Yang Zhaocong, Zhang Yanfeng, Tang Tingting, Zhu Qinhua, Shi Wanyue, Yin Xin, Xing Yun, Shen Yumeng, Pan Yi, Jin Liang
Cell Physiol Biochem. 2018;47(5):2109-2125. doi: 10.1159/000491479. Epub 2018 Jul 5.
BACKGROUND/AIMS: Pancreatic cancer remains one of the deadliest human malignancies, the lethality of which may be attributed to the presence of pancreatic cancer stem cells (PCSCs), a small subpopulation of cells existing within pancreatic tumor with high carcinogenesis. Therefore, it is crucial to establish an efficient enrichment and culture system of PCSCs and identify the key genes involved in the regulation of PCSCs. The three-dimensional (3D) liquid suspension mammosphere culture system has been established for enrichment and culture of PCSCs in vitro as the cell spheres are likely to originate from individual cell clone, but it has been challenged because the cell spheroids could be a result of cell aggregation.
We optimized the existing culture system by adding methylcellulose to create a 3D semi-solid system which prevented the non-specific aggregation. Then we identified the CSC properties of Panc-1 spheroid cells cultured by this system by detecting the genes associated with stemness and by evaluation of the tumorigenicity in vitro and in vivo through invasion, migration and xenograft experiments methods. Subsequently, we performed high-throughput sequencing (HTS) of the Panc-1 spheroid cells.
We confirmed the PCSCs properties and high malignancy of the Panc-1 spheroid cells enriched by our novel 3D semi-solid system both in vitro and in vivo. Hundreds of mRNA, microRNA (miRNA) and dozens of long non-coding RNA (LncRNA) were identified to be differentially regulated in PCSCs-like Panc-1 spheroid cells compared with their parental cells by HTS.
Our results demonstrate an efficient enrichment and culture system for Panc-1 spheroid cells with the PCSCs properties. The differentially expressed genes and their targets identified by the HTS of the Panc-1 spheroid cells can serve as new potential biomarkers for pancreatic cancer diagnosis and targeted therapy.
背景/目的：胰腺癌仍然是人类最致命的恶性肿瘤之一，其致死性可能归因于胰腺癌干细胞（PCSCs）的存在，PCSCs是胰腺肿瘤内存在的一小部分具有高致癌性的细胞亚群。因此，建立高效的PCSCs富集和培养系统并鉴定参与PCSCs调控的关键基因至关重要。三维（3D）液体悬浮乳腺球培养系统已被用于体外富集和培养PCSCs，因为细胞球可能起源于单个细胞克隆，但该系统受到了挑战，因为细胞球体可能是细胞聚集的结果。
我们通过添加甲基纤维素优化了现有的培养系统，创建了一个防止非特异性聚集的3D半固体系统。然后，我们通过检测与干性相关的基因，并通过侵袭、迁移和异种移植实验方法评估体外和体内的致瘤性，来鉴定用该系统培养的Panc-1球体细胞的CSC特性。随后，我们对Panc-1球体细胞进行了高通量测序（HTS）。
我们在体外和体内均证实了通过我们新型的3D半固体系统富集的Panc-1球体细胞具有PCSCs特性和高恶性。通过HTS鉴定出与亲代细胞相比，在类PCSCs的Panc-1球体细胞中有数百种mRNA、微小RNA（miRNA）和数十种长链非编码RNA（LncRNA）受到差异调控。
我们的结果证明了一种用于具有PCSCs特性的Panc-1球体细胞的高效富集和培养系统。通过对Panc-1球体细胞进行HTS鉴定出的差异表达基因及其靶点可作为胰腺癌诊断和靶向治疗的新潜在生物标志物。
