Abstract 3775: Multi-omic spatial analysis with simultaneous detection of small RNAs, mRNAs and proteins using the novel RNAscope™ Plus technology
Authors: Dikshit Anushka , Basak Sayantani , Deshpande Sonali , Sambhi Manvir , Wang Li-Chong , Srinivasan Maithreyan 
Published: April 2023

Abstract
Regulatory RNA molecules such as microRNAs (miRNA) and long non-coding RNAs play critical roles in regulating translation of mRNA to protein. Several miRNAs have been implicated in disease initiation and progression, especially in cancer. The regulatory mechanisms of miRNAs and other small RNAs such as antisense oligos (ASOs) and silencing RNAs (siRNA) have been exploited to develop oligonucleotide therapies for 1. undruggable targets, 2. achieving longer-term effects 3. rapid production of therapeutics and 4. lower drug development costs. These therapeutics have the promise to treat debilitating neurodegenerative diseases, rare and inherited disorders. RNA therapies are typically delivered as a part of nanoparticles or a viral vector. Regardless of the delivery mechanism, methods to understand the biodistribution of the vector, transgene expression and cell type identification in a spatial context is crucial to study the safety and efficacy of these therapies. Built on the flagship RNAscope technology, the RNAscope Plus assay can detect 1 small RNA and 3 mRNA targets using TSA-based fluorescent readouts and is compatible with the new fluorescent Vivid™ dyes. Fixed, fresh frozen samples and formalin fixed paraffin embedded (FFPE) tissues are supported by manual and automated workflows using the Leica Bond Rx system. We have leveraged this technology to investigate spatial expression profile of miRNA and associated RNA targets across different tissue types and applications. We can also combine protein detection using a target antibody to visualize cell-type specific markers. This assay was used to demonstrate expression of miRNAs and target genes implicated in tumor initiation, progression, and angiogenesis. Expression of miR-205, associated tumor target genes such as PanCK, PTEN and tumor suppressor TP53 was visualized in head and neck cancer tumors. Downregulation of tumor-suppressor, TP53 resulted in upregulation of miR-205 which downregulates PTEN expression. Pan-CK stained the tumor region in the tissue. Similarly, miR-155 expression was observed in niche areas within breast cancer, head and neck cancer and cervical cancer tumors. Expression of miR-155 demonstrated correlation with high VEGF expression suggesting its role in angiogenesis. This novel platform will enable researchers to visualize regulatory RNA simultaneously with target RNAs, cell-type and morphology markers in intact cells/tissues with single cell resolution. This technology can provide meaningful insights into disease pathology driven by miRNAs as well as assess biodistribution and efficacy of oligonucleotide therapeutics.
Citation Format: Anushka Dikshit, Sayantani Basak, Sonali Deshpande, Manvir Sambhi, Li-Chong Wang, Maithreyan Srinivasan. Multi-omic spatial analysis with simultaneous detection of small RNAs, mRNAs and proteins using the novel RNAscope™ Plus technology. [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 3775.