In VivoVisualization and Characterization of Epithelial–Mesenchymal Transition in Breast Tumors

Abstract

The activation of the epithelial-to-mesenchymal transition (EMT) program is a critical step in cancer progression and metastasis, but visualization of this process at the single cell level, especially in vivo, remains challenging. We established an in vivo approach to track the fate of tumor cells based on a novel EMT-driven fluorescent color switching breast cancer mouse model and intravital two-photon laser scanning microscopy. Specifically, the MMTV-PyMT, Rosa26-RFP-GFP, and Fsp1-Cre triple transgenic mouse model was used to monitor the conversion of RFP-positive epithelial cells to GFP-positive mesenchymal cells in mammary tumors under the control of the Fsp1 (ATL1) promoter, a gate-keeper of EMT initiation. RFP-positive cells were isolated from the tumors, sorted, and transplanted into mammary fat pads of SCID mice to monitor EMT during breast tumor formation. We found that the conversion from RFP to GFP-positive and spindle-shaped cells was a gradual process, and that GFP-positive cells preferentially localized close to blood vessels, independent of tumor size. Furthermore, cells undergoing EMT expressed high levels of the HGF receptor, c-Met, and treatment of RFP-positive cells with the c-Met inhibitor, cabozantinib, suppressed the RFP-to-GFP conversion in vitro. Moreover, administration of cabozantinib to mice with palpable RFP-positive tumors resulted in a silent EMT phenotype whereby GFP-positive cells exhibited reduced motility, leading to suppressed tumor growth. In conclusion, our imaging technique provides a novel opportunity for visualizing tumor EMT at the single cell level and may help to reveal the intricacies underlying tumor dynamics and treatment responses.

Identification of clinically predictive metagenes that encode components of a network coupling cell shape to transcription by image-omics

“…6A). That genes associated with high NF and protrusions have high expression in more aggressive tumors is in line with the idea that aggressive tumors have regions of high cell density driven by high rates of proliferation and have protrusive invasive fronts (Jögi et al 2012;Zhao et al 2016).…”

“…6A). Because high values of cell W/L metagene are indicative of epithelial shape, while lower values are indicative of a more mesenchymal shape (Zhao et al 2016), this suggests that genes associated with epithelial shapes are down-regulated in highly aggressive breast cancers. Moreover, this supports the idea that the activity of the shape-gene network contributes to disease progression.…”

Epithelial-to-mesenchymal transition confers pericyte properties on cancer cells

“…Carcinoma cells undergoing spontaneous EMT have been identified in transgenic mouse mammary tumors and in human breast cancer specimens (4)(5)(6). As EMT may enable carcinoma cells to overcome cell-cell adhesion and to invade neighboring tissue, EMT has been proposed to be a critical event initiating tumor invasion and metastasis (7).…”

Molecular mobility and activity in an intravital imaging setting – implications for cancer progression and targeting

“…Zhao et al (2016) simultaneously observed in vivo EMT alongside changes in cell migration and morphology in the MMTV-PyMT model of invasive and metastatic breast cancer, in which expression of the oncoprotein polyoma middle T antigen (PyMT) under the control of the mouse mammary tumour virus long terminal repeat (MMTV-LTR) drives cancer progression (Lin et al, 2003). Here, IVM can provide insight into single-cell EMT events in their native environment, as well as distinct real-time readouts of drugs targeting the EMT process or cells that have undergone EMT (Zhao et al, 2016). De-regulated expression of the cell-cell junction protein Ecadherin is also often associated with EMT and cell invasiveness (Gregory et al, 2008;Lehmann et al, 2016b).…”