Short-term expansion of breast circulating cancer cells predicts response to anti-cancer therapy
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Bee Luan Khoo1,*, Soo Chin Lee2,3,*, Prashant Kumar4,*, Tuan Zea Tan3, Majid Ebrahimi Warkiani5,6, Samuel GW. Ow2, Sayantani Nandi4, Chwee Teck Lim4,5,7,8,*, Jean Paul Thiery3,4,9,*
1Mechanobiology Institute, National University of Singapore, Singapore
2Department of Hematology-Oncology, National University Cancer Institute, National University Hospital, Singapore
3Cancer Science Institute of Singapore, National University of Singapore, Singapore
4Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore
5BioSystems and Micromechanics (BioSyM) IRG, Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore
6School of Mechanical and Manufacturing Engineering, University of New South Wales, Sydney, Australia
7Department of Biomedical Engineering, National University of Singapore, Singapore
8Department of Mechanical Engineering, National University of Singapore, Singapore
9Department of Biochemistry Yong Loo Lin School of Medicine, National University of Singapore, Singapore
*These authors have contributed equally to this work
Jean Paul Thiery, e-mail: [email protected]
Keywords: breast cancer, circulating tumor cells, CTCs, enrichment
Received: February 16, 2015 Accepted: April 24, 2015 Published: May 06, 2015
Circulating tumor cells (CTCs) are considered as surrogate markers for prognosticating and evaluating patient treatment responses. Here, 226 blood samples from 92 patients with breast cancer, including patients with newly diagnosed or metastatic refractory cancer, and 16 blood samples from healthy subjects were cultured in laser-ablated microwells. Clusters containing an increasing number of cytokeratin-positive (CK+) cells appeared after 2 weeks, while most blood cells disappeared with time. Cultures were heterogeneous and exhibited two distinct sub-populations of cells: ‘Small’ (≤ 25 μm; high nuclear/cytoplasmic ratio; CD45-) cells, comprising CTCs, and ‘Large’ (> 25 μm; low nuclear/cytoplasmic ratio; CD68+ or CD56+) cells, corresponding to macrophage and natural killer-like cells. The Small cell fraction also showed copy number increases in six target genes (FGFR1, Myc, CCND1, HER2, TOP2A and ZNF217) associated with breast cancer. These expanded CTCs exhibited different proportions of epithelial–mesenchymal phenotypes and were transferable for further expansion as spheroids in serum-free suspension or 3D cultures. Cluster formation was affected by the presence and duration of systemic therapy, and its persistence may reflect therapeutic resistance. This novel and advanced method estimates CTC clonal heterogeneity and can predict, within a relatively short time frame, patient responses to therapy.
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