Background: Several markers have been shown to define subpopulations enriched for cancer stem cells (CSCs) and correlate with poor clinical outcomes in oral squamous cell carcinoma (OSCC). Objective: To investigate the pattern of expression and correlation with clinical parameters of two CSC markers, namely p75NTR and ALDH1A1, in both patient samples and cell lines. Methods: Archival formalin-fixed paraffin-embedded samples from normal human oral mucosa (NHOM, n = 31), oral dysplasia (OD, n = 10) and OSCC (n = 177) were subjected to multiple immunohistochemistry and some to qRT-PCR for expression of CSC and proliferation-related markers, BMI1 and Ki67. Correlations between CSC marker expression and clinical parameters were investigated. Primary cells and cell lines derived from NHOM, OD or OSCC were FACS-analyzed for the same markers. Results: A higher frequency of cells positive for CSC markers was detected in OD and OSCC compared to NHOM. Co-localization of the two markers was a rare finding in OSCC as compared to NHOM or OD and was more heterogeneous in OSCC cell lines than in OD and NHOM cells. Cells positive for p75NTR exhibited higher expression of proliferative and self-renewal markers in comparison to ALDH1A1+ or double ALDH1A1+/p75NTR+ cells. Cells positive for p75NTR were more frequent in small-size tumors and poorly to moderately differentiated tumors and correlated with poor survival of patients otherwise (clinically) deemed as of better prognosis. Higher frequency of ALDH1A1+ cells was found to be associated with lymph node metastasis. Both p75NTR+ cells and ALDH1A1+ cells could emerge de novo from the respective negative subpopulation after FACS and in vitro growth, but with different kinetics. Conclusions: Here, we show that several stem cell subpopulations with distinct phenotypes co-exist in a tumor, each having impact on different clinical parameters. The cell subpopulations identified by the use of different CSC markers were found to be dynamic populations, able to switch between phenotypes. In addition, our data suggest that the stem cell heterogeneity is acquired and evolves parallel with carcinoma progression.
背景：多项标志物已被证实可用于富集癌症干细胞亚群，并与口腔鳞状细胞癌的不良临床预后相关。目的：本研究旨在探讨两种癌症干细胞标志物（p75NTR与ALDH1A1）在患者样本及细胞系中的表达模式及其与临床参数的相关性。方法：对存档的正常人口腔黏膜组织、口腔上皮异常增生组织及口腔鳞癌组织的石蜡包埋样本进行多重免疫组化检测，部分样本通过定量逆转录聚合酶链反应检测癌症干细胞标志物及增殖相关标志物BMI1与Ki67的表达水平，并分析癌症干细胞标志物表达与临床参数的相关性。同时通过流式细胞术对源自上述组织的原代细胞及细胞系进行相同标志物的检测。结果：与正常口腔黏膜组织相比，口腔上皮异常增生及口腔鳞癌组织中癌症干细胞标志物阳性细胞出现频率更高。在口腔鳞癌组织中，两种标志物的共定位现象较正常组织及异常增生组织更为罕见，且口腔鳞癌细胞系中的表达异质性高于异常增生及正常黏膜细胞。相较于ALDH1A1阳性或ALDH1A1/p75NTR双阳性细胞，p75NTR阳性细胞表现出更高的增殖及自我更新标志物表达水平。p75NTR阳性细胞在小体积肿瘤及中低分化肿瘤中出现频率更高，并与临床预后较好患者的较差生存率相关。ALDH1A1阳性细胞的高频出现与淋巴结转移相关。流式分选后的体外培养实验表明，p75NTR阳性与ALDH1A1阳性细胞均可从相应阴性亚群中重新产生，但呈现不同的动力学特征。结论：本研究证实肿瘤中存在多种表型各异的干细胞亚群，不同亚群对临床参数产生差异化影响。通过不同癌症干细胞标志物鉴定的细胞亚群具有动态转化特性，能够在不同表型间转换。此外，研究数据提示干细胞异质性是在癌变进程中逐步获得并随疾病进展同步演化的。
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