背景:磁球形成测定已成为定量非贴壁体外培养物中推定的乳腺癌干细胞活性的通用工具。然而,优化悬浮培养体系对建立乳腺原发肿瘤的乳腺球培养至关重要。
方法:本研究旨在确定来自人原发性浸润性导管癌和正常乳腺组织样本的乳腺癌干细胞样细胞的自我更新和球体形成潜力,和MCF-7乳腺癌细胞系使用最佳悬浮培养系统。比较了从组织样品和细胞系产生的乳腺球的形成效率。我们通过流式细胞术评估了CD44/CD24-/low和CD49f/EpCAM-/low表型在干细胞样细胞中的表达。CK-18,CK-19,α-SMA,使用免疫组织化学染色评估EpCAM标记物的表达。
结果:从三个样品中分离的乳腺上皮细胞在悬浮培养物中形成二维球体。有趣的是,由患者来源的原发性乳腺肿瘤形成的乳腺球富集了表型为CD44+/CD24-/low的乳腺癌干细胞样细胞,与正常乳腺干细胞相比,其球体数量相对较多.MCF-7来源的SCs更具侵袭性,并导致形成明显更高数量的球状体。证实了CK-18/CK-19和α-SMA/EpCAM蛋白在乳腺癌组织中的表达。
结论:因此,使用乳腺球培养系统验证了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的用途.
BACKGROUND: 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.
METHODS: 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-/low and CD49f+/EpCAM-/low phenotypes in the stem-like cells by flow cytometry. CK-18, CK-19, α-SMA, and EpCAM marker expression was assessed using immunohistochemical staining.
RESULTS: 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-/low 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.
CONCLUSIONS: 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.