Detecting oncogenic protein-coding messenger RNA (mRNA) in tumor tissues is a key assay for accurate cancer diagnosis. Along with cancer protein biomarker analysis, mRNA identification is critical for matching the appropriate treatment to individual patients. Likewise, cytokine mRNA localization in on-treatment biopsies enables evaluation of the antitumor response to immunotherapy by identifying the types and functional states of lymphocytes, for example distinguishing activated cytotoxic T-cells from naive T-cells. To assess mRNA localization, a biopsy is typically formalin fixed and paraffin embedded (FFPE), thin sections are cut, and then are stained for mRNA by chromogenic In situ hybridization (CISH). Analyzing samples by mRNA CISH typically takes a few days and requires considerable technical expertise. Recent progress in multiplexed fluorescence In situ hybridization (FISH) has enabled simultaneous analysis of multiple mRNA types in a single tissue section. Nonetheless, even if fully characterized, individual tissue sections cannot adequately represent the complex three-dimensional (3D) architecture of the tumor microenvironment. There remains a pressing need for a rapid sample-to-answer technology for multiplex 3D imaging of mRNA at sub-cellular resolution that is able to fully characterize the tumor microenvironment in intact biopsy tissues for cancer diagnosis and immunotherapeutic response analysis. To address these challenges, we have adapted Transparent Tissue Tomography (T3), a simple and fast tissue clearing and multiplex 3D imaging method, to analyze mRNA distribution in the tumor microenvironment. With T3, oncogenic and immunoregulatory cytokine mRNAs are readily localized in the context of cell types and other features in murine, patient derived xenograft (PDX), and head and neck tumors. This work establishes T3 as a tool for a quantitative, 3D spatial analysis of mRNA and protein distribution in the tumor microenvironment.

Citation Format: Steve Seung-Young Lee, David Scholten, Vytautas P. Bindokas, Stephen Kron. Multiplex three-dimensional mapping of mRNA and protein in the tumor microenvironment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B025.