Characterizing epithelial‐mesenchymal transition‐linked heterogeneity in breast cancer circulating tumor cells at a single‐cell level

Epithelial–mesenchymal transition (EMT) generates heterogeneity in circulating tumor cells (CTCs), affecting their biological properties and hampering their detection. This limits our understanding of the mechanisms underlying hematogenous dissemination, especially in early breast cancer (BC), where CTCs are rare. Here, we aimed to detect CTCs with different EMT statuses from BC patients. CTCs in blood samples from 107 BC patients were evaluated using immunomagnetic depletion and multi‐marker immunofluorescence (EpCAM, E‐cadherin, MCAM, cell surface vimentin, CD31, CD45), followed by single‐cell transcriptomics. CTCs were detected in 51.9% of therapy‐naïve early BC cases, with 3.8% showing only epithelial CTCs (eCTCs), 5.8% epithelial‐mesenchymal (emCTCs), 26.0% mesenchymal (mCTCs), and 16.3% mixed phenotypes. CTC heterogeneity was more frequent in triple‐negative (86%) than in luminal BC (17%, P = 0.008). Lymph node involvement strongly predicted dissemination of all CTC phenotypes, while tumor size correlated with mCTC abundance. Single‐cell RNA sequencing revealed downregulation of ribosomal genes and translation inhibition in CTCs with mesenchymal features, linked to mTORC1 signaling. Findings were also validated in an independent dataset, highlighting vulnerabilities in CTCs during dissemination.