Abstract 1537: Understanding role of uniquely enriched RNAs carried in non-small cell lung cancer derived extracellular vesicles and dynamics of their selective export
Authors: Hasan Humna , Lanman Nadia A. , Utturkar Sagar , Kasinski Andrea L. 
Published: June 2022

Abstract
The dynamic interaction mediated by extracellular vesicles (EVs) between cancer cells and microenvironment has been shown to regulate cancer progression. Specifically, tumor derived EVs alter the phenotypes of recipient cells by delivering functional biomolecules including RNAs (EV-RNA). Studies from our lab reveal the impact of NSCLC derived EVs (Calu6 and H358) in promoting invasion of non-tumorigenic cells (BEAS-2B) and disruption of an epithelial barrier. We further determined that of all the major macromolecules contained in EVs, EV-RNA is a significant contributor to the observed phenotypes. Additionally, our data supports dysregulation of RNA subsets enclosed in NSCLC cell-derived EVs in comparison with non-tumorigenic EVs. Interestingly, RNA sequencing analysis revealed preferential enrichment of several small RNAs (&lt;200nt), especially miRNAs, in EVs isolated from NSCLC cell lines which suggest the presence of precise mechanisms involved in loading and export of RNAs into EVs. We shortlisted a cohort of uniquely enriched miRNAs in EVs derived from Calu6 and H358 cell lines to understand their combinatorial effects on non-tumorigenic recipient cells. Amongst the shortlisted candidates miR-100, miR-10b, miR-21, miR-155 and miR-486 stand out as potential contributors to NSCLC-EV mediated function. To understand the dynamics of export of miRNAs into EVs, we transfected Calu6 cells with one of the uniquely enriched fluorescently labelled candidate miRNA (miR-451) and tracked its export into EVs. We first verified that inclusion of a fluorophore to the RNA does not impair RNA loading into EVs. To set the experiment up, one EV-enriched RNA and one Cell-enriched RNA (as a control) conjugated with different fluorophores were transfected into cells, and cellular and EV fluorescence were monitored. Interestingly, cellular fluorescence corresponding to the EV-enriched RNA diminished, while fluorescence corresponding to the Cell-enriched RNA was retained. Fluorescence signal for EV-enriched RNA showed retention in EVs isolated 48 hours after transfection. Validation of release of candidate miRNA into EVs was performed by inhibiting biosynthesis of EVs after treatment with nSMase inhibitor Gw4869. Inhibition of EV release caused EV-enriched RNA to be retained by the cells which was confirmed following flow cytometry analysis of treated cells. Despite clear evidence that dysregulated EV-RNA subsets enclosed in cancer cell-derived EVs can modulate the cellular microenvironment, research showing dynamics and mechanisms of their export is limiting. Hence, there is a critical need to reveal mechanistic details of RNA export into EVs. Resulting data from this study is expected to not only reveal functional EV-RNA subsets and dynamics of their loading but will ultimately point to novel targets for future therapeutic intervention.
Citation Format: Humna Hasan, Nadia A. Lanman, Sagar Utturkar, Andrea L. Kasinski. Understanding role of uniquely enriched RNAs carried in non-small cell lung cancer derived extracellular vesicles and dynamics of their selective export [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1537.