Profiling Memory and Plasticity of Mesenchymal-Epithelial Transitions in Multicellular Spheroids

Introduction: : Mesenchymal cells typically exhibit minimal cell-cell adhesion and aggressive invasion, but transitions towards more epithelial phenotypes are believed to be advantageous for tissue regeneration and tumor metastasis. However, the relative memory or plasticity of an epithelial or mesenchymal cell state remains poorly understood and varies across different cell lines. In particular, MDA-MB-231 is a highly mesenchymal breast cancer line with minimal E-cadherin expression, and it is challenging to induce epithelial states in monolayer culture. Here, we investigate the (ir)reversibility of E-cadherin induction and more epithelial states in MDA-MB-231 cells prepared as multicellular spheroids. We utilize a dual fluorescent reporter line for continuous, real-time visualization of epithelial and mesenchymal marker dynamics at the single-cell level. This innovative approach provides a robust platform for dissecting temporal phenotypic changes. It establishes a quantitative framework for studying mesenchymal-to-epithelial plasticity, thereby providing a more accurate reflection of the complexities of cancer progression.

Materials and

Methods: : Dual Z-cadherin reporter MDA-MB-231 breast cancer cells were used to monitor epithelial and mesenchymal phenotypes. Cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. To induce mesenchymal–epithelial transition (MET), doxycycline was added to the medium at 2 µg/mL for either 3 days (short-term) or 10 days (long-term) prior to 3D spheroid formation. Spheroids were formed by seeding approximately 7,200 cells/spheroid into 2% agarose microwell molds, followed by centrifugation at 300 × g for 3 minutes. Doxycycline was introduced just before centrifugation, and spheroids were maintained in agarose molds for either 4 or 10 days, with media changes every 2 days. Spheroids were then transferred to 48-well culture plates and imaged every 24 hours for 3 days. Imaging was performed using confocal microscopy with z-stack acquisition in GFP and Cy3 channels to capture reporter expression. Image analysis was conducted in Fiji (ImageJ). Statistical analysis was performed using Python.

Results, Conclusions, and Discussions:: To investigate MDA-MB-231 cell line’s preference for E-cadherin expression in cellular aggregate formation, we utilized an inducible expression system, designed to reduce heterogeneity within the cellular population. In 2D culture, short-term doxycycline treatment effectively induces a strong epithelial signal, significantly increasing the population of RFP-positive (E-cadherin-expressing) cells (p=0.002) while minimizing the number of GFP-positive (N-cadherin-expressing) cells (p < 0.001), all without affecting cell proliferation. These cells were harvested to form 3D spheroids in agarose microwells and cultured for 10 days in either doxycycline-containing or normal DMEM. We hypothesized that the induced expression of E-cadherin would enhance cell-cell adhesion, thereby promoting more compact spheroids. However, contrary to our expectations, both groups displayed similar size trajectories, constantly expanding due to proliferation, and did not show any notable differences in compaction (p=0.283), despite the significant difference in RFP expression (p < 0.001), as shown in Fig.1. Additionally, spheroids from both conditions dispersed, indicating a lack of sufficient mechanical cohesion despite forced E-cadherin expression.
To investigate whether a longer induction period could foster more stable epithelial characteristics, we preconditioned cells in doxycycline for 10 days prior to spheroid formation. This approach resulted in spheroids that exhibited rapid and sustained compaction (Fig. 2), remaining structurally intact during subsequent handling. This suggests an establishment of mechanical phenotypic memory, which may indicate the necessity for sustained to overcome the stable mesenchymal program of MDA-MB-231 cells. Upon being transferred to 2D surfaces, these spheroids maintained a circular morphology, though thinning over time, and exhibited a donut-like pattern of RFP expression (Fig. 3). Importantly, epithelial-to-mesenchymal ratio remained no different regardless of media conditions post-transfer (p=0.338), suggesting a secondary niche from simple anchorage. However, further in-depth studies, particularly in biomaterials or extracellular matrix, will be essential.
In summary, these findings establish a temporally controllable platform for studying EMT/MET plasticity and phenotypic memory in mesenchymal cancer cells. Notably, transient E-cadherin induction reverses MDA-MB-231 cells to a mesenchymal state, whereas sustained E-cadherin induction results in a more sustained epithelial state. We will further interrogate this phenotypic memory or plasticity in more complex microenvironments to elucidate the consequences for invasion and drug resistance.

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