Nishiyama Mitsuo, Tsunedomi Ryouichi, Yoshimura Kiyoshi, Hashimoto Noriaki, Matsukuma Satoshi, Ogihara Hiroyuki, Kanekiyo Shinsuke, Iida Michihisa, Sakamoto Kazuhiko, Suzuki Nobuaki, Takeda Shigeru, Yamamoto Shigeru, Yoshino Shigefumi, Ueno Tomio, Hamamoto Yoshihiko, Hazama Shoichi, Nagano Hiroaki
Department of Gastroenterological, Breast and Endocrine Surgery, Yamaguchi University Graduate School of Medicine, Ube, Japan.
Division of Cancer Immunotherapy, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Tokyo, Japan.
Cancer Sci. 2018 Apr;109(4):1101-1109. doi: 10.1111/cas.13527. Epub 2018 Mar 6.
Cancer stem cells (CSCs) are thought to play important roles in cancer malignancy. Previously, we successfully induced sphere cancer stem-like cells (CSLCs) from several cell lines and observed the property of chemoresistance. In the present study, we examined the metastatic potential of these induced CSLCs. Sphere cancer stem-like cells were induced from a human hepatoma cell line (SK-HEP-1) in a unique medium containing neural survival factor-1. Splenic injection of cells into immune-deficient mice was used to assess hematogenous liver metastasis. Transcriptomic strand-specific RNA-sequencing analysis, quantitative real-time PCR, and flow cytometry were carried out to examine the expression of epithelial-mesenchymal transition (EMT)-related genes. Splenic injection of CSLCs resulted in a significantly increased frequency of liver metastasis compared to parental cancer cells (P < .05). In CSLCs, a mesenchymal marker, Vimentin, and EMT-promoting transcription factors, Snail and Twist1, were upregulated compared to parental cells. Correspondingly, significant enrichment of the molecular signature of the EMT in CSLCs relative to parental cancer cells was shown (q < 0.01) by RNA-sequencing analysis. This analysis also revealed differential expression of CD44 isoforms between CSLCs and parental cancer cells. Increasing CD44 isoforms containing an extra exon were observed, and the standard CD44 isoform decreased in CSLCs compared to parental cells. Interestingly, another CD44 variant isoform encoding a short cytoplasmic tail was also upregulated in CSLCs (11.7-fold). Our induced CSLCs possess an increased liver metastatic potential in which promotion of the EMT and upregulation of CD44 variant isoforms, especially short-tail, were observed.
癌症干细胞（CSCs）被认为在癌症恶性发展中起重要作用。此前，我们成功地从几种细胞系中诱导出球形癌干细胞样细胞（CSLCs），并观察到其化疗耐药特性。在本研究中，我们检测了这些诱导产生的CSLCs的转移潜能。在含有神经存活因子-1的独特培养基中，从人肝癌细胞系（SK-HEP-1）诱导出球形癌干细胞样细胞。将细胞经脾脏注射到免疫缺陷小鼠体内，以评估血行性肝转移情况。进行转录组链特异性RNA测序分析、定量实时PCR和流式细胞术，以检测上皮-间质转化（EMT）相关基因的表达。与亲代癌细胞相比，脾脏注射CSLCs导致肝转移频率显著增加（P < 0.05）。与亲代细胞相比，在CSLCs中，间充质标志物波形蛋白以及促进EMT的转录因子Snail和Twist1上调。相应地，RNA测序分析显示，与亲代癌细胞相比，CSLCs中EMT分子特征显著富集（q < 0.01）。该分析还揭示了CSLCs和亲代癌细胞之间CD44异构体的差异表达。观察到含有额外外显子的CD44异构体增加，与亲代细胞相比，CSLCs中的标准CD44异构体减少。有趣的是，另一种编码短细胞质尾巴的CD44变异异构体在CSLCs中也上调（11.7倍）。我们诱导产生的CSLCs具有增加的肝转移潜能，其中观察到EMT的促进以及CD44变异异构体尤其是短尾异构体的上调。
