Triple-negative breast cancer metastasis involves complex epithelial-mesenchymal transition dynamics and requires vimentin

Triple-negative breast cancer (TNBC) is an aggressive subtype associated with early metastatic recurrence and worse patient outcomes. TNBC tumors express molecular markers of the epithelial-mesenchymal transition (EMT), but its requirement during spontaneous TNBC metastasis in vivo remains incompletely understood. We demonstrated that spontaneous TNBC tumors from a genetically engineered mouse model (GEMM), multiple patient-derived xenografts, and archival patient samples exhibited large populations in vivo of hybrid E/M cells that lead invasion ex vivo while expressing both epithelial and mesenchymal characteristics. The mesenchymal marker vimentin promoted invasion and repressed metastatic outgrowth. We next tested the requirement for five EMT transcription factors and observed distinct patterns of utilization during invasion and colony formation. These differences suggested a sequential activation of multiple EMT molecular programs during the metastatic cascade. Consistent with this model, our longitudinal single- cell RNA analysis detected three different EMT-related molecular patterns. We observed cancer cells progressing from epithelial to hybrid E/M and strongly mesenchymal patterns during invasion and from epithelial to a hybrid E/M pattern during colony formation. We next investigated the relative epithelial versus mesenchymal state of cancer cells in both GEMM and patient metastases. In both contexts, we observed heterogeneity between and within metastases in the same individual. We observed a complex spectrum of epithelial, hybrid E/M, and mesenchymal cell states within metastases, suggesting that there are multiple successful molecular strategies for distant organ colonization. Together, our results demonstrate an important and complex role for EMT programs during TNBC metastasis.

Automated summary

Triple-negative breast cancer (TNBC) is an aggressive subtype associated with early metastatic recurrence and worse patient outcomes. Our study demonstrates the important and complex role of epithelial-mesenchymal transition (EMT) programs during TNBC metastasis. We observed the presence of a large population of hybrid epithelial-mesenchymal cells in TNBC tumors, which express both epithelial and mesenchymal characteristics and promote invasion in vitro. The mesenchymal marker vimentin promotes invasion and represses metastatic outgrowth. We also identified distinct patterns of EMT transcription factor utilization during invasion and colony formation. Our findings suggest a sequential activation of multiple EMT molecular programs during the metastatic cascade. Longitudinal single-cell RNA analysis detected three different EMT-related molecular patterns, indicating a complex spectrum of epithelial, hybrid E/M, and mesenchymal cell states within metastases. Our results highlight the heterogeneity and multiple molecular strategies for successful distant organ colonization in TNBC.