Abstract 3761: Mediators of RNA sorting and export in non-small cell lung cancer derived extracellular vesicles

Extracellular vesicle (EV) mediated transfer of biologically functional molecules between cancer cells and the surrounding environment significantly impacts cancer progression. Our previous study established that non-small cell lung cancer (NSCLC) cell-derived EV-RNA is an important mediator of this communication, since it significantly augments the invasive potential of recipient non-tumorigenic cells (BEAS2Bs). This led us to determine the EV-RNA cargo that is specific to NSCLC EVs. Our RNA sequencing analysis revealed preferential enrichment of specific repertoires of RNA in NSCLC EVs amongst which miR-10b, miR-100 and miR-155 show synergistic effects with regard to an invasive phenotype elicited in recipient BEAS2B cells. Interestingly, our RNA sequencing analysis also revealed a global decrease in specific subsets of microRNAs (miRNAs) in NSCLC (H358 and Calu6) cells in comparison to non-tumorigenic BEAS2B cells. In contrast, the dysregulated miRNAs were found to be exclusively abundant in the respective NSCLC cell derived EVs. This suggests a precise and active mechanism of RNA sorting and loading into EVs. A cohort of dysregulated miRNAs was shortlisted, and their cellular and EV expression was validated using RT-qPCR and flow cytometry analysis by conjugating each one of the candidates to a fluorophore. After confirming that inclusion of a fluorophore to the RNA does not disrupt RNA sorting into the EVs, we transfected the cohort of shortlisted miRNAs including miR-122, miR-451a, miR-486, miR-150, miR-200b, and miR-142 each conjugated with a separate fluorophore and evaluated the dynamics of export. As a control, one cell-retained miRNA was also transfected with the pool of candidate miRNAs and the fluorescence of transfected cells was monitored at different times. Our results indicate that of all miRNAs, the fluorescence specific to miR-122 and miR-150 diminishes in less than 72 hours while others are retained longer, indicating that all miRNAs are not sorted or exported similarly. EV-RNA release was validated by treating the cells with an inhibitor of EV biogenesis i.e., nSMase inhibitor Gw4869. The inhibitor caused the cells to retain the candidate fluorescence corresponding to the respective EV-RNAs. To investigate sequence similarities between the miRNAs exclusively enriched in EVs of NSCLC cells, multiple sequence alignment analysis (STREME) was conducted, and specific motif sequences overrepresented in EVs and lost in the cells were shortlis