Department of Pediatrics, Hematology/Oncology Division, University of Nebraska Medical Center, Omaha, NE, USA.
Fred and Pamela Buffett Cancer Center, Omaha, NE, USA.
Methods Mol Biol. 2023;2701:253-259. doi: 10.1007/978-1-0716-3373-1_17.
Cancer is a heterogeneous disease, comprising of a mixture of different cell populations. Cancer stem cells (CSCs), also known as tumor-initiating cells (TICs), are a subpopulation of multipotent cells within the cancer that has self-renewing capability, tumor-initiating ability, multi-differentiation potential, and an inherent capacity for drug and chemoresistance. Sphere-formation assay is commonly used for enrichment and analysis of CSC properties in vitro and is typically used as a metric for testing the viability of tumor cells to anticancer agents. This model is based on the ability of CSCs to grow under ultralow-attachment conditions in serum-free medium supplemented with growth factors. In contrast to the adherent 2D culture of cancer cells, the 3D culture of tumorsphere assay exploits inherent biologic features of CSCs such as anoikis resistance and self-renewal. We describe here the detailed methodology for the generation and propagation of spheres generated from pediatric brain tumor medulloblastoma (MB) cells. As signal transducer and activator of transcription (STAT3) is known to play an important role in maintaining cancer stem cell properties, we accessed the effect of depleting or inhibiting STAT3 on MB-sphere sizes, numbers, and integrity. This may serve as a promising platform for screening potential anti-CSC agents and small-molecule inhibitors.
癌症是一种异质性疾病，由不同细胞群体的混合物组成。癌症干细胞（CSC），也称为肿瘤起始细胞（TIC），是癌症中具有自我更新能力、肿瘤起始能力、多向分化潜能和固有药物及化疗耐药性的多能细胞亚群。球形成实验常用于体外富集和分析 CSC 特性，通常用作测试肿瘤细胞对抗癌药物活力的指标。该模型基于 CSCs 在无血清培养基中添加生长因子在超低附着条件下生长的能力。与癌细胞的贴壁 2D 培养相比，肿瘤球体实验的 3D 培养利用了 CSCs 的固有生物学特征，如抗凋亡和自我更新。我们在这里描述了从小儿脑肿瘤髓母细胞瘤（MB）细胞生成和增殖球体的详细方法。由于信号转导和转录激活因子 3（STAT3）被认为在维持癌症干细胞特性方面发挥重要作用，我们研究了耗尽或抑制 STAT3 对 MB 球体大小、数量和完整性的影响。这可能成为筛选潜在的抗 CSC 药物和小分子抑制剂的有前途的平台。
