Protein and phosphoprotein levels in glioma and adenocarcinoma cell lines grown in normoxia and hypoxia in monolayer and three-dimensional cultures

Abstract

Background: Three dimensional (3D) growths of cancer cells in vitro are more reflective of in situ cancer cell growth than growth in monolayer (2D). The present study is designed to determine changes in protein and phosphoprotein that reflect adaptation of tumor cells to 3D as compared to 2D. Since relative hypoxia is a common feature of most solid tumors, the present study also aims to look at the impact of transition from normoxia to hypoxia in these two growth conditions.

Results: Using reverse-phase protein arrays, we compared levels of 121 different phosphorylated and non-phosphorylated proteins in 5 glioma and 6 adenocarcinoma lines under conditions of 3D and monolayer culture in normoxia and hypoxia. A three-way analysis of variance showed levels of 82 antibodies differed between media (2D vs. 3D) and 49 differed between treatments (hypoxia vs. normoxia). Comparing 2D to 3D growth, 7 proteins were commonly (i.e., > 50% of tumors) elevated in 3D: FAK, AKT, Src, GSK3αβ, TSC2, p38, and NFκβp65. Conversely, 7 other proteins are commonly decreased: ATRIP, ATR, β-catenin, BCL-X, cyclin B1, Egr-1, and HIF-1α. Comparing normoxia to hypoxia, only NCKIPSD was commonly elevated in hypoxia; 6 proteins were decreased: cyclin B1, 4EBP1(Ser65), c-Myc, SMAD3(Ser423), S6(Ser235), and S6(Ser240). Hypoxia affected glioma cell lines differently from adenocarcinoma cell lines: 8 proteins were increased in gliomas (BAX, caspase 7, HIF-1α, c-JUN, MEK1, PARP 1 cleaved, Src, and VEGFR2) and none in adenocarcinomas.

Conclusions: We identified subsets of proteins with clearly concordant/discordant behavior between gliomas and adenocarcinomas. In general, monolayer to 3D culture differences are clearer than normoxia to hypoxia differences, with anti-apoptotic, cytoskeletal rearrangement and cell survival pathways emphasized in the former and mTOR pathway, transcription, cell-cycle arrest modulation, and increased cell motility in the latter.

Figure 1

Histogram showing the distribution of p-values from the feature-by-feature three-way ANOVAs. Superimposed curves represent fits of the BUM models for (A) medium (2D and 3D), (B) treatment (hypoxia and normoxia), and (C) interaction between medium and treatment.

Figure 2

Protein changes from ANOVA of 3D-2D (monolayer to 3D) medium by cell line whether normoxic or hypoxic conditions for proteins with p-value < 0.05. Proteins are ordered from based on the FC (fold change) and scored -1, 0, or +1, depending on quartile distribution of raw antibody difference values (-1, blue, lowest quartile; +1, red, highest quartile; 0, green, others). Also shown is the score when > 50% of the total for glioma (GL; 6/10) and adenocarcinoma (AC; 7/12) values move in the same direction. The data is divided into six groupings based on > 50% score (+1, -1, and 0) for gliomas (GL) and adenocarcinomas (AC) together and separately as well as neutral and ill-defined scores.

Figure 3

Protein changes from ANOVA of 3D-2D (monolayer to 3D) medium by cell line whether normoxic or hypoxic conditions for proteins with p-value < 0.05. Proteins are ordered from based on the FC (fold change) and scored -1, 0, or +1, depending on quartile distribution of raw antibody difference values (-1, blue, lowest quartile; +1, red, highest quartile; 0, green, others). Also shown is the score when > 50% of the total for glioma (GL; 6/10) and adenocarcinoma (AC; 7/12) values move in the same direction. The data is divided into six groupings based on > 50% score (+1, -1, and 0) for gliomas (GL) and adenocarcinomas (AC) together and separately as well as neutral and ill-defined scores. The Sum Score at the bottom of the figure is the summed values for columns in figures 2 and 3.

Figure 4

Protein changes from ANOVA of the hypoxia-normoxia by cell line, whether grown in 2D or 3D conditions for proteins with p-values < 0.05. The proteins are ordered based on the FC (fold change) and scored -1, 0, or +1 depending on quartile distribution of raw antibody difference values (-1, blue, lowest quartile; +1, red, highest quartile;0, green, others). Also shown is the score when > 50% of the total for glioma (GL; 6/10) and adenocarcinoma (AC; 7/12) values move in the same direction. The data is divided into five groupings based on > 50% score (+1, -1, and 0) for gliomas and adenocarcinomas together and separately as well as neutral and ill-defined scores.

Figure 5

This is a cartoon of the shared partial pathways involved in the transition of > 50% of glioma and adenocarcinoma cell lines from monolayer to 3D growth and are consistent with in situ tumors seeking to survive the more difficult 3D growth that will lead to genetic stress and part of the angiogenesis cascade. The red color indicates the protein increased and the blue color that the protein decreased.

Figure 6

This is a cartoon of the partial shared pathways involved in the transition of > 50% of glioma and adenocarcinoma cell lines from normoxia to hypoxia. The red color indicates the protein increased and the blue color that the protein decreased.

Figure 7

This is a cartoon of the partial pathways involved in the transition of > 50% of glioma cell lines from normoxia to hypoxia. The red color indicates the protein increased and the blue color that the protein decreased.