Introduction Metastasis is the leading cause of death for cancer patients. As cancer cells metastasize, they interact with various extracellular molecules and stromal cells such as macrophages. Macrophages have been shown to promote invasion and change multicellular organisation of cancer cells. Breast cancer cells (BCC) and macrophages are known to interact via epidermal growth factor (EGF) produced by macrophages and colony stimulating factor-1 (CSF-1) produced by BCC. Despite contradictory

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... ndings, this interaction is perceived as a paracrine loop. Yet, an in-depth understanding of the mechanistic basis of this interaction is lacking: It is not known whether the interactions between breast cancer cells and macrophages are based on chemotaxis or haptotaxis or direct contact. Material and methods We used a multidisciplinary approach including classical and up-to-date techniques such as cell-on-achip devices. MDA-MB-231 and RAW264.7 cell lines were used to represent BCC and macrophages, respectively. Secretion of EGF and CSF-1 was determined using ELISA. Cell adhesion and motility were assayed via live cell imaging in the presence and absence of iressa. Classical 3D cell culture, cellon-a-chip 3D co-cultures and custom designed 3D cell-on-achip cultures were performed to determine migration of cells in matrigel and collagen. Immunofluorescence and live cell imaging were used to determine endocytosis of EGF receptors. Results and discussions BCC did not show chemotaxis to macrophages in custom designed 3D cell-on-a-chip devices, which was in agreement with ELISA results showing that macrophage-derived-EGF was not secreted into macrophage-conditioned-medium. Live cell imaging of BCC in the presence and absence of iressa showed that macrophages but not macrophage-derived-matrix modulated adhesion and motility of BCC in 2D. 3D co-culture experiments in matrigel and collagen showed that BCC changed their multicellular organisation in the presence of macrophages. In custom designed 3D co-culture cell-on-a-chip devices, macrophages reduced and promoted migration of BCC in matrigel and collagen, respectively. Furthermore, adherent but not suspended BCC endocytosed their EGF receptors when in contact with macrophages. Conclusion Collectively, our data revealed that macrophages showed chemotaxis towards BCC-derived-CSF-1 whereas BCC required direct contact to interact with macrophage-derived-EGF. We propose that the interaction between cancer cells and macrophages is a paracrine-juxtacrine loop of CSF-1 and EGF, respectively. Introduction The transcription factor PRH/HHEX (Proline-Rich Homeodomain/Haematopoietically Expressed Homeobox) controls cell proliferation, cell differentiation and cell migration/invasion in a diverse range of cell types. Our recent work showed that PRH expression is down-regulated in breast and prostate cancer cells and that this results in increased cell proliferation and cancer cell invasion. We also showed that PRH inhibits breast and prostate cancer cell migration and invasion in part at least by activating the transcription of Endoglin, a Transforming Growth Factor beta1 (TGFb1) co-receptor. Here we show that TGFb1 down-regulates PRH expression in prostate cells and thereby up-regulates cell migration. Material and methods Normal immortalised prostate epithelial cells (PNT2-C2 cells) and prostate adenocarcinoma cells (PC3 cells) were treated with TGFb1 (5 ng/ml) or vehicle for 48 hours. mRNA expression was then assayed using quantitative PCR. Protein expression was assayed using western blotting and immunofluorescence microscopy. Cell migration was analysed using chemotaxis assays. pSMAD3 binding was examined using quantitative chromatin immunoprecipitation assays. Results and discussions The treatment of immortalised prostate epithelial cells and prostate cancer cells with TGFb1 increases cell migration. In immortalised prostate epithelial cells this is accompanied by morphological changes suggestive of an epithelial to mesenchymal transition (EMT). In both immortalised prostate epithelial cells and prostate cancer cells TGFb1 treatment results in the down-regulation of E-cadherin expression and the up-regulation of SNAIL expression, two established hallmarks of EMT. In both cell lines TGFb1 treatment results in increased levels of the TGFb1 effector pSMAD3, increased binding of pSMAD3 to the PRH/HHEX gene, and down-regulation of PRH expression at both the protein and mRNA level. Interestingly, PRH over-expression increases E-cadherin levels and represses cell migration. Conclusion TGFb1 treatment increases prostate cell migration in part at least by the down-regulation of PRH expression. PRH up-regulates E-cadherin expression and down-regulates cell migration suggesting that TGFb1 alters E-cadherin levels and increases cell migration via its effects on PRH. Since PRH also regulates TGFb1 signalling this creates a feedback loop that enables a more precise control of cell behaviour. Changes in PRH levels or activity during tumourigenesis disrupt this control mechanism.