Figure 1. Post-chemotherapy sera from BC patients and mice stimulate a CSC phenotype through elevated MCP levels.

(A) Twenty pairs of pre- and post-NT sera from TNBC (n=8; black) or HER2⁺ BC (n=12; blue) patients were analyzed for the activity to stimulate the ALDEFLUOR^bright population of BC cells. MDA-MB-231 cells were cultured for 48 h in base medium supplemented with 10% human serum before ALDEFLUOR assays by flow cytometry. Wilcoxon test was performed. (B) ELISA to determine the levels of human CCL2/7/8 in the 20 pairs of sera. Wilcoxon tests were performed. (C) ALDEFLUOR assays using 6 pairs of sera (3 cases for each BC subtype) were performed as in A except that CCL2/7/8 neutralizing antibodies (NAb; 30 ng/ml; alone or all 3 combined) or control IgG were added during serum treatment. (D) ALDEFLUOR assays of MDA-MB-231 cells treated with patient sera in the presence of a CCR2 inhibitor (MK-0812; 600 nM) or vehicle. (E) NSG mice with or without MDA-MB-231 xenograft tumors and tumor-free BALB/c mice received 3 weekly injections with doxorubicin (DOXO; 4 mg/kg) or docetaxel (DTX; 25 mg/kg) (n=3). Six days later, serum was collected to treat MDA-MB-231 cells for ALDEFLUOR assays as in A. (F) ELISA to determine the serum levels of mouse CCL2/7/8. (G) ALDEFLUOR assays of MDA-MB-231 cells treated with mouse sera in the presence of the CCR2 inhibitor MK-0812 or vehicle. (H) Sera from chemotherapy-treated patients and mice induce tumorigenicity. MDA-MB-231 cells pre-treated with human (case T1) or mouse (tumor-free BALB/c) sera for 48 h were injected into the mammary fat pad of NSG mice (n=10) at the indicated numbers. Tumor incidence after 4 weeks was shown. The estimated TIC frequency was estimated by ELDA. *P<0.05, **P<0.01, ***P<0.001 (compared to the corresponding IgG group in C or as indicated).