Brain Korea 21 Plus Project for Biomedical Science, Korea University College of Medicine, Seoul, 02841, Republic of Korea.
Division of Oncology/Hematology, Department of Medicine, Korea University Anam Hospital, Korea University College of Medicine, 73 Inchon-ro, Seongbuk-gu, Seoul, 02841, Republic of Korea.
Gastric Cancer. 2019 Sep;22(5):967-979. doi: 10.1007/s10120-019-00935-x. Epub 2019 Feb 6.
Three-dimensional in vitro spheroid models are unique because they are considered for enrichment of specific cell populations with self-renewal ability. In this study, we explored the different mechanisms of gastric cancer spheroid-forming cells according to the Lauren classification.
We isolated and enriched cells with self-renewal ability using spheroid-forming methods from gastric cancer cell lines. The expression of candidate target genes was investigated using western blot and qRT-PCR analysis. Lentiviral shRNA knockdown of target gene expression was performed and the effects on spheroid, colony forming, and tumorigenic ability were analyzed.
The SNU-638, SNU-484, MKN-28, and NCI-N87 successfully formed spheroid from single cell and enriched for self-renewal ability from 11 gastric cancer cell lines, including diffuse and intestinal types. The expression of SOX2 and E-cadherin increased in spheroid-forming cells in a diffuse-type cell line (SNU-638 and SNU-484), but not in the intestinal type (MKN-28 and NCI-N87). In contrast, ERBB3 expression was only increased in intestinal-type spheroid cells. The depletion of each candidate target gene expression suppressed self-renewal ability to grow as spheroids and colonies in a soft agar assay. In particular, down-regulated ERBB3 in the intestinal-type cell lines inhibited tumor growth in a mouse xenograft model. We found that high ERBB3 gene expression correlates with decreased survival in the intestinal type of gastric cancer.
Our results suggest that diffuse- and intestinal-type spheroid-forming cells express genes differently. Our data suggest that these candidate genes from spheroid-forming cells can be used in applications in targeted therapy.
三维体外球体模型是独特的，因为它们被认为可以富集具有自我更新能力的特定细胞群体。在这项研究中，我们根据 Lauren 分类法探索了胃癌球体形成细胞的不同机制。
我们使用球体形成方法从胃癌细胞系中分离和富集具有自我更新能力的细胞。使用 Western blot 和 qRT-PCR 分析研究候选靶基因的表达。通过慢病毒 shRNA 敲低靶基因表达，并分析对球体、集落形成和致瘤能力的影响。
SNU-638、SNU-484、MKN-28 和 NCI-N87 成功地从单细胞中形成球体，并从包括弥漫型和肠型在内的 11 种胃癌细胞系中富集具有自我更新能力的细胞。在弥漫型细胞系（SNU-638 和 SNU-484）中，球体形成细胞中 SOX2 和 E-钙黏蛋白的表达增加，但在肠型（MKN-28 和 NCI-N87）中则没有。相比之下，肠型球体细胞中仅 ERBB3 的表达增加。每个候选靶基因表达的耗尽均抑制自我更新能力，使球体和软琼脂集落生长。特别是，下调肠型细胞系中的 ERBB3 抑制了小鼠异种移植模型中的肿瘤生长。我们发现，高 ERBB3 基因表达与肠型胃癌患者的生存率降低相关。
我们的结果表明，弥漫型和肠型球体形成细胞表达不同的基因。我们的数据表明，这些来自球体形成细胞的候选基因可用于靶向治疗的应用。
