Characterization of Changes in the Proteome in Different Regions of 3D Multicell Tumor Spheroids

K. M. McMahon, M. Volpato, H. Y. Chi, P. Musiwaro, K. Poterlowicz, Y. Peng, A. J. Scally, L. H. Patterson, R. M. Phillips, C. W. Sutton

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

Three dimensional multicell tumor spheroids (MCTS) provide an experimental model where the influence of microenvironmental conditions on protein expression can be determined. Sequential trypsin digestion of HT29 colon carcinoma MCTS enabled segregation into four populations comprising proliferating cells from the surface (SL), an intermediate region (IR), nonproliferating hypoxic cells from the perinecrotic region (PN), and a necrotic core (NC). Total protein was extracted from each population and subjected to iTRAQ-based quantitative proteomics analysis. From a total of 887 proteins identified, 209 were observed to be up-regulated and 114 were down-regulated in the PN and NC regions relative to the SL. Among the up-regulated proteins, components of glycolysis, TCA cycle, lipid metabolism, and steroid biosynthesis increased progressively toward the PN and NC regions. Western blotting, immunohistochemistry, and enzyme assays confirmed that significant changes in the expression of proteins involved in cellular metabolism occur in the nonproliferating fraction of cells within the viable rim. The presence of full length, functional proteins within the NC was unexpected, and further analysis demonstrated that this region contains cells that are undergoing autophagy. This study has identified possible targets that may be suitable for therapeutic intervention, and further studies to validate these are required.

LanguageEnglish
Pages2863-2875
Number of pages13
JournalJournal of Proteome Research
Volume11
Issue number5
Early online date14 Mar 2012
DOIs
Publication statusPublished - 4 May 2012
Externally publishedYes

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Proteome
Tumors
Neoplasms
Proteins
Biosynthesis
Autophagy
Enzyme Assays
Glycolysis
Lipid Metabolism
Metabolism
Proteomics
Trypsin
Population
Digestion
Assays
Colon
Theoretical Models
Western Blotting
Immunohistochemistry
Steroids

Cite this

McMahon, K. M., Volpato, M., Chi, H. Y., Musiwaro, P., Poterlowicz, K., Peng, Y., ... Sutton, C. W. (2012). Characterization of Changes in the Proteome in Different Regions of 3D Multicell Tumor Spheroids. Journal of Proteome Research, 11(5), 2863-2875. https://doi.org/10.1021/pr2012472
McMahon, K. M. ; Volpato, M. ; Chi, H. Y. ; Musiwaro, P. ; Poterlowicz, K. ; Peng, Y. ; Scally, A. J. ; Patterson, L. H. ; Phillips, R. M. ; Sutton, C. W. / Characterization of Changes in the Proteome in Different Regions of 3D Multicell Tumor Spheroids. In: Journal of Proteome Research. 2012 ; Vol. 11, No. 5. pp. 2863-2875.
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McMahon, KM, Volpato, M, Chi, HY, Musiwaro, P, Poterlowicz, K, Peng, Y, Scally, AJ, Patterson, LH, Phillips, RM & Sutton, CW 2012, 'Characterization of Changes in the Proteome in Different Regions of 3D Multicell Tumor Spheroids', Journal of Proteome Research, vol. 11, no. 5, pp. 2863-2875. https://doi.org/10.1021/pr2012472

Characterization of Changes in the Proteome in Different Regions of 3D Multicell Tumor Spheroids. / McMahon, K. M.; Volpato, M.; Chi, H. Y.; Musiwaro, P.; Poterlowicz, K.; Peng, Y.; Scally, A. J.; Patterson, L. H.; Phillips, R. M.; Sutton, C. W.

In: Journal of Proteome Research, Vol. 11, No. 5, 04.05.2012, p. 2863-2875.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Characterization of Changes in the Proteome in Different Regions of 3D Multicell Tumor Spheroids

AU - McMahon, K. M.

AU - Volpato, M.

AU - Chi, H. Y.

AU - Musiwaro, P.

AU - Poterlowicz, K.

AU - Peng, Y.

AU - Scally, A. J.

AU - Patterson, L. H.

AU - Phillips, R. M.

AU - Sutton, C. W.

PY - 2012/5/4

Y1 - 2012/5/4

N2 - Three dimensional multicell tumor spheroids (MCTS) provide an experimental model where the influence of microenvironmental conditions on protein expression can be determined. Sequential trypsin digestion of HT29 colon carcinoma MCTS enabled segregation into four populations comprising proliferating cells from the surface (SL), an intermediate region (IR), nonproliferating hypoxic cells from the perinecrotic region (PN), and a necrotic core (NC). Total protein was extracted from each population and subjected to iTRAQ-based quantitative proteomics analysis. From a total of 887 proteins identified, 209 were observed to be up-regulated and 114 were down-regulated in the PN and NC regions relative to the SL. Among the up-regulated proteins, components of glycolysis, TCA cycle, lipid metabolism, and steroid biosynthesis increased progressively toward the PN and NC regions. Western blotting, immunohistochemistry, and enzyme assays confirmed that significant changes in the expression of proteins involved in cellular metabolism occur in the nonproliferating fraction of cells within the viable rim. The presence of full length, functional proteins within the NC was unexpected, and further analysis demonstrated that this region contains cells that are undergoing autophagy. This study has identified possible targets that may be suitable for therapeutic intervention, and further studies to validate these are required.

AB - Three dimensional multicell tumor spheroids (MCTS) provide an experimental model where the influence of microenvironmental conditions on protein expression can be determined. Sequential trypsin digestion of HT29 colon carcinoma MCTS enabled segregation into four populations comprising proliferating cells from the surface (SL), an intermediate region (IR), nonproliferating hypoxic cells from the perinecrotic region (PN), and a necrotic core (NC). Total protein was extracted from each population and subjected to iTRAQ-based quantitative proteomics analysis. From a total of 887 proteins identified, 209 were observed to be up-regulated and 114 were down-regulated in the PN and NC regions relative to the SL. Among the up-regulated proteins, components of glycolysis, TCA cycle, lipid metabolism, and steroid biosynthesis increased progressively toward the PN and NC regions. Western blotting, immunohistochemistry, and enzyme assays confirmed that significant changes in the expression of proteins involved in cellular metabolism occur in the nonproliferating fraction of cells within the viable rim. The presence of full length, functional proteins within the NC was unexpected, and further analysis demonstrated that this region contains cells that are undergoing autophagy. This study has identified possible targets that may be suitable for therapeutic intervention, and further studies to validate these are required.

KW - autophagy

KW - cancer

KW - hypoxia

KW - lipid metabolism

KW - metabolic processes

KW - necrosis

KW - proteomics

KW - spheroids

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UR - http://pubs.acs.org/journal/jprobs

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DO - 10.1021/pr2012472

M3 - Article

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SP - 2863

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JO - Journal of Proteome Research

T2 - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

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ER -