Mishra Amrendra, Sriram Harshini, Chandarana Pinal, Tanavde Vivek, Kumar Rekha V, Gopinath Ashok, Govindarajan Raman, Ramaswamy S, Sadasivam Subhashini
1 Institute for Stem Cell Biology and Regenerative Medicine, National Centre for Biological Sciences, UAS-GKVK Campus, Bengaluru, India.
2 Hannover Biomedical Research School, Hannover Medical School, Hannover, Germany.
Tumour Biol. 2018 May;40(5):1010428318780859. doi: 10.1177/1010428318780859.
The goal of this study was to isolate cancer stem-like cells marked by high expression of CD44, a putative cancer stem cell marker, from primary oral squamous cell carcinomas and identify distinctive gene expression patterns in these cells. From 1 October 2013 to 4 September 2015, 76 stage III-IV primary oral squamous cell carcinoma of the gingivobuccal sulcus were resected. In all, 13 tumours were analysed by immunohistochemistry to visualise CD44-expressing cells. Expression of CD44 within The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma RNA-sequencing data was also assessed. Seventy resected tumours were dissociated into single cells and stained with antibodies to CD44 as well as CD45 and CD31 (together referred as Lineage/Lin). From 45 of these, CD44Lin and CD44Lin subpopulations were successfully isolated using fluorescence-activated cell sorting, and good-quality RNA was obtained from 14 such sorted pairs. Libraries from five pairs were sequenced and the results analysed using bioinformatics tools. Reverse transcription quantitative polymerase chain reaction was performed to experimentally validate the differential expression of selected candidate genes identified from the transcriptome sequencing in the same 5 and an additional 9 tumours. CD44 was expressed on the surface of poorly differentiated tumour cells, and within the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma samples, its messenger RNA levels were higher in tumours compared to normal. Transcriptomics revealed that 102 genes were upregulated and 85 genes were downregulated in CD44Lin compared to CD44Lin cells in at least 3 of the 5 tumours sequenced. The upregulated genes included those involved in immune regulation, while the downregulated genes were enriched for genes involved in cell adhesion. Decreased expression of PCDH18, MGP, SPARCL1 and KRTDAP was confirmed by reverse transcription quantitative polymerase chain reaction. Lower expression of the cell-cell adhesion molecule PCDH18 correlated with poorer overall survival in the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma data highlighting it as a potential negative prognostic factor in this cancer.
本研究的目的是从原发性口腔鳞状细胞癌中分离出以假定的癌症干细胞标志物CD44高表达为特征的癌症干细胞样细胞，并确定这些细胞中独特的基因表达模式。2013年10月1日至2015年9月4日，对76例III-IV期牙龈颊沟原发性口腔鳞状细胞癌进行了切除。总共对13个肿瘤进行了免疫组织化学分析，以观察表达CD44的细胞。还评估了癌症基因组图谱-头颈部鳞状细胞癌RNA测序数据中CD44的表达。将70个切除的肿瘤解离成单细胞，并用抗CD44以及CD45和CD31的抗体（统称为谱系/Lin）进行染色。从其中45个样本中，使用荧光激活细胞分选成功分离出CD44Lin和CD44Lin亚群，并从14对这样分选的样本中获得了高质量的RNA。对5对样本的文库进行了测序，并使用生物信息学工具分析了结果。进行逆转录定量聚合酶链反应，以实验验证从转录组测序中鉴定出的选定候选基因在相同的5个肿瘤以及另外9个肿瘤中的差异表达。CD44在低分化肿瘤细胞表面表达，在癌症基因组图谱-头颈部鳞状细胞癌样本中，其信使RNA水平在肿瘤中高于正常组织。转录组学显示，与CD44Lin细胞相比，在至少5个测序肿瘤中的3个中，CD44Lin中有102个基因上调，85个基因下调。上调的基因包括参与免疫调节的基因，而下调的基因则富集了参与细胞黏附的基因。通过逆转录定量聚合酶链反应证实了PCDH18、MGP、SPARCL1和KRTDAP的表达降低。细胞间黏附分子PCDH18的低表达与癌症基因组图谱-头颈部鳞状细胞癌数据中较差的总生存率相关，突出了其作为该癌症潜在负性预后因素的地位。
