The Drug Sensitivity Research Of Breast Cancer Stem Cells On Targeted-, Chemo-and Endocrine-Therapy

Objective:1. To test the antitumor effects of the mTOR inhibitor everolimus in breast cancer stem cells and total cells in vitro and in vivo.2. To test the combining effects of everolimus and docetaxel in vitro and in vivo in stem cells of primary breast cancer cells and two breast cancer models.3. To find the role of trastuzumab in stem cells of HER2+breast cancer and the combining effects of everolimus and trastuzumab in stem cells in vitro and in vivo.4. To evaluate the role of letrozole in stem cells of MCF-7/Aro and the combining effects of everolimus and letrozole in stem cells of MCF-7/Aro in vitro and in vivo.Methods:1. In vitro studies, we sorted ESA+CD44+CD24-/low cells as stem cells using flow cytometry from primary breast cancer cells, MCF-7, MDA-MB-231and BT474cell lines. MTT assays were used to quantify the inhibitory effect of the drugs on total cells and stem cells. Apoptosis and the cell cycle distributions of stem cells were examined by flow cytometry. The tumorigenicity of stem cells after treatment was investigated by soft agar colony formation assays.2. In vivo studies, the BALB/c mice were injected with stem cells and the different treatments were administered.After necropsy, the expression of KI67, CD31, AKT1, and phospho-AKT (Thr308) was analyzed by immunohistochemistry.Results:1. Treatment with everolimus resulted in growth inhibition of all stem cells in a dose-dependent manner. An increase in G0-G1cell cycle arrest and an increased population of cells in early apoptosis were seen in everolimus treatment.In vivo, the volumes of the xenograft tumors significantly decreased in everolimus alone group compared to control group.2. The combining effects of everolimus and docetaxel in stem cells:An additive growth inhibitory effect of the combination treatment on the three stem cells was observed in vitro compared with treatment with alone. In addition, an increase in G2/M cell cycle arrest and an increased population of cells in early apoptosis were seen in the combination treatment group compared with either single-agent group. In vivo, everolimus plus docetaxel therapy was much more effective at reducing tumor volume in mice compared with either single-agent alone. Compared with everolimus alone, the combination of everolimus and docetaxel reduced the expression of KI67, CD31, AKT1and phospho-AKT (Thr308).3. The combining effects of everolimus and trastuzumab in stem cells:Compared with single-agent therapy, the combination of everolimus with trastuzumab was more effective in the inhibition of cell growth and tumorigenicity. In vivo, the volumes of the xenograft tumors significantly decreased in everolimus alone group compared to untreated group, and everolimus plus trastuzumab therapy was much more effective at reducing tumor volume in mice compared with either single-agent alone. Compared with everolimus alone, the combination of everolimus and trastuzumab reduced the expression of KI67, AKT1and phospho-AKT (Thr308).4. The combining effects of everolimus and letrozole in stem cells:The combination of everolimus with letrozole was more effective in the inhibition of cell growth and tumorigenicity than single-agent therapy.An increase in G1cell cycle arrest and an increased population of cells in early apoptosis were seen in the combination treatment group.In vivo,everolimus plus letrozole therapy was much more effective at reducing tumor volume in mice compared with either single-agent by reducing the expression of K167and phospho-AKT (Thr308).Conclusions:1. Everolimus could inhibit the growth of both total breast cancer cells and sorted stem cells in vitro and in vivo.2. The combination treatment of everolimus and docetaxel can inhibit the growth of stem cells in vitro and in vivo by inhibiting cell proliferation, inducing apoptosis, cell cycle arrest and reducing the expression of K167, CD31, AKT1and pAKT.3. Everolimus plus trastuzumab has effective inhibitory effects on HER2+breast cancer stem cells in vitro and in vivo, which is a rational combination treatment that may be promising in human clinical trials.4. The combination treatment of everolimus and letrozole can inhibit the growth of MCF-7/Aro stem cells in vitro and in vivo.