Abstract
Three-dimensional models are considered a powerful tool for improving the concordance between in vitro and in vivo phenotypes. However, the duration of spheroid culture may influence the degree of correlation between these counterparts. When using immortalised cell lines as model systems, the assumption for consistency and reproducibility is often made without adequate characterization or validation. It is therefore essential to define the biology of each spheroid model by investigating proteomic dynamics, which may be altered relative to culture duration. As an example, we assessed the influence of culture duration on the relative proteome abundance of HepG2 cells cultured as spheroids, which are routinely used to model aspects of the liver. Quantitative proteomic profiling of whole cell lysates labelled with tandem-mass tags was conducted using liquid chromatography-tandem mass spectrometry (LC–MS/MS). In excess of 4800 proteins were confidently identified, which were shared across three consecutive time points over 28 days. The HepG2 spheroid proteome was divergent from the monolayer proteome after 14 days in culture and continued to change over the successive culture time points. Proteins representing the recognised core hepatic proteome, cell junction, extracellular matrix, and cell adhesion proteins were found to be continually modulated.
Highlights
Three-dimensional models are considered a powerful tool for improving the concordance between in vitro and in vivo phenotypes
Phenotypic transitions as a result of growth in 3D have been investigated at the gene transcription level[24] which while informative, does not account for the fact that only 20–50% of transcribed genes result in expression of functional proteins[25], which would limit the perceived utility of these model systems
Spheroids undergo dynamic proteomic transitions based on time spent in culture
Summary
Three-dimensional models are considered a powerful tool for improving the concordance between in vitro and in vivo phenotypes. Since proteomic changes which occur as cells arrange and mature within complex 3D structure are often o verlooked[27], we sought to monitor the relative quantitative changes within spheroids over a long-term culture of 28 days using isobaric tagging This was done to determine whether cells maintained in spheroids for more than 10 days undergo reproducible, significant, proteomic changes related to their altered culture architecture. These data suggest that there is a continual divergence of the HepG2 proteome as a consequence of long-term spheroid culture which impacts cellular functionality. We provide new insights into the dynamic nature of 3D culture systems and highlight important considerations essential to the field of biological modelling
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