Abstract P6-04-22: Metformin is an effective drug to target the bevacizumab driven cancer stem cell stimulation in murine 4T1 breast cancer cell line
Authors: Antonio HMR , Sicchieri RD , Andrade JM , Tiezzi DG 
Published: August 2014

Abstract
Hypoxia-driven cancer stem cell (CSC) stimulation limits the effectiveness of antiangiogenic agents in breast cancer. The use of antiangiogenic drugs in combination with CSC-targeting drugs is a reasonable strategy to optimize cancer treatment. Metformin has been demonstrated to be efficient against multiple forms of cancer. Recent reports have demonstrated metformin can inhibit CSCs proliferation. We analyzed the influence of metformin and its combination with bevacizumab on CSC population in breast cancer. The MDA-MB-231, MCF7, 67nr and 4T1 cell lines were exposed to different concentration of metformin (0 mM, 1 mM, 5mM and 10 mM) in vitro during 72 hours. Using the trypan blue exclusion assay, we estimated the percentage of dead cells in culture. We observed a dose-time dependent citotoxicity of metformin among all cell lines. Using a dose of 10 mM of metformin, the 4T1 cells were cultured for 48 hours and the flow cytometry was performed to analyze the PI / Anexin expression. The mean percentage of PI+ cells in the control group was 12.8 ± 1.2%. The mean percentage of PI+ in metformin treated group was 22.7 ± 2.2% (p = 0.005). We did not observe any difference in anexin expression. To determine the role of ALDH expression as a marker for breast CSCs in 4T1 cell line, we sorted Aldefluorhigh 4T1 cells and performed a limiting dilution assay. We observed the prevalence of CSCs in Aldefluorhigh 4T1 cells was 1/455 cells (CI 95% from 1,948 to 107). The prevalence in Aldefluor- and the Aldefluorwt 4T1 cells was 1/5,361 cells (CI 95% from 21,557 to 1,333) and 1/2,086 cells (CI 95% from 7,466 to 583), respectively (p = 0.06). The pairwise test for differences in stem cell frequency between Aldefluorhigh and Aldefluor- cells was significant (p = 0.01). In order to determine whether metformin interferes with breast CSC population in vitro and in vivo, we treated 4T1 cells in culture (10 nM) and 4T1 tumor-bearing mice with intraperitoneal (n = 4) metformin (2,100 mg/kg/day) alone or in combination with bevacizumab (5mg/kg s.c. twice a week). In the in vitro assay, the mean percentage of Aldefluorhigh 4T1 cells was 28.8 ± 4.06% in the control group. After 48 hours of metformin exposure, the mean percentage of Aldefluorhigh 4T1 cells was 15.6 ± 3.5% (p = 0.01). In the in vivo assay, tumors were smaller in mice treated with metformin or bevacizumab alone or in combination. However, there is a significant increase in Aldefluorhigh cell population (p = 0.04) and ALDH1A1 gene expression (real time RT-PCR; p&lt; 0.0001) in bevacizumab treated tumors. We did not observed an increase in Aldefluorhigh cell population and ALDH1A1 gene expression in tumors from mice treated with metformin/bevacizumab combination. Our results demonstrate metformin is an effective drug to target the bevacizumab driven cancer stem cell stimulation in murine 4T1 breast cancer cell line.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-22.