Department of Gynecologic Oncology, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
Laboratory of Cell and Molecular Biology & State Key Laboratory of Molecular Oncology, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
Am J Reprod Immunol. 2020 Aug;84(2):e13251. doi: 10.1111/aji.13251. Epub 2020 May 31.
Since not too many human uterus cervical squamous cell carcinoma (CSCC) cell lines in existence, efficient isolation, culture, and purification protocols for primary CSCC cells were optimized as a tool for the study of uterus CSCC.
The protocols for partial multiple enzymatic digestion and explant cell culture were combined and then the resulting mixed cell component cultures were purified by magnetic-activated cell sorting. Colony-forming assay was utilized for detection of cell carcinogenesis potential, and immunofluorescence was used to detect protein expression of CSCC. Finally, flow cytometry (FCM) was performed to analyze cancer stem cells (CSCs) phenotypic markers as well as programmed cell death ligand 1(PD-L 1).
Freshly isolated cells containing tumor cells and cancer-associated fibroblasts (CAFs) efficiently proliferate to 85% confluence on a 6 cm petri dish in 5-7 days. Anti-epithelial cell adhesion molecule antibody (EpCAM) microbeads were used to successfully separate a homogeneous subpopulation of epithelial tumor cells. Both EpCAM+ and EpCAM- cell subpopulations were able to be passaged more than 30 times. Proportions of tumor cell populations expressed CSCs markers such as CD133, CD24, aldehyde dehydrogenase 1 (ALDH1), and CD44. The vimentin+ & EpCAM- population, defined with CAFs, could express CD146 mesenchymal stem cells marker. Meanwhile, PD-L 1 was identified in most subpopulation of CD44+ cells at low passage numbers.
Efficient isolation, culture, and purification protocols for primary CSCC cells were successfully built. Additionally, the profiling of CSCs cell markers might provide promising therapeutic targets and clinic strategies.
由于存在的人子宫颈鳞状细胞癌 (CSCC) 细胞系不多，因此优化了用于研究子宫 CSCC 的原发性 CSCC 细胞的有效分离、培养和纯化方案。
将部分多重酶消化和组织块细胞培养方案结合起来，然后通过磁激活细胞分选对所得混合细胞成分培养物进行纯化。集落形成测定法用于检测细胞致癌潜能，免疫荧光法用于检测 CSCC 的蛋白表达。最后，通过流式细胞术 (FCM) 分析癌症干细胞 (CSCs) 表型标志物以及程序性细胞死亡配体 1(PD-L 1)。
新鲜分离的细胞含有肿瘤细胞和癌症相关成纤维细胞 (CAFs)，在 6cm 培养皿中在 5-7 天内以 85%的汇合度有效增殖。使用抗上皮细胞黏附分子抗体 (EpCAM) 微珠成功分离出同质的上皮肿瘤细胞亚群。EpCAM+和 EpCAM-细胞亚群均能传代 30 多次。肿瘤细胞群体表达 CSCs 标志物，如 CD133、CD24、醛脱氢酶 1 (ALDH1) 和 CD44。定义为 CAFs 的 vimentin+&EpCAM-群体能够表达间充质干细胞标志物 CD146。同时，在低传代数时，大多数 CD44+细胞亚群中都鉴定出 PD-L 1。
成功建立了用于原发性 CSCC 细胞的有效分离、培养和纯化方案。此外，CSC 细胞标志物的分析可能为有前途的治疗靶点和临床策略提供依据。
