Abstract
For studying cellular communications ex-vivo, a two-dimensional (2D) cell culture model is currently used as the “gold standard”. 2D culture models are also widely used in the study of RNA expression profiles from tumor cells secreted extracellular vesicles (EVs) for tumor biomarker discovery. Although the 2D culture system is simple and easily accessible, the culture environment is unable to represent in vivo extracellular matrix (ECM) microenvironment. Our study observed that 2D- culture derived EVs showed significantly different profiles in terms of secretion dynamics and essential signaling molecular contents (RNAs and DNAs), when compared to the three-dimensional (3D) culture derived EVs. By performing small RNA next-generation sequencing (NGS) analysis of cervical cancer cells and their EVs compared with cervical cancer patient plasma EV-derived small RNAs, we observed that 3D- culture derived EV small RNAs differ from their parent cell small RNA profile which may indicate a specific sorting process. Most importantly, the 3D- culture derived EV small RNA profile exhibited a much higher similarity (~96%) to in vivo circulating EVs derived from cervical cancer patient plasma. However, 2D- culture derived EV small RNA profile correlated better with only their parent cells cultured in 2D. On the other hand, DNA sequencing analysis suggests that culture and growth conditions do not affect the genomic information carried by EV secretion. This work also suggests that tackling EV molecular alterations secreted into interstitial fluids can provide an alternative, non-invasive approach for investigating 3D tissue behaviors at the molecular precision. This work could serve as a foundation for building precise models employed in mimicking in vivo tissue system with EVs as the molecular indicators or transporters. Such models could be used for investigating tumor biomarkers, drug screening, and understanding tumor progression and metastasis.
Highlights
Extracellular vesicles (EVs) are a heterogeneous mixture of membranous structures, derived from either the cellular endosomal pathway or shed from the plasma membrane[1]
extracellular vesicles (EVs)-derived miRNAs showed significantly different expression profile than their parent cells in both 2D and 3D conditions. Their parent cells cultured between 2D and 3D systems exhibited similar miRNA expression profile
We observed that 3D cell-derived EV sample was clustered together with two in vivo cervical cancer patient plasma sample derived EV miRNAs
Summary
Extracellular vesicles (EVs) are a heterogeneous mixture of membranous structures, derived from either the cellular endosomal pathway or shed from the plasma membrane[1]. We observed that 2D- culture derived EVs showed significantly different profiles in terms of secretion dynamics and essential signaling molecular contents (RNAs), compared to the 3D- culture derived EVs. By next-generation sequencing (NGS) analysis of cervical cancer cells and cervical cancer patient plasma-derived EV RNA, we observed that 3D- culture derived EV small RNAs may undergo specific sorting process that differs from their parent cells.
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