Colorectal cancer is highly prevalent in New Zealand, with an increasing incidence of early-onset disease. Effective and earlier diagnosis would improve CRC treatment and mortality. Extracellular vesicles (EVs) are circulating communicative vesicles, containing bioactive miRNA. Cancer cell models are a convenient source of tumour-EVs for research. However, cell models must accurately represent physiological conditions to study EVs with diagnostically relevant cargo.
This study aims to compare EVs released from colorectal cancer cells grown in monolayer versus spheroid cultures. Spheroids were formed using a Matrigel-Collagen matrix to mimic the in vivo extracellular environment. Initial optimisation focused on dome volume and cell seeding density. Colorectal cancer cell lines, DLD-1 and SW837, were seeded at various concentrations in 20, 45 and 75 μL-sized domes. Reliable spheroid formation and growth were observed in 45 μL domes with the following concentrations. DLD-1 spheroids at 3.8⨉105 cells/mL showed a 1212 μm2 mean increase from day 3 to day 5 (p < 0.001; Welch’s t-test). SW837 spheroids at 1.3⨉106 cells/mL showed a 1911 μm2 mean increase from day 3 to day 7 (p = 0.003; one-way ANOVA with Games-Howell post hoc test), with no significant differences at day 5 (n=10 spheroids per group). Therefore, different culture periods will be used to adjust for cell doubling rates.
Conditioned media (48-hour incubation) will be collected to isolate EVs, using size exclusion chromatography and ultrafiltration. Tunable resistance pulse sensing and real-time quantitative PCR methods will be used to characterise EV size distribution, concentration and miRNA cargo differences between the conditions.