Mammosphere Formation Assay Optimization in the Characterization of Cancer Stem Cells of the Primary Breast Tumor

Authors : Gizem USLU, Zehra Seda ÜNAL HALBUTOĞULLARI, Gülçin GACAR, Nihat Zafer UTKAN, Yusufhan YAZIR

Pages : 108-118

Doi:10.31067/acusaglik.1225228

View : 24 | Download : 11

Publication Date : 2023-03-17

Article Type : Research Paper

Abstract :Background/Purpose: Breast cancer is the most frequently diagnosed cancer and the leading cause of death from cancer among females worldwide. Breast cancer tumors that feature breast cancer stem cells insert ignore into journalissuearticles values(BCSCs); are known to cause drug resistance and metastasis. Culturing BCSCs from primary tumors as mammospheres is both difficult and costly. Therefore, the ability to form BCSC mammospheres in-vitro has become essential in assessing their characterization. Flow cytometric analysis of surface markers and measurement of aldehyde dehydrogenase insert ignore into journalissuearticles values(ALDH); activity are among other methods used to evaluate cancer cells\` stem cell activity. Methods:The research material consisted of BCSCs isolated from the tumor tissues collected from two patients with invasive ductal carcinoma breast tumors. Subsequently, several stem cell surface markers, i.e., cluster of differentiation 44 insert ignore into journalissuearticles values(CD44);, CD24, and CD133, were analyzed using flow cytometry during the third passaging of the cells. ALDH assay is performed with negative control verapamil incubated cells. Two mammosphere forming methods, i.e., low attachment and agar-coated wells together with medium seeded in three different cell concentrations, were compared. Results: CD44+, CD24- and CD133+antibody expressions showed that these cells could be tumor-initiating CSCs. ALDH assay results also indicated that these cells possessed stem cell features. In addition, the results of the mammosphere assay revealed that agar-coated wells at a concentration of 7000 cells/cm2 had more prominent floating features and viable spheres. Conclusion: The findings of this study supported the hypothesis that agar-coated culture dishes in mammosphere culture would increase the mammosphere formation efficiency insert ignore into journalissuearticles values(MFE); value and revealed the importance of the number of cells in elucidating the nature of BCSCs.
Keywords : Breast cancer, mammosphere formation assay, cancer stem cell