Abstract 3494: Dissecting the cellular expression and subcellular localization of lncRNAs implicated as prognostic biomarkers in non-small cell lung carcinomas by RNA ISH
Authors: Anderson Courtney M. , Li Na , Ma Xiao-Jun , Park Emily 
Published: July 2017

Abstract
Background: Long non-coding RNAs (lncRNAs) are involved in many biological processes, including epigenetic modification, cell differentiation, and apoptosis. More recently, lncRNAs have emerged as unique biomarkers that may be associated with a physiological or diseased state, and several lncRNAs have been identified as prognostic markers in a wide variety of human cancers. Because lncRNAs do not translate into protein, their discovery in the tumor is entirely dependent upon RNA detection. The majority of lncRNAs are also expressed at very low levels compared to mRNAs and may be more heterogeneously expressed than mRNAs. Therefore, detection of lncRNAs in tumor biopsies requires a highly sensitive and specific detection method that can discern single-cell and subcellular localization of lncRNA expression.
Methods: Single-molecule RNA in situ hybridization (ISH) is a well-suited method for the detection of lncRNAs in tumor biopsies because it allows for the visualization of single RNA molecules with morphological context. To interrogate the expression pattern of lncRNAs within the tumor and its microenvironment, we performed RNA ISH using the RNAscope assay on FFPE tissue microarrays consisting of archived tissue samples of 53 primary non-small cell lung carcinoma (NSCLC) tumors and 4 adjacent normal lung tissues. We examined the expression of 4 lncRNAs which have been implicated as potential prognostic markers of lung cancer: AFAP-AS1, ANRIL, HOTAIR, and UCA1.
Results: These lncRNAs were detected in approximately 40-60% of the 53 NSCLC tumor cores and in none of the 4 adjacent normal lung tissues. Expression of these 4 lncRNAs was observed predominantly in tumor cells, with little to no detection in the stroma. Some lncRNAs displayed a heterogeneous expression pattern, with some tumor cell foci displaying strong lncRNA signal while other foci in the same tumor did not display signal. Furthermore, some lncRNAs displayed heterogeneous cell expression in the same foci, with some cells expressing very high levels of lncRNA while other cells in the same foci had little to no lncRNA expression. An evaluation of each lncRNA in serial sections revealed that 6 of the 53 NSCLC tumors expressed all 4 lncRNAs in the same tumor foci. Lastly, little to no signal was observed for the prostate cancer-specific lncRNA PCA3 in the NSCLC tumors and no PCA3 expression was detected in adjacent normal lung tissues.
Conclusions: These results demonstrate the ability of the RNAscope ISH assay to detect potential lncRNA biomarkers in lung cancer samples in situ, allowing for the identification of expression heterogeneity within the tissue environment and precise tumor expression patterns of lncRNAs. Identification of the subcellular localization and cell-to-cell expression patterns of lncRNAs can facilitate greater understanding about their specific biological roles in cancer and other diseases.
Citation Format: Courtney M. Anderson, Na Li, Xiao-Jun Ma, Emily Park. Dissecting the cellular expression and subcellular localization of lncRNAs implicated as prognostic biomarkers in non-small cell lung carcinomas by RNA ISH [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3494. doi:10.1158/1538-7445.AM2017-3494