala Smriti , Dikshit Anushka , Hupple Clinton 
Published: April 2023

Abstract
Purpose 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). 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. 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.
Methods The RNAscope HiPlex v2 assay was combined with protein detection on the Hyperion+™ Imaging System to evaluate expression of both RNA and protein targets in formalin-fixed, paraffin-embedded (FFPE) tumor tissue microarray (TMA). The RNAscope assay was used with 12 target RNA marker probes and associated metal-labeled detection probes, suitable for use in IMC. The RNAscope HiPlex v2 assay workflow was followed as recommended up until applying the fluoros. Instead of using the fluoros, metal-conjugated probes were used for RNA detection. Metal-conjugated antibodies were used for protein detection in the same tissue.
Results Target protein markers identified a range of immune cells, tumor cells, stromal cells, endothelial cells, and extracellular matrix. Co-detection of RNA and protein allowed visualization of several cytokines and chemokines, such as CXCL13, CXCL9, CXCL10, IFNγ, IL10, IL8, and IL1B, enabling the identification of the cellular source of these secreted factors. Additionally, immune cell subpopulations and their activation states were visualized using marker-specific antibodies. Immune cell hubs associated with anti-tumor immune responses were detected in tumor niches across the TMA.
Conclusions Overall, combining RNAscope with protein co-detection on the IMC platform allowed simultaneous visualization of RNA and protein targets to interrogate the tumor microenvironment. The single-cell resolution offered by the RNAscope assay provides superior sensitivity for RNA detection in addition to the existing protein-detection capability of the IMC platform. This workflow can be applied to FFPE tissues to study therapeutic efficacy, stratify patients based on inflammatory signature, and study cell-cell interactions within the TME.
Citation Format: James Pemberton, Smriti Kala, Anushka Dikshit, Clinton Hupple. High-plex co-detection of RNA and protein to explore tumor-immune interactions utilizing RNAscope with Imaging Mass Cytometry. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4636.