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.

Biotechnology Centre Silesian University of Technology Gliwice Poland

Centre for Experimental Cardiooncology Medical University of Gdańsk Poland

Centre of Biostatistics and Bioinformatics Medical University of Gdańsk Poland

Department of Oncological Surgery Faculty of Health Sciences with the Institute of Maritime and Tropical Medicine Medical University of Gdańsk Gdynia Poland

Department of Oncological Surgery Gdynia Oncology Centre Pomeranian Hospitals Gdynia Poland

Department of Oncology and Radiotherapy Faculty of Medicine Medical University of Gdańsk Poland

Department of Systems Biology and Engineering Faculty of Automatic Control Electronics and Computer Science Silesian University of Technology Gliwice Poland

Interdisciplinary Pomeranian Center for Digital Medicine Medical University of Gdańsk Poland

International Centre for Cancer Vaccine Science University of Gdańsk Poland

Laboratory of Translational Oncology Intercollegiate Faculty of Biotechnology University of Gdańsk and Medical Univeristy of Gdańsk Medical University of Gdańsk Poland

Research Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute Brno Czech Republic

Scientific Computing Group IT Division University of Bergen Norway

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