A high-content EMT screen identifies multiple receptor tyrosine kinase inhibitors with activity on TGFß receptor.

An epithelial to mesenchymal transition (EMT) enables epithelial tumor cells to break out of the primary tumor mass and to metastasize. Understanding the molecular mechanisms driving EMT in more detail will provide important tools to interfere with the metastatic process. To identify pharmacological modulators and druggable targets of EMT, we have established a novel multi-parameter, high-content, microscopy-based assay and screened chemical compounds with activities against known targets. Out of 3423 compounds, we have identified 19 drugs that block transforming growth factor beta (TGFß)-induced EMT in normal murine mammary gland epithelial cells (NMuMG). The active compounds include inhibitors against TGFß receptors (TGFBR), Rho-associated protein kinases (ROCK), myosin II, SRC kinase and uridine analogues. Among the EMT-repressing compounds, we identified a group of inhibitors targeting multiple receptor tyrosine kinases, and biochemical profiling of these multi-kinase inhibitors reveals TGFBR as a thus far unknown target of their inhibitory spectrum. These findings demonstrate the feasibility of a multi-parameter, high-content microscopy screen to identify modulators and druggable targets of EMT. Moreover, the newly discovered "off-target" effects of several receptor tyrosine kinase inhibitors have important consequences for in vitro and in vivo studies and might beneficially contribute to the therapeutic effects observed in vivo.

A high throughput system for long term application of intermittent cyclic hydrostatic pressure on cells in culture.

The process of bone remodeling is governed by mechanical stresses and strains. Studies on the effects of mechanical stimulation on cell response are often difficult to compare as the nature of the stimuli and differences in parameters applied vary greatly. Experimental systems for the investigation of mechanical stimuli are mostly limited in throughput or flexibility and often the sum of several stimuli is applied. In this work, a flexible system that allows the investigation of cell response to isolated intermittent cyclic hydrostatic pressure (icHP) on a high throughput level is shown. Human bone derived cells were cultivated with or without mechanical stimulus in the presence or absence of chemical cues triggering osteogenesis for 7-10 days. Cell proliferation and osteogenic differentiation were evaluated by cell counting and immunohistochemical staining for bone alkaline phosphatase as well as collagen 1, respectively. In either medium, both cell proliferation and level of differentiation were increased when the cultures were mechanically stimulated. These initial results therefore qualify the present system for studies on the effects of isolated icHP on cell fate and encourage further investigations on the details behind the observed effects.