Novel Nanobodies and Chromobodies to Study Biomarkers of Epithelial-Mesenchymal Transition (EMT)

Abstract

The epithelial-mesenchymal transition (EMT) is a reversible cellular reprogramming process that originally occurs during embryonic development and is strongly involved in the initiation of metastases and cancer progression. During EMT, single cells of an epithelial layer lose their characteristic epithelial features, detach from their neighbor cells and acquire a mesenchymal phenotype with increased migratory and invasive potential. Targeting EMT is of particular interest for the development of novel compounds in the anti-metastatic cancer therapy. Thereby, certain epithelial and mesenchymal proteins, including the tight junction component occludin, the actin cytoskeleton, the transcriptional repressor SNAI1 and the intermediate filament vimentin, serve as specific biomarkers or even as target structures for compounds. To date, there is no approach available to study these endogenous markers in living cells in a physiological context. In this thesis, novel single-domain antibodies (nanobodies) were selected to trace EMT biomarkers in a cancer-relevant living cell system. Nanobodies (~15 kDa), derived from heavy-chain-only antibodies of camelids, represent the smallest naturally occurring antigen binding reagent. For intracellular target visualization, they can be fused to fluorescent proteins (referred to as chromobodies) and introduced into cells on DNA level. Specific nanobodies against occludin, SNAI1 and vimentin were selected via the phage display technology and respective binding properties of nanobodies and chromobodies were analyzed in biochemical and cell biological assays. The vimentin specific and intracellular functional chromobody VB6-CB and a previously described chromobody specific for actin (Actin-CB) were stably introduced in cellular models. Based on the chromobody fluorescence, dynamic changes of endogenous actin and vimentin upon induction of EMT with the transforming growth factor β (TGF-β) were monitored and quantified for the first time by high-content-imaging. Moreover, by treatment with the vimentin modifying compound Withaferin A (WFA) time- and dose- dependent alterations of vimentin were observed and analyzed. Taken together, this versatile approach allows detailed studies of the spatiotemporal organization of relevant EMT-biomarkers in living cells. This may provide the basis for novel screening technologies for anti-metastatic therapeutics affecting EMT.