A reducible comparison of 2D vs. 3D HepG2 culture-derived sEV characteristics and cancer pathway-related miRNA content.

Hepatocellular carcinoma cells (HCCs) produce small extracellular vesicles (sEVs or exosomes) to enable tumor survival, including inactivation of anti-tumor macrophage immune responses. For sEV studies in vitro, 2D adherent culture cells are typically used to manufacture sEVs. In contrast, 3D matrix-based culture systems closely simulate in vivo tissues but produce difficult to isolate sEVs. To address this issue, we developed a reducible 3D suspension HCC spheroid culture system to generate easily obtainable sEVs for investigations. The objective was to evaluate biophysical and cancer pathway-focused miRNA content differences between HCC HepG2 sEVs produced in 2D adherent vs. 3D spheroid suspension culture. To summarize methods, sEVs were isolated from 2D and 3D cell culture using differential ultracentrifugation and size and zeta potential biophysical characteristics were determined by nanoparticle tracking analysis. Fold regulation of 2D and 3D derived sEV miRNAs, compared to their source cells and one another, were determined by qRT-PCR, and statistically significant differences were determined by Student's t-test. Results indicated that HepG2 sEVs generated from 2D adherent or 3D spheroid suspension culture do not differ significantly in terms of size (~130 nm) and zeta potential (< -30 mV) characteristic of sEVs. A comparison of 2D vs. 3D sEVs revealed differences in the enrichment of let-7a-5p (decreased in HCC in vivo), miR-21-5p (enables HCC drug resistance), and miR-126-3p (impairs HCC tumor volume in vivo). let-7a-5p and miR-21-5p were downregulated, while miR-126-3p was upregulated in 3D sEVs compared to 2D sEVs (control) respectively. Overall, similarities and differences between 2D and 3D sEV miRNA content were observed relevant to HCC pathogenesis. In conclusion, the 3D HepG2 spheroid suspension model provides an additional reducible level of HCC sEV investigation to augment traditional 2D sEV studies while better simulating an in vivo 3D source of easily obtainable HCC sEVs. Our 3D model could be used in conjunction with typical 2D EV culture systems to streamline the identification of candidate sEV-based biomarkers and therapeutic targets for HCC and other cancers.