Trajectories of partial and full EMT of mammary tumor cells in the MMTV-PyMT mouse model

Epithelial-mesenchymal transition (EMT) is a multistep process of cell de-differentiation which confers carcinoma cells with a variety of malignant characteristics. Due to the transient and reversible nature of the process, cancer cells may transit between various stages of an EMT continuum, including epithelial, partial EMT or full EMT, and the actual status of cancer cells, the kinetics of cellular state transition in vivo and the consequences thereof have remained elusive. We have established tamoxifen inducible dual recombinase lineage tracing systems combined with live cell imaging and 5-cell RNA-sequencing analysis to track cancer cells undergoing a partial or a full EMT in the MMTV-PyMT mouse model of metastatic breast cancer. In primary tumours, cancer cells infrequently undergo an EMT, and most of these cells transition between epithelial and partial EMT states, but rarely undergo a full EMT. Consequently, cells that have ever undergone a partial EMT dynamically transit between various EMT states, and they contribute to lung metastasis and chemoresistance. In contrast, full EMT cells mostly retain a mesenchymal phenotype and stably reside in perivascular regions, but fail to colonize the lungs. However, full EMT cancer cells are enriched in recurrent tumors upon chemotherapy. Our findings suggest that cancer cells in various stages of the EMT continuum contribute differentially to hallmarks of breast cancer malignancy, such as cell migration, invasion, metastasis and chemoresistance. Partial and full EMT mammary tumor cells were sorted by flow cytometry from tumors of Tnc-CreERT2;MMTV-Flpo;RC::FrePe;MMTV-PyMT (Tnc-CreERT2) and Cdh2-CreERT2;MMTV-Flpo;RC::FrePe;/MMTV-PyMT (Cdh2-CreERT2) mice. The dual recombinase lineage tracing system allows to label all breast cancer cells as mCherry expressing cells (red). Upon tamoxifen administration cancer cells that have undergone either partial or full EMT switch to the expression of GFP (green). This red to green color switching is irreversible, which allows to trace all cancer cells that have ever undergone an EMT by the expression of GFP, even if they have reverted back to an epithelial state by undergoing a MET. In consequence, mCherry+ cells are breast cancer cells, GFP+ cells are breast cancer cells that have ever undergone either partial or full EMT and double positive cells are cells undergoing an EMT (transtioning cells). Based on the expression of GFP or mCherry in combination with the epithelial marker EpCAM expression, 3 populations of mCherry+ cells (P5, P17 and P18), 4 populations of GFP+ cells (P15, P10, P16 and P9) and 1 population of doble positive cells (P22) were collected from Tnc-CreERT2 mice, and 3 populations of mCherry+ cells (P5, P17 and P18) and 1 population of GFP+ cells (P15) were collected from Cdh2-CreERT2 mice. mCherry populations, P05: mCherry+ EpCAM-, P17: mCherry+ EpCAMhigh, P18: mCherryhigh EpCAMhigh. GFP populations, P15:GFP+ EpCAM-, P10: GFP+ EpCAMlow, P16: GFP+ EpCAMmed and P18: GFPhigh EpCAMmed. Double positive cells: P22: mCherry+ GFP+ EpCAMhigh