背景:基底细胞(BCs)是支气管祖细胞/干细胞,可以再生受损的气道,吸烟者,可能会发生恶性转化。作为肺癌早期阶段的模型,我们着手描述从未吸烟者和从未吸烟者没有癌症的细胞学正常BC生长(对照),以及来自患有解剖学上遥远癌症的吸烟者的正常上皮“领域”,包括肺腺癌(LUAD)和鳞状细胞癌(LUSC)(例)。
方法:从远离临床或可见病变/肿瘤部位的支气管内冲洗中培养和扩增原代BCs。供体亚组进行了生长测试,形态学,通过qRT-PCR和潜在的分子特征,RNAseq,流式细胞术,免疫荧光,和免疫印迹。
结果:(a)BC群体包括上皮细胞粘附分子(EpCAM)阳性和阴性细胞亚群;(b)吸烟降低了总体BC增殖,相应地,EpCAMpos/ITGA6pos/CD24pos干细胞分数减少了2.6倍;(c)LUSC供体细胞显示出高达2.8倍的异常BCs增加;(d)AD细胞从LUS期细胞增殖增加。这些差异对应于:(i)不同的NOTCH1/NOTCH2转录物表达和潜在下游(ii)E-cadherin(CDH1)的表达改变,肿瘤蛋白-63(TP63),分泌球蛋白家族1a成员1(SCGB1A1),和毛状/分裂增强子与YRPW基序1(HEY1)相关;(iii)在LUAD供体BCs中减少EPCAM并增加NK2同源盒1(NKX2-1)mRNA的表达。
结论:这些和其他发现证明了供体年龄的影响,吸烟,和肺癌病例对照状态对BC表型和分子特征的影响,可能提示早期人类肺癌发病过程中Notch信号通路的失调。
BACKGROUND: Basal cells (BCs) are bronchial progenitor/stem cells that can regenerate injured airway that, in smokers, may undergo malignant transformation. As a model for early stages of lung carcinogenesis, we set out to characterize cytologically normal BC outgrowths from never-smokers and ever-smokers without cancers (controls), as well as from the normal epithelial \"field\" of ever-smokers with anatomically remote cancers, including lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) (cases).
METHODS: Primary BCs were cultured and expanded from endobronchial brushings taken remote from the site of clinical or visible lesions/tumors. Donor subgroups were tested for growth, morphology, and underlying molecular features by qRT-PCR, RNAseq, flow cytometry, immunofluorescence, and immunoblot.
RESULTS: (a) the BC population includes epithelial cell adhesion molecule (EpCAM) positive and negative cell subsets; (b) smoking reduced overall BC proliferation corresponding with a 2.6-fold reduction in the EpCAMpos/
ITGA6 pos/CD24pos stem cell fraction; (c) LUSC donor cells demonstrated up to 2.8-fold increase in dysmorphic BCs; and (d) cells procured from LUAD patients displayed increased proliferation and S-phase cell cycle fractions. These differences corresponded with: (i) disparate NOTCH1/NOTCH2 transcript expression and altered expression of potential downstream (ii) E-cadherin (CDH1), tumor protein-63 (TP63), secretoglobin family 1a member 1 (SCGB1A1), and Hairy/enhancer-of-split related with YRPW motif 1 (HEY1); and (iii) reduced EPCAM and increased NK2 homeobox-1 (NKX2-1) mRNA expression in LUAD donor BCs.
CONCLUSIONS: These and other findings demonstrate impacts of donor age, smoking, and lung cancer case-control status on BC phenotypic and molecular traits and may suggest Notch signaling pathway deregulation during early human lung cancer pathogenesis.