Metastatic cells from pleural effusions isolated from breast cancer patients can form mammospheres. (a) Cells were isolated from pleural effusions (PEs) and placed in non-differentiating medium in non-adherent culture flasks (see Materials and methods). PE14, PE6, PE21, and PE8 show four representative cultures. (b) PE mammospheres were disrupted and the cells plated onto glass coverslips in medium supplemented with 1% foetal calf serum. After 5 days of adherent culture, the cells were stained with antibodies to MUC1 (HMFG2), CK5 (D5/6), CK14 (LL002), and CK19 (BA17). (c) Breast cancer cell lines were also placed in non-differentiating medium in non-adherent culture flasks. Mammospheres could be seen developing in MCF7 and SKBR3 cultures, while MDAMB231 produced loosely adhered clumps of cells.