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.