Maletzki Claudia, Gock Michael, Randow Martin, Klar Ernst, Huehns Maja, Prall Friedrich, Linnebacher Michael
Claudia Maletzki, Martin Randow, Michael Linnebacher, Section of Molecular Oncology and Immunotherapy, University of Rostock, D-18057 Rostock, Germany.
World J Gastroenterol. 2015 Jan 7;21(1):164-76. doi: 10.3748/wjg.v21.i1.164.
To generate novel tumor models for preclinical validation of biomarkers that allow drug response prediction and individual therapeutic decisions.
Cell line establishment was conducted by both direct in vitro culturing and in vivo xenografting followed by in vitro culturing procedure. A comprehensive characterization was subsequently performed. This included quality control, consisting of the confirmation of human and colorectal cancer (CRC) origin by DNA fingerprint and epithelial cell adhesion molecule (EpCAM) staining, as well as mycoplasma and human virus testing. Phenotypic analysis was done by light microscopy and multicolor flow cytometry. Histopathological examination (β-catenin and cytokeratin staining) was conducted in direct comparison to parental tumor tissues. Extensive molecular-pathological profiling included mutation analysis for CRC-associated driver mutations, assessment of chromosomal and microsatellite instability, and the grade of CpG island methylation. Additionally, an array-based comparative genomic hybridization analysis was performed. Drug responsiveness was assessed for a panel of classical and novel substances in clinical use for the treatment of solid cancers. Finally, tumorigenicity of the cell lines was tested by xenografting into immunocompromised nude mice.
Herein we describe the establishment of three ultra-low passage cell lines from two individual patients suffering from sporadic CRC. One cell line was derived directly from an early stage case (HROC18), whereas two cell lines could be established both direct from patient material and after xenografting from a late stage tumor (HROC32). All cell lines were free of contaminating mycoplasma and viruses. Molecular-pathological analysis allowed all cell lines to be classified as chromosomal instable (CIN(+)). They were aneuploid, with CpG island promoter methylation and microsatellite instability being absent. The following mutational profile was observed both in the cell lines and the parental tumor tissue: HROC18: APC(mut), p53(mut), K-ras(wt); HROC32: APC(wt), p53(mut), K-ras(mut). All cell lines were characterized as epithelial (EpCAM(+)) cells, showing distinct morphology and migration speed, but comparable growth kinetics. The cell lines showed different patterns of response towards clinically approved and novel drugs, with HROC18 being more resistant than HROC32 cells. Finally, in vivo tumorigenicity was demonstrated.
We successfully established and characterized novel ultra-low passage patient-derived CRC models as useful instruments for analyzing biological characteristics associated with the CIN(+) phenotype.
生成新的肿瘤模型，用于生物标志物的临床前验证，以实现药物反应预测和个体化治疗决策。
通过直接体外培养和体内异种移植后再进行体外培养的程序来建立细胞系。随后进行全面的特征分析。这包括质量控制，即通过DNA指纹图谱和上皮细胞粘附分子（EpCAM）染色来确认人源和结直肠癌（CRC）起源，以及支原体和人类病毒检测。通过光学显微镜和多色流式细胞术进行表型分析。与亲本肿瘤组织直接对比进行组织病理学检查（β-连环蛋白和细胞角蛋白染色）。广泛的分子病理学分析包括对CRC相关驱动基因突变的分析、染色体和微卫星不稳定性的评估以及CpG岛甲基化程度。此外，还进行了基于阵列的比较基因组杂交分析。评估了一组用于治疗实体癌的临床常用经典和新型物质的药物反应性。最后，通过将细胞系异种移植到免疫缺陷裸鼠中来测试其致瘤性。
在此我们描述了从两名散发性CRC患者中建立的三个超低传代细胞系。一个细胞系直接来源于早期病例（HROC18），而两个细胞系既可以直接从患者材料中建立，也可以从晚期肿瘤经异种移植后建立（HROC32）。所有细胞系均无支原体和病毒污染。分子病理学分析使所有细胞系被归类为染色体不稳定（CIN(+)）。它们为非整倍体，不存在CpG岛启动子甲基化和微卫星不稳定性。在细胞系和亲本肿瘤组织中均观察到以下突变谱：HROC18：APC（突变），p53（突变），K-ras（野生型）；HROC32：APC（野生型），p53（突变），K-ras（突变）。所有细胞系均被表征为上皮细胞（EpCAM(+)），具有独特的形态和迁移速度，但生长动力学相当。细胞系对临床批准的药物和新型药物表现出不同的反应模式，HROC18比HROC32细胞更具抗性。最后，证明了其体内致瘤性。
我们成功建立并表征了新型超低传代患者来源的CRC模型，作为分析与CIN(+)表型相关生物学特征的有用工具。
