New Zealand has one of the highest incidences of colorectal cancer (CRC) in the world, resulting in over 1200 deaths per year. Current screening options are either invasive/expensive (e.g. colonoscopy), or lack sensitivity for early stage disease (faecal immunochemical tests). Recent research highlights the role of extracellular vesicles (EVs) as potential diagnostic or predictive biomarkers for CRC. Previous studies on CRC-derived EVs have relied on plasma/serum from CRC patients and two-dimensional (2D) immortalised cell lines, which poorly represent primary tumour characteristics. Recently, three-dimensional (3D) cultures have been developed with CRC cells, which simulate primary tumours to a greater extent than 2D cultures. This study aims to compare EVs between 2D and 3D models in CRC cell lines, by analysing EV concentrations and sizes by tunable resistive pulse sensing and characterizing protein and RNA content.
Standard 2D and Matrigel-based 3D culture models have been developed using HCT116, DLD1 and SW837 CRC cell lines. EVs were isolated from culture media using size exclusion columns (qEV, Izon). RT-qPCR has been performed on total RNA from 2D cultured cells, and EV RNA isolated from 2D culture media, for small non-coding RNA (sncRNA) markers known to be overexpressed in CRC. EVs are currently being isolated from 3D culture media and RT-qPCR will be performed on EVs and cells for the same markers. The fold-change of sncRNAs will be compared between 2D and 3D cultures for all the cell lines, normalised to miR-16. Western blotting will determine protein expression of known markers overexpressed in CRC, including EpCam and CD147.
We have demonstrated expression of miR-21 and miR-29a in cells and EVs isolated from 2D cell culture media. The expression of miR-21 and miR-29a was similar across the 3 cell lines for total RNA. Interestingly, in HCT116 cultures, miR-21 was enriched in EVs over cells by 2-fold, however no other cell lines showed the same enrichment.
The results from this study will provide insight into the influence of cellular architecture on the production and output of EVs and whether 3D cultures can be utilised for ongoing EV studies in CRC.