Quantitative Proteomic and Phosphoproteomic Comparison of 2D and 3D Colon Cancer Cell Culture Models

Cell cultures are
widely used model systems. Some immortalized
cell lines can be grown in either two-dimensional (2D) adherent monolayers
or in three-dimensional (3D) multicellular aggregates, or spheroids.
Here, the quantitative proteome and phosphoproteome of colon carcinoma
HT29 cells cultures in 2D monolayers and 3D spheroids were compared
with a stable isotope labeling of amino acids (SILAC) labeling strategy.
Two biological replicates from each sample were examined, and notable
differences in both the proteome and the phosphoproteome were determined
by nanoliquid chromatography tandem mass spectrometry (LC–MS/MS)
to assess how growth configuration affects molecular expression. A
total of 5867 protein groups, including 2523 phosphoprotein groups
and 8733 phosphopeptides were identified in the samples. The Gene
Ontology analysis revealed enriched GO terms in the 3D samples for
RNA binding, nucleic acid binding, enzyme binding, cytoskeletal protein
binding, and histone binding for their molecular functions (MF) and
in the process of cell cycle, cytoskeleton organization, and DNA metabolic
process for the biological process (BP). The KEGG pathway analysis
indicated that 3D cultures are enriched for oxidative phosphorylation
pathways, metabolic pathways, peroxisome pathways, and biosynthesis
of amino acids. In contrast, analysis of the phosphoproteomes indicated
that 3D cultures have decreased phosphorylation correlating with slower
growth rates and lower cell-to-extracellular matrix interactions.
In sum, these results provide quantitative assessments of the effects
on the proteome and phosphoproteome of culturing cells in 2D versus
3D cell culture configurations.