The most common in vitro cancer-cell culture methods rely on flat, 2-dimensional (2D) plastic culturing surfaces which are highly artificial and lack the 3-dimensional (3D) complexity of the tumor environment in the body. The metastatic potential of tumor cells is underpinned partly by their interactions with the extracellular matrix (ECM) whose properties differ between organs and is absent in 2D culturing. The reconstruction of organ-specific environments and the ECM support provided by them has shown promise in studying drug interactions in cultured cells1. By decellularizing native organs, the ECM remains intact and produces a more realistic biomaterial scaffold on which to grow cells. In this study, we used a human ovarian adenocarcinoma cell line (BG1) to compare traditional 2D monolayer culturing with two 3D methods: cells grown as multicellular spheroids in an agarose support that lacks ECM support, and on de-cellularised pig ovary scaffolds that are comprised of naturally occurring proteoglycan ECM proteins.
Membrane proteins were isolated from cells grown under the 2D and the two different 3D growth conditions, and the protein N-glycosylation profiled using PGC-LC coupled with ESI-MS/MS. The 3D spheroid-grown cells were found to be highly contaminated with serum glycans from the culture media, rendering this method of growth unfeasible for ‘omics analysis. The 2D-grown cells showed relatively high levels of protein oligo-mannosylation compared to the 3D-scaffold grown cells which produced more complex-type products of the N-glycan biosynthetic pathway. The de-N-glycosylated samples from 2D- and 3D on-scaffold grown cells were subsequently digested with trypsin and the peptides analysed using RP-LC-ESI-MS/MS. Of the 1389 proteins identified, 354 were differentially expressed between the two conditions with nucleic and cytoplasmic proteins generally higher in the 3D culture, while several mitochondrial proteins were lower in abundance. The results of this study demonstrate the markedly altered proteome and glycome produced by different in vitro culturing environments and the potential impacts on the outcomes and conclusions from different in vitro based studies of diseases.
(1) Guller, A.; Kuschnerus, I.; Rozova, V.; Nadort, A.; Yao, Y.; Khabir, Z.; Garcia-Bennett, A.; Liang, L.; Polikarpova, A.; Qian, Y.; Goldys, E. M.; Zvyagin, A. V. Biomedicines 2021, 9, 1578.