Isolation and Characterization of Murine Breast Cancer Stem Cells

Carnella M. Lee; Anandi Sawant, Ph.D.; Ha-Ram Cha; and Selvarangan Ponnazhagan, Ph.D. University of Alabama at Birmingham, Birmingham, AL

CarnellaLeeABRCMS

Breast Cancer (BCa) is the second leading cause of cancer deaths in the United States accounting for about 40,000 deaths each year. One of the common complications in BCa patients is the bone metastasis, which is associated with increased morbidity and mortality. Although surgery, chemotherapy, and radiation therapies provide good prognosis for early stage disease, the metastatic disease possess a bigger challenge for treatment. Recent studies have identified the presence of Cancer Stem Cells (CSC) and attributed the mechanism of treatment resistance to these cells. Breast cancer stem cells are typically characterized by a CD24 (low) and CD44 (high) phenotype with mesenchymal properties, which differ from the epithelial CD23+ and CD44+ phenotype expressed in the majority of BCa cells. Thus, the identification of CSC and their role in BCA relapse provides new clues towards developing treatment strategies for long-term remission. In order to develop such strategies it becomes important first to characterize the CSC and understand their plasticity in detail. Towards this goal, the present study utilized a murine BCa cell line 4T1, syngenic to the immunocompetent BALB/c mice. Isolation of stem cell population from 4T1 cell pool was performed by flow cytometry using CSC markers CD24 (low) and CD44 (high). However, upon in vitro culture, the stem cell population reverted back to non-stem cells with CD24+ and CD44+. In order to better understand the influence of chemotherapy on the stemness of these cells, clonal populations of 4T1 cells are were treated using docetaxel. Interestingly, the cells retained stem cell characteristics in the presence of docetaxel. 4T1 cells were grown in MEGM stem cell media and the expression of CSC surface markers was assessed every 6 hrs. Results of this analysis show that the expression of stem cell markers peaked every 24 hrs and remained highest for additional 24 hrs. This data supports a possible reprogramming of BCa stem cells based on cell cycle regulation. Further confirmation of stem cell characteristics of this population are being performed with addition stem CSC markers including: TWIST, ALDH1, Snail, E-Cadherin and Sp 1, 3, and 4. Ongoing identification of this phenomenon would allow us to test novel therapies targeting BCa stem cells in a preclinical mouse model for possible clinical translation.