背景:组织细胞病是罕见的疾病,表现为与巨噬细胞或树突状细胞具有组织学特征并在各种器官中积累的致病性骨髓细胞的增殖增加,i.a.,骨头和皮肤。可用于确定疾病分子途径的临床前体外模型是有限的,因此,对组织细胞病的研究具有挑战性。本研究比较了祖细胞的细胞生理学特征,来自三个具有不同组织细胞类型和结局的儿科患者的组织细胞病变(sl-pHCs)的基质样细胞。表征的细胞可以在药物测试中找到潜在的应用。
方法:细胞的分子表型,即CD1a和CD207(langerin)的表达,使用流式细胞术确定。细胞遗传学分析包括GTG条带中期和微阵列(aCGH)评估。此外,使用共聚焦和扫描电子显微镜评估细胞的形态和超微结构。来自共聚焦成像的显微照片用于重建线粒体网络及其形态。基本细胞学参数,比如生存能力,线粒体活性,和扩散,使用多种细胞测定法进行分析,包括膜联蛋白V/7-AAD染色,线粒体电位分析,BrdU试验,克隆性分析,以及细胞周期内细胞的分布。可能与组织细胞进展相关的生物标志物使用RT-qPCR在mRNA,miRNA和lncRNA水平。用蛋白质印迹检测组织细胞增生特异性蛋白的细胞内积累。用MTS测定法测定维罗非尼和曲美替尼的细胞毒性和IC50。
结果:获得了细胞模型,即RAB-1、HAN-1和CHR-1在分子表型和形态方面是异源性的。细胞表达CD1a/CD207标记的树突状细胞的特征,而且还显示了间充质起源细胞特征性标记的细胞内积累,即波形蛋白(VIM)和骨桥蛋白(OPN)。在后来的文化中,细胞保持活力和代谢活跃,线粒体网络很发达,在每个细胞系中都有一些独特的形态类型。注意到细胞特异性转录组谱,提供有关具有诊断和预后特征的潜在新生物标志物(非编码RNA)的信息。细胞对vemurafenib和trametinib表现出不同的敏感性。
结论:从组织细胞病变获得并表征的基质样细胞模型可用于组织细胞增生症生物学和药物测试的研究。
BACKGROUND: Histiocytoses are rare disorders manifested by increased proliferation of pathogenic myeloid cells sharing histological features with macrophages or dendritic cells and accumulating in various organs, i.a., bone and skin. Pre-clinical in vitro models that could be used to determine molecular pathways of the disease are limited, hence research on histiocytoses is challenging. The current study compares cytophysiological features of progenitor, stromal-like cells derived from histiocytic lesions (sl-pHCs) of three pediatric patients with different histiocytoses types and outcomes. The characterized cells may find potential applications in drug testing.
METHODS: Molecular phenotype of the cells, i.e. expression of CD1a and CD207 (langerin), was determined using flow cytometry. Cytogenetic analysis included GTG-banded metaphases and microarray (aCGH) evaluation. Furthermore, the morphology and ultrastructure of cells were evaluated using a confocal and scanning electron microscope. The microphotographs from the confocal imaging were used to reconstruct the mitochondrial network and its morphology. Basic cytophysiological parameters, such as viability, mitochondrial activity, and proliferation, were analyzed using multiple cellular assays, including Annexin V/7-AAD staining, mitopotential analysis, BrdU test, clonogenicity analysis, and distribution of cells within the cell cycle. Biomarkers potentially associated with histiocytoses progression were determined using RT-qPCR at mRNA, miRNA and lncRNA levels. Intracellular accumulation of histiocytosis-specific proteins was detected with Western blot. Cytotoxicyty and IC50 of vemurafenib and trametinib were determined with MTS assay.
RESULTS: Obtained cellular models, i.e. RAB-1, HAN-1, and CHR-1, are heterogenic in terms of molecular phenotype and morphology. The cells express CD1a/CD207 markers characteristic for dendritic cells, but also show intracellular accumulation of markers characteristic for cells of mesenchymal origin, i.e. vimentin (VIM) and osteopontin (OPN). In subsequent cultures, cells remain viable and metabolically active, and the mitochondrial network is well developed, with some distinctive morphotypes noted in each cell line. Cell-specific transcriptome profile was noted, providing information on potential new biomarkers (non-coding RNAs) with diagnostic and prognostic features. The cells showed different sensitivity to vemurafenib and trametinib.
CONCLUSIONS: Obtained and characterized cellular models of stromal-like cells derived from histiocytic lesions can be used for studies on histiocytosis biology and drug testing.