A study published in Science (2013) reveals that circulating tumor cells (CTCs) from breast cancer patients exhibit dynamic changes in epithelial and mesenchymal characteristics. The research team characterized EMT in CTCs from breast cancer patients and found that mesenchymal CTCs were highly enriched in CTCs. Serial monitoring of 11 patients suggested an association between mesenchymal CTCs and disease progression. In an index patient, reversible shifts between epithelial and mesenchymal cell fates occurred with each cycle of therapy and disease progression. Mesenchymal CTCs were found as single cells and multicellular clusters, expressing EMT regulators such as TGF-β and FOXC1. These findings support a role for EMT in the bloodborne dissemination of human breast cancer. Most cancer-related deaths are caused by metastasis, the spread of cancer cells from the primary tumor to new sites. Aberrant activation of EMT has been implicated in this process. However, identifying EMT in tumors is complicated by the presence of reactive mesenchymal stromal cells. Analysis of CTCs has been hindered by reliance on epithelial markers to separate cancer cells from surrounding hematopoietic cells. To address these challenges, the researchers optimized microfluidic capture of CTCs with epithelial- and tumor-specific antibodies and used this technology to analyze EMT in CTCs from breast cancer patients. The study established a dual-colorimetric RNA-in situ hybridization (ISH) assay to examine tumor cells for expression of seven epithelial and three mesenchymal transcripts. These probes were validated in cell lines and confirmed differential expression in epithelial versus mesenchymal cancer cells. The assay was applied to primary human breast cancer specimens, revealing that most cancer cells were epithelial, while reactive stromal cells were mesenchymal. In contrast, invasive breast cancer contained rare tumor cells with epithelial morphology that stained with both E and M markers. The study found that mesenchymal CTCs were more abundant in triple-negative breast cancer. The researchers used microfluidic HB chips to capture CTCs from blood and found that CTCs from patients with lobular type cancers were predominantly epithelial, while those from the TN subtype were predominantly mesenchymal. CTCs from patients with HER2+ breast cancer were also predominantly mesenchymal. The study also found that CTC clusters, ranging from 4 to 50 cells, were more common in patients with advanced cancer. These clusters were strongly positive for M markers and weakly positive for E markers by ISH. RNA sequencing of CTCs revealed that EMT-related gene expression was enriched in CTCs, with significant overlap with a core EMT signature. The study also found that TGF-β was strongly expressed in mesenchymal CTC clusters, many of which carriedA study published in Science (2013) reveals that circulating tumor cells (CTCs) from breast cancer patients exhibit dynamic changes in epithelial and mesenchymal characteristics. The research team characterized EMT in CTCs from breast cancer patients and found that mesenchymal CTCs were highly enriched in CTCs. Serial monitoring of 11 patients suggested an association between mesenchymal CTCs and disease progression. In an index patient, reversible shifts between epithelial and mesenchymal cell fates occurred with each cycle of therapy and disease progression. Mesenchymal CTCs were found as single cells and multicellular clusters, expressing EMT regulators such as TGF-β and FOXC1. These findings support a role for EMT in the bloodborne dissemination of human breast cancer. Most cancer-related deaths are caused by metastasis, the spread of cancer cells from the primary tumor to new sites. Aberrant activation of EMT has been implicated in this process. However, identifying EMT in tumors is complicated by the presence of reactive mesenchymal stromal cells. Analysis of CTCs has been hindered by reliance on epithelial markers to separate cancer cells from surrounding hematopoietic cells. To address these challenges, the researchers optimized microfluidic capture of CTCs with epithelial- and tumor-specific antibodies and used this technology to analyze EMT in CTCs from breast cancer patients. The study established a dual-colorimetric RNA-in situ hybridization (ISH) assay to examine tumor cells for expression of seven epithelial and three mesenchymal transcripts. These probes were validated in cell lines and confirmed differential expression in epithelial versus mesenchymal cancer cells. The assay was applied to primary human breast cancer specimens, revealing that most cancer cells were epithelial, while reactive stromal cells were mesenchymal. In contrast, invasive breast cancer contained rare tumor cells with epithelial morphology that stained with both E and M markers. The study found that mesenchymal CTCs were more abundant in triple-negative breast cancer. The researchers used microfluidic HB chips to capture CTCs from blood and found that CTCs from patients with lobular type cancers were predominantly epithelial, while those from the TN subtype were predominantly mesenchymal. CTCs from patients with HER2+ breast cancer were also predominantly mesenchymal. The study also found that CTC clusters, ranging from 4 to 50 cells, were more common in patients with advanced cancer. These clusters were strongly positive for M markers and weakly positive for E markers by ISH. RNA sequencing of CTCs revealed that EMT-related gene expression was enriched in CTCs, with significant overlap with a core EMT signature. The study also found that TGF-β was strongly expressed in mesenchymal CTC clusters, many of which carried