Epithelial-mesenchymal transition (EMT) is a process that shifts cells from an epithelial (E) to a mesenchymal (M) state. This plays a pivotal role in cancer where mesenchymal phenotype is known to contribute to therapy resistance and metastatic progression. The cytokine TGF-β is the most studied activator of epithelial-mesenchymal transition (EMT), however, it is currently unknown to what extent the TGF-β increases the ratio of M to E states by directing actual plasticity (cells changing state) as opposed to selection of M cells via increased proliferation or repression of E cells.
To answer this question, we performed Cylic IF Immunofluorescence on a basal-like breast cancer cell line (HCC38) treated with varying duration of exposure to TGF-β, enabling us to examine the changes in expression and localization of 66 markers including epithelial (E) and mesenchymal (M), cell cycle, signalling, stemness and cell fate proteins across time at single-cell resolution.
Using this approach, we found that TGF-β differentially decreases proliferation of epithelial cells and increases proliferation of mesenchymal cells, evidenced by decreased cell numbers and differential expression of the proliferative marker Ki67. Finally, we found that TGF-β treatment induces expression of the negative cell-cycle regulator, p21, prominently in epithelial cells after long-term exposure highlighting a potential explanatory mechanism.
Our findings show that TGF-β-induced regulation of EM-phenotypes is not driven exclusively by plasticity but indicates that that selective proliferation holds a significant contribution to controlling the balance of E and M subpopulations.