Abstract 537: Evaluation of immune function in the tumor microenvironment by RNA <i>in situ</i> hybridization to reveal spatial information on immune cell infiltration and the expression of functional mRNA biomarkers
Authors: Ming-Xiao He, Courtney Anderson, Na Li, Xiao-Jun Ma, Emily Park
Journal: Cancer Research (2016)

Abstract Growing evidence for the antitumor activity of the immune system has supported recent success in cancer immunotherapy. Therapeutic approaches such as anti-cancer vaccines and suppression of immune checkpoints have been successfully applied in various cancers to raise specific immune responses and to enhance antitumor immune responses. In particular, blocking immune checkpoint pathways such as CTLA-4 and PD-1/PD-L1 is transforming cancer therapeutics. Based on these successes, clinical trials of combinatorial strategies are in progress to target multiple pathways. While many biomarker analysis technologies are employed to guide specific therapeutic approaches, spatially mapped biomarker expression data at the single-cell level is crucial to understanding the composition and activities of various immune cells in the tumor microenvironment. In this study, in order to visualize gene expression of key immune biomarkers in the tumor microenvironment, we have applied the RNAscope® technology, an RNA in situ hybridization (ISH) method that provides single molecule RNA detection with spatial and morphological context. This highly sensitive ISH presents a unique advantage to the visualization of mRNA biomarkers for secreted proteins such as cytokines and chemokines in individual cells. Specific probes for key cytokines (e.g., IL6, IL12A, TGF-β, IFN-γ, TNF-α), chemokines (e.g., CCL5, CXCL9, CXCL10, CXCL13), and immune subset markers (e.g., CD3, CD4, CD8A, FOXP3) were designed in addition to key receptor/ligand pairs in immune check points (e.g., PD1/PD-L1, OX40/OX40L). Expression of each target and combinations of multiple targets were assessed on formalin-fixed paraffin embedded (FFPE) tissue sections derived from multiple tumor types. Using this approach, the various functional mRNA biomarker profiles of the immune cell types infiltrated in the tumor are directly visualized as punctate dots. The visible mRNA dots are readily quantifiable, so that enumeration of immune cells is feasible. RNAscope® based ISH is well-suited to study immune function in the tumor microenvironment by visualizing gene expression. By applying this method, it is feasible to evaluate the relationship of specific biomarkers in different cell types. With the emerging effort towards combinatorial approaches to target multiple pathways, the method presented here can be applied as a universal platform to study both cancer specific molecular pathways as well as immune function. Citation Format: Ming-Xiao He, Courtney Anderson, Na Li, Xiao-Jun Ma, Emily Park. Evaluation of immune function in the tumor microenvironment by RNA in situ hybridization to reveal spatial information on immune cell infiltration and the expression of functional mRNA biomarkers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 537.