Department of Research, Panimalar Medical College Hospital & Research Institute, Varadharajapuram, Poonamallee, Chennai, Tamil Nadu, 600 123, India.
Department of Biochemistry, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences (SIMATS), Saveetha University, Velappanchavadi, Chennai, Tamil Nadu, 600 077, India.
J Egypt Natl Canc Inst. 2022 Dec 12;34(1):51. doi: 10.1186/s43046-022-00152-1.
Mammosphere formation assay has become a versatile tool to quantify the activity of putative breast cancer stem cells in non-adherent in vitro cultures. However, optimizing the suspension culture system is crucial to establish mammosphere cultures from primary breast tumors.
This study aimed at determining the self-renewal and sphere-forming potential of breast cancer stem-like cells derived from human primary invasive ductal carcinoma and normal breast tissue samples, and MCF-7 breast cancer cell line using an optimal suspension culture system. Mammosphere-forming efficiency of the mammospheres generated from the tissue samples and cell line were compared. We evaluated the expression of CD44/CD24/ and CD49f/EpCAM/ phenotypes in the stem-like cells by flow cytometry. CK-18, CK-19, α-SMA, and EpCAM marker expression was assessed using immunohistochemical staining.
Breast epithelial cells isolated from the three samples formed two-dimensional spheroids in suspension cultures. Interestingly, mammospheres formed from patient-derived primary breast tumors were enriched in breast cancer stem-like cells with the phenotype CD44/CD24/ and exhibited a relatively more number of large spheres when compared to the normal breast stem cells. MCF-7-derived SCs were more aggressive and resulted in the formation of a significantly higher number of spheroids. The expression of CK-18/CK-19 and α-SMA/EpCAM proteins was confirmed in breast cancer tissues.
Thus, the use of primary tumor specimens and breast cancer cell lines as suitable models for elucidating the breast cancer stem cell activity was validated using mammosphere culture system.
乳腺球体形成分析已成为一种通用的工具，可用于量化非贴壁体外培养中假定的乳腺癌干细胞的活性。然而，优化悬浮培养系统对于从原发性乳腺癌建立乳腺球体培养至关重要。
本研究旨在使用优化的悬浮培养系统确定源自人原发性浸润性导管癌和正常乳腺组织样本以及 MCF-7 乳腺癌细胞系的乳腺癌类干细胞的自我更新和球体形成潜力。比较了从组织样本和细胞系生成的乳腺球体的乳腺球体形成效率。我们通过流式细胞术评估了类干细胞中 CD44/CD24/和 CD49f/EpCAM/表型的表达。通过免疫组织化学染色评估 CK-18、CK-19、α-SMA 和 EpCAM 标志物的表达。
从三个样本中分离的乳腺上皮细胞在悬浮培养中形成二维球体。有趣的是，源自患者源性原发性乳腺癌的乳腺球体富集了具有 CD44/CD24/表型的乳腺癌类干细胞，并且与正常乳腺干细胞相比，形成了相对较多的大球体。MCF-7 衍生的 SCs 更具侵袭性，导致形成的球体数量显著增加。在乳腺癌组织中证实了 CK-18/CK-19 和 α-SMA/EpCAM 蛋白的表达。
因此，使用原发性肿瘤标本和乳腺癌细胞系作为阐明乳腺癌干细胞活性的合适模型，通过乳腺球体培养系统得到了验证。
