H J Knowles1, R M Phillips. 1. Cancer Research Unit, University of Bradford, United Kingdom. Knowles@icrf.icnet.uk
Abstract
BACKGROUND: Genes upregulated within the tumour microenvironment represent potential targets for rational drug design. Most studies to date concentrate on the effects of hypoxia, although it is likely many genes are regulated by a more physiological combination of factors. MATERIALS & METHODS: Cells under conditions analogous to the normal and tumour microenvironments were isolated from the plateau-phase system and multicellular spheroids. Gene expression was analysed by differential display and confirmed by Northern blot or semiquantitative RT-PCR. RESULTS: p21-activated kinase (PAK1), a calmodulin-related mRNA, cytochrome oxidase subunit I and an H3.3 histone were upregulated within the in vitro tumour microenvironment, the last 3 within spheroids. CONCLUSIONS: Both models exhibit a range of microenvironmental parameters, although spheroids are more physiological with respect to the presence of extreme hypoxia and the formation of 3-dimensional interactions. We have shown that it is feasible to manipulate the spheroid system by serial trypsinisation to obtain reproducible cell populations for gene expression studies.
BACKGROUND: Genes upregulated within the tumour microenvironment represent potential targets for rational drug design. Most studies to date concentrate on the effects of hypoxia, although it is likely many genes are regulated by a more physiological combination of factors. MATERIALS & METHODS: Cells under conditions analogous to the normal and tumour microenvironments were isolated from the plateau-phase system and multicellular spheroids. Gene expression was analysed by differential display and confirmed by Northern blot or semiquantitative RT-PCR. RESULTS:p21-activated kinase (PAK1), a calmodulin-related mRNA, cytochrome oxidase subunit I and an H3.3 histone were upregulated within the in vitro tumour microenvironment, the last 3 within spheroids. CONCLUSIONS: Both models exhibit a range of microenvironmental parameters, although spheroids are more physiological with respect to the presence of extreme hypoxia and the formation of 3-dimensional interactions. We have shown that it is feasible to manipulate the spheroid system by serial trypsinisation to obtain reproducible cell populations for gene expression studies.
Authors: Richard B Keithley; Eric M Weaver; Andrea M Rosado; Mark P Metzinger; Amanda B Hummon; Norman J Dovichi Journal: Anal Chem Date: 2013-09-12 Impact factor: 6.986
Authors: Sadek A Nehmeh; Nancy Y Lee; Heiko Schröder; Olivia Squire; Pat B Zanzonico; Yusuf E Erdi; Carlo Greco; Gig Mageras; Hai S Pham; Steve M Larson; Clifton C Ling; John L Humm Journal: Int J Radiat Oncol Biol Phys Date: 2008-01-01 Impact factor: 7.038