Abstract 961: Comprehensive catalog of culture-derived exosomal microRNA cargo from 4 discrete head and neck carcinoma cell lines
Authors: Scott M. Langevin, Damaris Kuhnell, Tess L. Parry, Jacek Biesiada, Xiang Zhang, Mario Medvedovic, Susan Kasper
Journal: Cancer Research (2016)

Abstract Background: Exosomes are nano-sized (40-100 nm), membrane encapsulated vesicles that are released by cells into the extracellular space and function as intercellular signaling vectors through the horizontal transfer of biologic molecules, including non-coding RNA. MicroRNA (miRNA) are small (18-25 nucleotides in length), evolutionarily conserved, non-coding RNA molecules that are involved in regulation of gene expression in essentially all eukaryotic organisms through post-transcriptional degradation and translational inhibition of messenger RNA (mRNA). The purpose of this study was to catalog exosomal miRNA cargo that are differentially secreted from head and neck squamous cell carcinoma (HNSCC) cells in vitro using Next Generation miRNA-sequencing. Methods: We obtained 4 commercially available HNSCC cell lines: (1) H413 (female, buccal); (2) Detroit 562 (female, pharynx metastatic to pleura); (3) FaDu (male, hypopharynx) and (4) Cal 27 (male, tongue). We additionally obtained HGEPp primary human gingival epithelial cells (pooled from 3 healthy female donors) for comparison. Cells were cultivated in supplier-recommended media with 10% fetal bovine serum (FBS) that was super-depleted of exosomes via 18 hour ultracentrifugation at 100,000g and 1% penicillin/streptomycin at 37°C with 5% CO2 in 150 cm2 flasks with 25mL media. To achieve adequate volume for exosome isolation (50mL), cells were cultured in 2-pair sets of flasks in triplicate (6 flasks total per cell line). Media was collected when cells reached 80-90% confluence. Exosomes were isolated and purified from exosome-depleted cell culture media by differential ultracentrifugation and the presence of purified exosomes was verified via nanoparticle tracking analysis (NTA) using a NanoSight NS300 instrument. Total RNA was extracted from each exosome pellet using the miRNeasy Micro kit, and Next Generation miRNA-sequencing was subsequently performed by the University of Cincinnati Genomics, Epigenomics and Sequencing Core (Cincinnati, OH) using the Illumina HiSeq platform. Differential exosomal miRNA secretion was assessed between each HNSCC cell line and the non-pathologic gingival epithelial control cells, adjusting for false-discovery rate using the Benjamini and Hochberg method. Results: We observed 93 miRNA transcripts that were differentially secreted (Q &amp;lt; 0.05) in exosomes derived from at least one of the HNSCC cell lines relative to the non-pathologic epithelial control cells; 23 miRNA transcripts were differentially secreted by all 4 cell-lines. Conclusions: This provides evidence for differential secretion of miRNA in HNSCC-derived exosomes. These exosomal miRNA warrant further evaluation as potential biomarkers for HNSCC. Citation Format: Scott M. Langevin, Damaris Kuhnell, Tess L. Parry, Jacek Biesiada, Xiang Zhang, Mario Medvedovic, Susan Kasper. Comprehensive catalog of culture-derived exosomal microRNA cargo from 4 discrete head and neck carcinoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 961.