Poster Presentation Australasian Extracellular Vesicles Conference 2020

Understanding the role of Extracellular Vesicles in microenvironment manipulation in Merkel Cell Carcinoma (#57)

Priscila Dauros-Singorenko 1 2 , Vanessa Chang 3 , Cherie Blenkiron 1 3
  1. Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand
  2. Surgery, University of Auckland, Auckland, New Zealand
  3. Obstetrics and Gynecology , University of Auckland, Auckland, New Zealand

Introduction. Merkel Cell Carcinoma (MCC) is a rare, aggressive, skin neuroendocrine tumour. A Polyoma virus (MCPyV) has been identified as irreversibly integrated into the DNA of MCC tumour cells in ~80% of Northern hemisphere cases, in contrast to Southern hemisphere, where most prevalent cases are MCPyV negative and associated with a more aggressive clinical course. MicroRNAs (miRNAs) are known regulators of many cellular functions and often play important role in cancer pathophysiology. To better understand the role of miRNA and Extracellular Vesicle (EV) signalling in MCC pathology of these two aetiologies, we characterised the miRNA content in EVs from MCC cell-lines. Method. MCC13, MCC14/2 and MCC26 (virus negative, adherent lines) and MS-1 and MKL-1 (virus positive, suspension lines) were grown in Advanced RPMI media with minimal EV-depleted FBS. Cells were counted and EVs isolated from media after at least 2 doubling times. EVs were isolated using sequential centrifugation (2,000xg, 10,000xg) and concentration (100KDa filter) before purifying using size exclusion chromatography. NanoEV rich fractions were chosen based on Nanoparticle Tracking Analysis and protein quantity data. RNA was extracted from purified NanoEVs and their matching donor cells. RNA was quantified by Qubit HS RNA assay and analysed by miRNA Affymetrix microarray to assess presence, range and abundance of miRNAs. Results. Although virus positive cell-lines are slow to proliferate and grow as spheroids, they released significant numbers of EVs (966-4678/cell) in comparison to the rapidly proliferating adherent virus negative lines (163-993/cell). There was no distinctive trend in protein amount per EV. Extracted RNA from all nanoEV preparations was below the limit of detection of the quantification assay. miRNA profiling showed a wider distribution in EVs from the two MCPyV positive lines, with 2 to 5 times more detectable miRNAs than MCPyV negative lines, including miR-375 which has been reported as a valuable plasma biomarker for MCC diagnosis and monitoring. Conclusions. Virus positive MCC cell-lines release significant quantities of EVs that are associated with protein and packaged with selected miRNAs that in part reflect the cellular content. These EV cargos will be further investigated for their function in microenvironment regulation in MCCs.