Cell Populations Expressing Stemness-Associated Markers in Lung Adenocarcinoma

<b>Abstract:</b><br>The stemness-associated markers OCT4, NANOG, SOX2, KLF4 and c-MYC are expressed in numerous cancer types suggesting the presence of cancer stem cells (CSCs). Immunohistochemical (IHC) staining performed on 12 lung adenocarcinoma (LA) tissue samples showed protein expression of OCT4, NANOG, SOX2, KLF4 and c-MYC, and the CSC marker CD44. In situ hybridization (ISH) performed on six of the LA tissue samples showed mRNA expression of OCT4, NANOG, SOX2, KLF4 and c-MYC. Immunofluorescence staining performed on three of the tissue samples showed co-expression of OCT4 and c-MYC with NANOG, SOX2 and KLF4 by tumor gland cells, and expression of OCT4 and c-MYC exclusively by cells within the stroma. RT-qPCR performed on five LA-derived primary cell lines showed mRNA expression of all the markers except SOX2. Western blotting performed on four LA-derived primary cell lines demonstrated protein expression of all the markers except SOX2 and NANOG. Initial tumorsphere assays performed on four LA-derived primary cell lines demonstrated 0–80% of tumorspheres surpassing the 50 µm threshold. The expression of the stemness-associated markers OCT4, SOX2, NANOG, KFL4 and c-MYC by LA at the mRNA and protein level, and the unique expression patterns suggest a putative presence of CSC subpopulations within LA, which may be a novel therapeutic target for this cancer. Further functional studies are required to investigate the possession of stemness traits.

**摘要：**
干性相关标志物OCT4、NANOG、SOX2、KLF4及c-MYC在多种癌症类型中均有表达，提示癌症干细胞（cancer stem cells, CSCs）的存在。本研究对12份肺腺癌（lung adenocarcinoma, LA）组织样本开展免疫组织化学（immunohistochemical, IHC）染色，检测到OCT4、NANOG、SOX2、KLF4、c-MYC及癌症干细胞标志物CD44的蛋白表达。对其中6份肺腺癌组织样本实施原位杂交（in situ hybridization, ISH），结果显示OCT4、NANOG、SOX2、KLF4及c-MYC的mRNA表达情况。对3份组织样本进行免疫荧光染色，结果显示肿瘤腺细胞可共表达OCT4、c-MYC与NANOG、SOX2及KLF4，而基质内细胞仅表达OCT4与c-MYC。对5株肺腺癌来源的原代细胞系开展实时定量逆转录聚合酶链反应（RT-qPCR）检测，结果显示除SOX2外其余标志物的mRNA均有表达。对4株肺腺癌来源的原代细胞系进行蛋白质免疫印迹（Western blotting）分析，证实除SOX2与NANOG外其余标志物均存在蛋白表达。对4株肺腺癌来源的原代细胞系开展初步肿瘤球形成实验，结果显示0~80%的肿瘤球直径超过50 μm阈值。肺腺癌在mRNA与蛋白水平均表达干性相关标志物OCT4、SOX2、NANOG、KLF4及c-MYC，其独特的表达模式提示肺腺癌内部存在癌症干细胞亚群，该亚群或可成为该癌症的新型治疗靶点。后续需开展更多功能研究以验证其干性特征。