Spatial analysis of protein or gene expression is vital to understanding the distribution, phenotypes, and interactions between cells within tumor microenvironment (TME). Traditionally, multiplexed spatial analysis has been performed using methods to detect either protein or RNA separately. Combining spatial analysis of protein-RNA on a single specimen is a powerful method to identify the cellular source of secreted proteins, characterize the cytokine signature, study gene expression in specific cell types as defined by protein biomarkers, or map the distribution of CAR-T+ cells within the TME. Here we present a validation study of a combined immunofluorescent (IF) and in situ hybridization (ISH) assay for co-detection of multiple protein and RNA markers on a single formalin fixed-paraffin embedded (FFPE) sample. The validation study was performed on 3 colorectal cancer samples using a 2plx-ISH and 17plx-IF integrated assay for detection of multiple RNA and protein biomarkers. Expression of each RNA and protein biomarker was quantified and inter/intra run coefficient of variation (CV) were calculated for the precision assessment. The results demonstrate robustness of all RNA and protein markers analyzed as well as the ability to spatially correlate RNA expression in different populations of the TME.

Learning Objectives:

1. Identify the advantages of a multiplexed spatial biology approach for immune-oncology research.

2. Demonstrate understanding of a multiplexed IF-ISH spatial assay workflow and the associated challenges.

3. Describe assay validation design and understand methods to analyze and assess results.