Continual proteomic divergence of HepG2 cells as a consequence of long-term spheroid culture

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 infuence 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 defne the biology of each spheroid model by investigating proteomic dynamics, which may be altered relative to culture duration. As an example, we assessed the infuence 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 profling of whole cell lysates labelled with tandem-mass tags was conducted using liquid chromatographytandem mass spectrometry (LC–MS/MS). In excess of 4800 proteins were confdently identifed, 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.