Abstract 4571: A comparison of proteins and their expression levels within different regions of multi-cellular spheroids (MCTS)
Authors: McMahon Kelly M. , Naal Sham , Phillips Roger M. , Sutton Chris W. 
Published: February 2011

Abstract
Introduction
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 physiological/biochemical changes. The occurrence of these features in combination, present a situation which is unique to tumors, thus represents an attractive target for drug development. MCTS's provide an in vitro model system which encapsulates many of these features. Using a quantitative proteomics approach to characterise specific regions of the MCTS, it is hoped that novel targets for therapeutic interventions will be revealed.
Methods
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) by serial trypsinisation and gentle homogenisation. Proteins from each fraction were extracted and equal amounts from each fraction were digested with trypsin. Resulting peptides were iTRAQ labelled and the fractions combined. Peptides were firstly separated according to their pI, using the Agilent OffGel system and further separated by nano-HPLC. Fractions were 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.
Result
Optimisation of the spheroid separation method revealed that 2 trypsin treatments were required to remove the AF of cells and 10-13 further trypsin treatments were required to reach the HF. Immunohistochemistry using the endogenous hypoxia marker Carbonic Anhydrase IX was used to confirm the hypoxic status of these cells following their isolation. Gentle homogenisation of the remaining spheroid shell, was shown to result in the optimal separation of the hypoxic cells from the non adherent cells present within the spheroid necrotic core. Confirmation of this separation was provided by hematoxylin and eosin staining, where the necrotic core cells can be identified by their significantly smaller size.
MS analysis resulted in 882 protein identifications, of which 12.6% and 20.9% were shown at a higher level in the HF and NC, respectively, relative to the AF. Further, 8.4% and 8.7% of proteins were shown to be present at lower levels in the HF and NC, respectively, when compared to the AF.
Conclusion
Many of those proteins identified as differentially expressed in the HF and NC represented established cancer associated proteins. Interestingly, a number of proteins were highlighted as being up regulated in both the HF and NC which have no previous association with cancer and may upon validation, provide attractive leads for therapeutic intervention.
Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4571.