Fig. 1

Chemo-residual triple-negative (TN) breast tumor cells emanating from a short-term chemotherapy treatment model do not exhibit cancer stem-like cell behavior. a In vitro model of TN breast cancer chemo-resistance. SUM159 and BT549 tumor cells were treated with doxorubicin (Dox) (1 μg/ml, 0.5 μg/ml, respectively) for 2 days, after which chemotherapy was removed and fresh medium was added. Between 7 and 10 days, a small number of chemo-residual cells (0.1 % of the original population) remained, and exhibited reduced proliferation compared to parental (untreated) cells. Approximately 2 weeks after chemotherapy withdrawal, chemo-residual cells resumed proliferation and established colonies. Pictures of parental (untreated) cells, chemo-residual cells, and colonies emanating from chemo-residual cells were taken on days 0, 7, 14 (SUM159 cells) or days 0, 10, 18 (BT549 cells), respectively. Magnification ×200. Similar data were obtained by treating SUM159 cells with docetaxel (100 nM) for 2 days [8]. b SUM159 cells were treated with Dox as described in Fig. 1a. Parental and chemo-residual cells were seeded at equal density in a 96-well plate. Left panel: proliferation was determined by ³H-thymidine incorporation. Right panel: cell viability was assessed by alamar blue. Error bars represent SD, n = 6, ***p <0.001, two-tailed Student’s t test. c SUM159 parental and chemo-residual cells harvested on day 7 after Dox treatment were seeded at the indicated numbers into a mammosphere assay. Mammosphere number was quantified after 4 days. Error bars represent SD, n = 3, **p <0.01, ***p <0.001, two-tailed Student’s t test. d Parental and chemo-residual cells harvested on day 8 after docetaxel treatment were seeded into a mammmosphere assay. Mammosphere number was quantified after 4 days. Error bars represent SD, n = 3, ***p <0.001, two-tailed Student’s t test. e RNA was extracted from SUM159 parental and chemo-residual cells harvested on day 7 after Dox treatment. Equivalent amounts were subjected to hypoxia inducible factor-1α (HIF-1α) real-time PCR. Results are expressed as the mean HIF-1α/β- actin ratio from triplicate wells (+/- SD). **p <0.01, two-tailed Student’s t test. f Total cellular extracts obtained from SUM159 parental and chemo-residual cells harvested on day 7 after Dox treatment were subjected to SDS-PAGE and immunoblotted with a HIF-1α or β-actin antibody, followed by secondary antibody