Abstract 4872: A comparison of proteins and their expression levels within different regions of multi-cellular spheroids (MCTS)
Authors: McMahon Kelly M. , Sutton Chris W. , Phillips Roger M. , Naal Sham 
Published: June 2012

Abstract
Hypoxia is a well established target for anticancer therapy. Within the tumor microenvironment, hypoxia does not reside alone; instead it exists in combination with multiple other pathophysiological features. The occurrence of these features in combination, presents a situation which is unique to tumors, and thus represents an attractive target for drug development. MCTS's provide an in vitro model system which encapsulates many of these features and enables the exploration of the molecular and biochemical changes mimicking those in vivo. Using a quantitative proteomics approach to characterise specific regions of the MCTS, it is hoped that novel targets for therapeutic interventions will be revealed.
HT29 (colon adenocarcinoma) cell's were cultured as MCTS using spinner flasks. Spheroids were separated into 3 main fractions; the outer aerobic fraction (AF), the hypoxic fraction (HF) and the necrotic core (NC). This was achieved using serial trypsin treatments and gentle homogenisation. Proteins from each fraction were extracted and equal amounts from each were digested with trypsin. Resulting peptides were iTRAQ labelled and the fractions combined. The peptides were firstly initially separated according to their pI, using the Agilent OffGel system and then further separated by nano-HPLC, with fractions collected off-line onto a MALDI plate. MALDI TOF-TOF mass spectrometry was used to screen fractions in MS mode and a list of signals compiled for MS/MS analysis. The resulting MS/MS data was searched against protein sequence databases for identification. Validation of the proteomics results was achieved through western blot and Immunohistochemistry (IHC) analysis. The activity of a selection of proteins was also investigated using spectrophotometric assays.
Confirmation of the spheroid separation methods was provided by hematoxylin and eosin staining and FAC analysis, where the necrotic core cells can be identified by their significantly smaller size. IHC using the endogenous hypoxia marker Carbonic Anhydrase IX was used to confirm the hypoxic status of these cells following their isolation. MS analysis resulted in 1031 protein identifications, of which 6.1 and 18.1% were shown at a higher level in the HF and NC, respectively, relative to the AF. Further, 4.9 and 9.8% of proteins were shown to be present at lower levels in the HF and NC, respectively, when compared to the AF. Activity assays, IHC and western blot analysis for selected proteins that significantly changed in expression showed good agreement with the MS-derived results.
Many of those proteins identified as differentially expressed in the HF and NC represented established cancer associated proteins. Interestingly, a number of proteins, with no previous association with cancer, were shown to be up-regulated in both the HF and NC and may upon validation provide attractive leads for therapeutic intervention.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4872. doi:10.1158/1538-7445.AM2011-4872