Aberrant expression of the oncogenic retrotransposon LINE-1 is a hallmark of various cancer types, including non-small cell lung cancers (NSCLCs). Here, we present proof-of-principle evidence that LINE-1 analytes in extracellular vesicles (EVs) serve as tools for molecular diagnostics of NSCLC, with LINE-1 status in tumor cells and tissues mirroring the LINE-1 mRNA and ORF1p cargos of EVs from lung cancer cell culture conditioned media or human plasma. The levels of LINE-1 analytes in plasma EVs from ostensibly healthy individuals were higher in females than males. While the profiles of LINE-1 mRNA and ORF1p in African Americans compared to Hispanics were not significantly different, African Americans showed slightly higher ORF1p content, and 2–3 times greater ranges of LINE-1 values compared to Hispanics. Whole plasma ORF1p levels correlated with EV ORF1p levels, indicating that most of the circulating LINE-1 protein is contained within EVs. EV LINE-1 mRNA levels were elevated in patients with advanced cancer stages and in select patients with squamous cell carcinoma and metastatic tumors compared to adenocarcinomas. The observed EV LINE-1 mRNA profiles paralleled the patterns of ORF1p expression in NSCLC tissue sections suggesting that LINE-1 analytes in plasma EVs may serve to monitor the activity of LINE-1 retroelements in lung cancer.

Background: Long INterspersed Element-1 (LINE-1) retrotransposons comprise 17–20% of the human genome. These retroelements are normally silenced early in embryonic development through epigenetic mechanisms and reawakened during oncogenesis, leading to transcriptional dysregulation, genomic instability, and immune evasion. Methods: In the present study, we categorized LINE-1 transcripts across 121 non-small cell lung cancer (NSCLC) cell lines from the Cancer Cell Line Encyclopedia (CCLE) by subfamily, length, orientation, chromosomal origin, and distribution. In addition, high-prevalence insertions were mapped to nearby genes to assess potential functional interactions. Results: LINE-1 transcript abundance and length in NSCLC were dominated by evolutionarily young subfamilies, particularly L1HS and L1PA2 through L1PA5. Chromosomal patterns were conserved across NSCLC subtypes, with modest enrichment of L1HS activity on Chromosome 4 and the X Chromosome. The lung squamous cell carcinoma (LSQCC) subtype exhibited the highest total levels of L1HS expression relative to other NSCLC subtypes. Race modestly influenced LINE-1 transcript abundance, with cell lines derived from self-identified African American individuals showing elevated overall LINE-1 and L1HS expression. Age showed a weak positive correlation with total LINE-1 abundance. Integrative analysis revealed recurrent hotspots at 22q12.1 and 20p11.21 that were transcriptionally active across subtypes and coincided with previously reported intact LINE-1 elements active in epithelial cancers. Recurrent insertions were located near cancer-associated genes, including RB1, NEDD4, FTO, LAMA2, NOD1, and KCNB2, implicating LINE-1 activity in cis-regulatory remodeling of oncogenic pathways. Conclusions: Together, these findings indicate that LINE-1 transcript heterogeneity in NSCLC is shaped by host genomic architecture and conserved functional hotspots, providing new insights into the mechanisms of genetic and epigenetic dysregulation associated with LINE-1 retroelements.