背景:涎癌多形性腺瘤(CXPA)被定义为由良性多形性腺瘤(PA)发展而来的癌。已知异常激活的雄激素信号通路和HER-2/neu(ERBB-2)基因的扩增与CXPA肿瘤发生有关。肿瘤微环境研究的最新进展已证明,细胞外基质(ECM)重塑和刚度增加在肿瘤致癌中起着至关重要的作用。这项研究检查了ECM修饰以阐明CXPA肿瘤发生的潜在机制。
结果:成功建立了PA和CXPA类器官。组织学观察,免疫组织化学(IHC),和全外显子组测序表明,类器官概括了其亲本肿瘤的表型和分子特征。RNA测序和类器官的生物信息学分析表明,差异表达的基因在ECM相关术语中高度富集,这意味着ECM的变化可能与癌变有关。手术样品的显微镜检查显示,在CXPA肿瘤发生过程中,过多的透明组织沉积在肿瘤中。透射电子显微镜证实这些透明组织本质上是肿瘤ECM。随后,通过picrosiriusred染色检查,液相色谱与串联质谱,交联分析表明肿瘤ECM主要由I型胶原纤维组成,与致密的胶原蛋白排列和胶原蛋白交联水平增加。免疫组化显示COL1A1蛋白和胶原合成相关基因过表达,DCN和IGFBP5(p<0.05)。通过原子力显微镜和弹性成像分析证明了CXPA比PA更高的刚度。我们利用水凝胶在体外模拟具有不同刚度程度的ECM。与较软的基质(5Kpa)相比,CXPA细胞系和PA原代细胞在较硬的基质中表现出更多的增殖和侵袭表型(50Kpa,p<0.01)。RNA测序数据的蛋白质-蛋白质相互作用(PPI)分析显示AR和ERBB-2表达与TWIST1相关。此外,手术标本显示TWIST1在CXPA中的表达高于PA。敲除CXPA细胞中的TWIST1后,细胞增殖,迁移,侵袭性受到显著抑制(p<0.01)。
结论:开发CXPA类器官为癌症生物学研究和药物筛选提供了一个有用的模型。ECM重塑,归因于胶原蛋白的过度生产,胶原蛋白排列的交替,和增加交联,导致ECM刚度增加。ECM修饰是CXPA肿瘤发生的重要因素。
BACKGROUND: Salivary carcinoma ex pleomorphic adenoma (CXPA) is defined as a carcinoma that develops from benign pleomorphic adenoma (PA). Abnormally activated Androgen signaling pathway and amplification of HER-2/neu(ERBB-2) gene are known to be involved in CXPA tumorigenesis. Recent progress in tumour microenvironment research has led to identification that extracellular matrix (ECM) remodelling and increased stiffness act as critical contributing role in tumour carcinogenesis. This
study examined ECM modifications to elucidate the mechanism underlying CXPA tumorigenesis.
RESULTS: PA and CXPA organoids were successfully established. Histological observation, immunohistochemistry (IHC), and whole-exome sequencing demonstrated that organoids recapitulated phenotypic and molecular characteristics of their parental tumours. RNA-sequencing and bioinformatic analysis of organoids showed that differentially expressed genes are highly enriched in ECM-associated terms, implying that ECM alternations may be involved in carcinogenesis. Microscopical examination for surgical samples revealed that excessive hyalinized tissues were deposited in tumour during CXPA tumorigenesis. Transmission electron microscopy confirmed that these hyalinized tissues were tumour ECM in nature. Subsequently, examination by picrosirius red staining, liquid chromatography with tandem mass spectrometry, and cross-linking analysis indicated that tumour ECM was predominantly composed of type I collagen fibers, with dense collagen alignment and an increased level of collagen cross-linking. IHC revealed the overexpression of COL1A1 protein and collagen-synthesis-related genes, DCN and IGFBP5 (p < 0.05). Higher stiffness of CXPA than PA was demonstrated by atomic force microscopy and elastic imaging analysis. We utilized hydrogels to mimic ECM with varying stiffness degrees in vitro. Compared with softer matrices (5Kpa), CXPA cell line and PA primary cells exhibited more proliferative and invasive phenotypes in stiffer matrices (50Kpa, p < 0.01). Protein-protein interaction (PPI) analysis of RNA-sequencing data revealed that AR and ERBB-2 expression was associated with
TWIST1. Moreover, surgical specimens demonstrated a higher
TWIST1 expression in CXPA over PA. After knocking down
TWIST1 in CXPA cells, cell proliferation, migration, and invasiveness were significantly inhibited (p < 0.01).
CONCLUSIONS: Developing CXPA organoids provides a useful model for cancer biology research and drug screening. ECM remodelling, attributed to overproduction of collagen, alternation of collagen alignment, and increased cross-linking, leads to increased ECM stiffness. ECM modification is an important contributor in CXPA tumorigenesis.