Identification of differentially expressed genes in experimental models ofthe tumor microenvironment using differential display

Background Genes upregulated within the tumour microenvironment represent p otential 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 ex pression was analysed by differential display and confirmed by Northern blo t or semiquantitative RT-PCR. Results. p21-activated kinase (PAK1), a calmo dulin-related mRNA, cytochrome oxidase subunit I and an H3.3 histone were u pregulated within the in vitro tumour microenvironment, the last 3 within s pheroids. Conclusions. Both models exhibit a range of microenvironmental pa rameters, although spheroids are more physiological with respect to the pre sence of extreme hypoxia and the formation of 3-dimensional interactions. W e have shown that it is feasible to manipulate the spheroid system by seria l trypsinisation to obtain reproducible cell populations for gene expressio n studies.