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

Abstract

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 nano-liquid chromatography tandem mass spectrometry (nLC-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.

Mass Spectrometry Analyses of Multicellular Tumor Spheroids

“…The application of mass spectrometry analysis techniques to this biological model promises to provide new insight into cancer progression and treatment response. This includes identification of altered metabolism, cell–cell interactions and cell–ECM connections as well as antigen expression . Although additional techniques are required to complement proteomics analysis of these structures, it represents a powerful foundation from which novel cancer research can be developed.…”

Analyzing Liposomal Drug Delivery Systems in Three-Dimensional Cell Culture Models Using MALDI Imaging Mass Spectrometry

“…For this reason, it would be beneficial to test drug efficacy using three-dimensional cell cultures. 13–14 Three-dimensional cell cultures or spheroids display pathophysiological gradients that are more complex than conventional two-dimensional cell cultures and are higher throughput than animal models. 17–18 These cultures have naturally occurring layers of cellular populations that are comparable to in vivo tumors.…”

Global and site‐specific analysis of protein glycosylation in complex biological systems with Mass Spectrometry

“…Comprehensive and site-specific analysis of protein modifications is normally beyond the reach of conventional biochemistry methods. Modern MS technology provides a unique opportunity to globally and site-specifically characterize protein modifications (Ficarro et al, 2002;Peng et al, 2003;Trinidad et al, 2006;Siuti and Kelleher, 2007;Witze et al, 2007;Yates et al, 2009;Mertins et al, 2013;Nwosu et al, 2013;Huang et al, 2015a;Ludwig et al, 2015;Gu and Robinson, 2016;Lossl et al, 2016;Yue et al, 2016;Cao et al, 2017;Simithy et al, 2017;Hogrebe et al, 2018;Wu et al, 2018). However, it is still extraordinarily challenging to globally analyze them because of the low abundance of many modified proteins, sub-stoichiometry of protein modifications, and the complexity of biological samples.…”