Han Dan, Rodriguez-Bravo Veronica, Izadmehr Sudeh, Domingo-Domenech Josep, Cordon-Cardo Carlos
Department of Pathology, Icahn School of Medicine at Mount Sinai.
Department of Pathology, Icahn School of Medicine at Mount Sinai;
J Vis Exp. 2019 Jun 13(148). doi: 10.3791/57011.
The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.
在过去十年中，越来越多的证据支持肿瘤起始细胞（TICs）的存在及其重要性。这些TICs已被证明与肿瘤的起始、转移和耐药性有关。因此，除了目前主要针对大多数非TICs的化疗策略外，开发针对TICs的特异性治疗方法也很重要。为了进一步了解TICs恶性肿瘤背后的机制，我们描述了一种在人类肉瘤中分离和鉴定TICs的方法。在此，我们展示了一个详细的方案，用于生成人类肉瘤的患者来源异种移植（PDXs），并使用人类白细胞抗原I类（HLA-1）作为阴性标记，通过荧光激活细胞分选（FACS）分离TICs。此外，我们还描述了如何对这些TICs进行功能鉴定，包括球体形成试验和肿瘤形成试验，以及诱导其沿间充质途径分化。PDX TICs的分离和鉴定为发现潜在的靶向治疗试剂提供了线索。此外，越来越多的证据表明，该方案可能会进一步扩展，以从其他类型的人类癌症中分离和鉴定TICs。
