Unlocking the Potential of 3D Spheroid Cultures in Breast Cancer Stem Cell Enrichment and Isolation

Objectives: The present research conducted a comparative analysis of the efficacy of three-dimensional (3D) culturing methods in enriching and isolating breast cancer stem cells (BCSCs). The study compared multicellular spheroids grown in Matrigel and in suspension with the commonly used two-dimensional (2D) monolayer culturing method. Methods: The experiment involved a 9-day 3D multicellular spheroid culture, followed by a 24-hour monolayer culture using two breast cancer cell lines, namely MCF7 and MDA-MB-231. To evaluate BCSCs, the study assessed the expression of various surface markers, including CD44/CD24, Vimentin, and ALDH1, along with pluripotent stem cell genes like SOX2, OCT4, KLF4, and Nanog. Additionally, the Doxorubicin resistance and the capacity of single cells derived from each method to form spheroids in serum-free suspension culture were measured. Results: The findings revealed that 3D-cultured multicellular spheroids grown in suspension exhibited a significant increase in stem cell markers and Doxorubicin resistance. Furthermore, these spheroids demonstrated a higher ability to form single-cell spheroids with a size of more than 50 mu m in a serum-free medium. Conclusion: Overall, this method of 3D culturing in suspension resulted in a substantial enrichment of BCSCs with enhanced self-renewal and proliferation capabilities when compared to both the 2D monolayer and 3D Matrigel methods. Consequently, this approach can serve as a crucial preliminary step in isolating BCSCs from cell lines using any available BCSCs isolation method.