Characterization of Extracellular Vesicle LINE‐1 mRNA, Protein, and Reverse Transcriptase Activity Profiles

Abstract

Lung cancer has the highest mortality rate among all cancers, making early detection of this malignancy imperative for survival. Ongoing studies in our laboratory have focused on developing Long Interspersed Nuclear Element‐1 (LINE‐1) in circulating lung extracellular vesicles (EVs) as a novel mechanism‐based ‘liquid biopsy’ for lung cancer. LINE‐1 is a mammalian retroelement that can ‘copy and paste’ itself and other DNAs into different loci via a reverse transcriptase‐mediated mechanism. While LINE‐1 is epigenetically silenced in most healthy cells, its activity can be initiated by harmful environmental exposures and is strongly associated with cancer risk and mortality. Evidence is presented here that LINE‐1 mRNA and protein are present in EVs isolated from H460 lung cancer cells and human plasma and are segregated into distinct EV populations. Because reverse transcription (RT) of LINE‐1 requires formation of a ribonucleoprotein complex containing LINE‐1 mRNA and proteins, we hypothesize that this segregation has been engineered to control RT. In support of this hypothesis, we found that RT activity is only present when these two EV populations are lysed and combined. We then isolated EVs from ostensibly healthy plasma donors and found substantial inter‐individual variability with respect to LINE‐1 protein, mRNA, and RT activity. The variability between individuals and the ease of measurement from clinical samples further supports the use of these LINE‐1 metrics as health‐related biomarkers. After characterizing EV LINE‐1, we sought to determine whether EV LINE‐1 levels could serve as a proxy of cellular LINE‐1 content, which is critical to our overall goal of using isolated lung EVs as a liquid biopsy of the lung epithelium. Using a panel of six different lung epithelial cell lines, we compared baseline LINE‐1 expression between cells and EVs. We also treated lung epithelial cells with different concentrations of benzo(a)pyrene (BaP), a cigarette smoke carcinogen known to induce LINE‐1, and compared LINE‐1 levels in cells and EVs. Thus far we discovered that, in both baseline and toxicant‐induced scenarios, EV LINE‐1 mRNA levels mirror cellular levels. Together, these data demonstrate that EV LINE‐1 content and RT activity can be quantified in cultured cells and clinical samples and supports the use of EV LINE‐1 cargo as a biomarker of lung epithelial cell status.Support or Funding InformationThis work was funded in part by grants from the University of Arizona Health Sciences, the Southwest Environmental Health Sciences Center (P30 ES006694), the National Cancer Institute Cancer Center Support Grant (P30CA023074), the UAZ Bio5 Fellowship and GURI Texas.