Kolostova Katarina, Spicka Jan, Matkowski Rafal, Bobek Vladimir
Department of Laboratory Genetics, Institute of Laboratory Diagnostics, University Hospital Kralovske Vinohrady Srobarova 50, Prague, Czech Republic.
Department of Biochemistry, Institute of Laboratory Diagnostics, University Hospital Kralovske Vinohrady Srobarova 50, Prague, Czech Republic.
Am J Transl Res. 2015 Jul 15;7(7):1203-13. eCollection 2015.
The focus of the study was to implement a new workflow for circulating tumor cells (CTCs) characterization that would allow the analysis of CTCs on a cytomorphological and molecular level in patients with diagnosed gynecological cancer. Our findings may be useful in future cancer patient management. The study introduces a size-based enrichment (MetaCell(®)) method for the separation of viable CTCs, followed by CTCs culturing in vitro and gene expression characterization. It is based on the observation of CTCs and DTCs (Disseminated Tumor Cells) in several case studies of ovarian, endometrial and cervical cancer by means of cytomorphology and gene expression profiling. The viability of the enriched CTCs was estimated using vital and lethal fluorescence nuclear staining. This type of staining may be predictive for the success rate of subsequent CTC growth in vitro. To identify CTCs in the enriched CTC fraction, cytomorphological evaluations based on vital fluorescence staining were followed by gene expression analysis of tumor-associated (TA) genes. Cytokeratin expression (KRT7, KRT19) was analyzed in combination with MUC1, MUC16, CD24, CD44 and ALDH1. Gene expression analysis has shown that short-term in vitro culture enhanced the differentiation process of the captured CTCs growing on a membrane. On the other hand, redundant white blood cells captured on the membrane were eliminated during a short-term culture. The most frequently elevated genes in ovarian cancer (serous type) are EPCAM, KRT19 and MUC1. It has been demonstrated that CTC presence revealed by cytomorphological evaluation may be usefully complemented by TA-gene expression analysis, to increase the sensitivity of the analysis.
本研究的重点是实施一种用于循环肿瘤细胞（CTC）表征的新工作流程，该流程能够在细胞形态学和分子水平上对已确诊的妇科癌症患者的CTC进行分析。我们的研究结果可能对未来癌症患者的管理有用。该研究引入了一种基于大小的富集（MetaCell®）方法来分离活的CTC，随后进行CTC的体外培养和基因表达表征。它基于通过细胞形态学和基因表达谱分析在卵巢癌、子宫内膜癌和宫颈癌的多个病例研究中对CTC和播散肿瘤细胞（DTC）的观察。使用活细胞和死细胞荧光核染色评估富集的CTC的活力。这种染色类型可能对后续CTC体外生长的成功率具有预测性。为了在富集的CTC组分中鉴定CTC，在基于活细胞荧光染色的细胞形态学评估之后，对肿瘤相关（TA）基因进行基因表达分析。联合分析细胞角蛋白表达（KRT7、KRT19）与MUC1、MUC16、CD24、CD44和ALDH1。基因表达分析表明，短期体外培养增强了在膜上生长的捕获CTC的分化过程。另一方面，在短期培养过程中，膜上捕获的多余白细胞被清除。卵巢癌（浆液性）中最常上调的基因是EPCAM、KRT19和MUC1。已经证明，通过细胞形态学评估揭示的CTC存在可以通过TA基因表达分析得到有效补充，以提高分析的灵敏度。
