Multicellular spheroids represent a well-established 3D model to study healthy and diseased cells in vitro. The use of conventional 3D cell culture platforms for the generation of multicellular spheroids is limited to cell types that easily self-assemble into spheroids because less adhesive cells fail to form stable aggregates. A high-precision micromoulding technique developed in our laboratory produces deep conical agarose microwell arrays that allow the cultivation of uniform multicellular aggregates, irrespective of the spheroid formation capacity of the cells. Such hydrogel arrays warrant a steady nutrient supply for several weeks, permit live volumetric measurements to monitor cell growth, enable immunohistochemical staining, fluorescence-based microscopy, and facilitate immediate harvesting of cell aggregates. This system also allows co-cultures of two distinct cell types either in direct cell–cell contact or at a distance as the hydrogel permits diffusion of soluble compounds. Notably, we show that co-culture of a breast cancer cell line with bone marrow stromal cells enhances 3D growth of the cancer cells in this system.

Transcriptional signatures are an indispensible source of correlative information on disease-related molecular alterations on a genome-wide level. Numerous candidate genes involved in disease and in factors of predictive, as well as of prognostic, value have been deduced from such molecular portraits, e.g. in cancer. However, mechanistic insights into the regulatory principles governing global transcriptional changes are lagging behind extensive compilations of deregulated genes. To identify regulators of transcriptome alterations, we used an integrated approach combining transcriptional profiling of colorectal cancer cell lines treated with inhibitors targeting the receptor tyrosine kinase (RTK)/RAS/mitogen-activated protein kinase pathway, computational prediction of regulatory elements in promoters of co-regulated genes, chromatin-based and functional cellular assays. We identified commonly co-regulated, proliferation-associated target genes that respond to the MAPK pathway. We recognized E2F and NFY transcription factor binding sites as prevalent motifs in those pathway-responsive genes and confirmed the predicted regulatory role of Y-box binding protein 1 (YBX1) by reporter gene, gel shift, and chromatin immunoprecipitation assays. We also validated the MAPK-dependent gene signature in colorectal cancers and provided evidence for the association of YBX1 with poor prognosis in colorectal cancer patients. This suggests that MEK/ERK-dependent, YBX1-regulated target genes are involved in executing malignant properties. The simultaneous analysis of gene expression in cancer using microarrays is a standard approach for monitoring disease-related modifications involved in tumorigenesis, triggering malignant properties and clinical behavior. However, the factors that drive these alterations most often remain elusive. We sought to identify transcription factors that mediate the transcriptional effects of the receptor tyrosine kinase/RAS oncoprotein pathway, a frequently activated oncogenic signaling system, in cultured colorectal cancer cells. We used an integrated approach combining molecular and functional assays, as well as computational tools, to identify regulatory factors that trigger the alterations of gene expression and modulate cellular growth. We identified the YBX1 protein, a member of the highly conserved family of cold shock domain transcription factors, as a regulator of signaling effects triggered by the RAS cancer gene. Then we assayed the messenger RNA expression of YBX1 and YBX1-responsive target genes by interrogating microarrays, and also expression of the YBX1 protein by immunohistochemistry in colorectal tumors. We found that YBX1 expression is correlated with a bad clinical outcome in colon cancer patients.

Most malignant features of cancer cells are triggered by activated oncogenes and the loss of tumor suppressors due to mutation or epigenetic inactivation. It is still unclear, to what extend the escape of emerging cancer cells from recognition and elimination by the immune system is determined by similar mechanisms. We compared the transcriptomes of HCT116 colorectal cancer cells deficient in DNA methyltransferases (DNMTs) and of cells, in which the RAS pathway as the major growth‐promoting signaling system is blocked by inhibition of MAPK. We identified the MHC Class I genes HLA‐A1/A2 and the ULBP2 gene encoding 1 of the 8 known ligands of the activating NK receptor NKG2D among a cluster of immune genes up‐regulated under the conditions of both DNMT‐deficiency and MEK‐inhibition. Bisulphite sequencing analyses of HCT116 with DNMT deficiency or after MEK‐inhibition showed that de‐methylation of the ULPB2 promoter correlated with its enhanced surface expression. The HLA‐A promoters were not methylated indicating that components of the HLA assembly machinery were also suppressed in DNMT‐deficient and MEK‐inhibited cells. Increased HLA‐A2 surface expression was correlated with enhanced recognition and lysis by A2‐specific CTL. On the contrary, elevated ULBP2 expression was not reflected by enhanced recognition and lysis by NK cells. Cosuppression of HLA Class I and NKG2D ligands and genes encoding peptide transporters or proteasomal genes mediates a strong functional link between RAS activation, DNMT activity and disruption of the antigen presenting system controlling immune recognition in colorectal cancer cells. © 2009 UICC