Targeting EMT-driven metastatic breast cancer with mitotic inhibitors

Background: Metastatic breast cancer is the leading cause of cancer death in women and remains an incurable disease. The epithelial-to-mesenchymal transition (EMT) is widely recognized as a key driver of metastasis, with intermediate or hybrid EMT states shown to exhibit greater plasticity and metastasis-initiating potential than cells that undergo a complete EMT. However, the inherent heterogeneity and plasticity of EMT states pose significant challenges for therapeutic targeting. In this study, we characterized EMT heterogeneity in the "aggressive front" of primary tumors and circulating tumor cells (CTCs) in the bloodstream and explored repurposing of mitotic inhibitors to target EMT-driven plasticity.

Methods: CTCs were isolated from whole blood of metastatic breast cancer patients (n=3) by gradient separation and enrichment with EpCAM beads and cultured in low-attachment plates with serum supplemented organoid media. Patient-derived organoids (PDOs) were developed from the center and periphery of primary breast tumors (n=3), by mechanical and enzymatic digestion, and cultured in an extracellular matrix in organoid media. RNA from CTCs and PDOs was purified for EMT marker analysis by RT-qPCR. CTCs and PDOs were formalin-fixed and paraffin-embedded for EMT marker evaluation by immunocytochemistry. The effect of an AURKA inhibitor on the growth and invasion of PDOs was analyzed with a live-cell imaging instrument.

Results: Invasive cells showed enrichment of the mesenchymal markers vimentin, Zeb1, and Twist, and reduced expression of the epithelial markers E-cadherin and EpCAM, compared to epithelial-like MCF-7 cells. Vimentin and N-cadherin levels were lower compared to mesenchymal-like MDA-MB-231 cells, and SNAI1/2 and fibronectin expression was reduced relative to both epithelial- and mesenchymal-like cells, indicating an intermediate EMT state. AURKA inhibition with alisertib effectively blocked the growth of PDOs generated from invasive cells.

Conclusions: These findings highlight the therapeutic potential of AURKA inhibitors for targeting hybrid EMT states present in CTCs and invasive regions of primary tumors, offering a strategy to combat metastatic disease and potentially transform current breast cancer treatment.