Isolation and biological identification of tumor stem cells from ovarian cancer ID8 cell lines

doi:10.3969/j.issn.2095-4344.0968

Abstract

Abstract:

BACKGROUND: Tumor stem cells play an important role in the initiation and development of tumor. However there is no report on whether tumor stem cells exist in mouse ovarian cancer cell line ID8.
OBJECTIVE: To sort tumor stem-like cells from murine ID8 ovarian cancer cell line and to investigate its biological characteristics.
METHODS: ID8 cells were cultured in the serum-free medium to derive tumor spheres. Cell counting kit 8, plate colony formation, Transwell assays and chemosensitivity testing were adopted to analyze the biological differences between suspending sphere cells and conventional cultured ID8 cells. Real-time PCR and western blot assays were performed to examine the expression of ALDH1. A mouse model of tumorigenicity was used to observe the tumorigenesis in vivo.
RESULTS AND CONCLUSION: Ovarian ID8 cells grew into stable suspending tumor spheres in the serum-free medium. Compared with conventional ID8 cells, suspending sphere cells had stronger abilities of proliferation, self-renewal, invasion, drug resistance and tumorigenesis. Moreover, ALDH1 expression in suspending spheres cells was higher than that in conventional cultured ID8 cells. These findings indicate that serum-free medium culture can effectively enrich ID8 suspending sphere cells which have the properties of ovarian cancer stem cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Neoplastic Stem Cells, Culture Media, Serum-Free, Tissue Engineering

CLC Number:

R394.2

Yu Xiao-yu, Wu Di, Wang Jing, Li Fei, Bai Xu-le, Zhao Feng-shu, Dou Jun. Isolation and biological identification of tumor stem cells from ovarian cancer ID8 cell lines[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(29): 4687-4691.

Figures/Tables 1

2.1 细胞形态倒置显微镜下可见，ID8细胞在含血清的培养基中呈梭形单层贴壁生长(图1)，而在含有细胞因子的无血清培养基中，部分ID8细胞悬浮增生，呈球形或者椭圆形，悬浮球内的细胞结合紧密、球体饱满且球内细胞折光性好(图1)。表明ID8细胞在无血清培养条件下能够存活并增殖形成具有自我更新能力的肿瘤细胞球。 2.2 细胞增殖和克隆形成能力 CCK8检测ID8细胞和悬浮球细胞增殖结果显示，从培养第4天开始，悬浮球细胞组的吸光度值明显高于常规培养的ID8细胞组(图2A)，表明相对于ID8细胞，悬浮球细胞具有较强的增殖能力。同时，在平板克隆实验中，2组细胞经14 d培养后，计数大于50个细胞的克隆，发现悬浮球形成的克隆数为(51.3±4.2)个，而ID8细胞为(26.3±8.4)个，差异有显著性意义(P=0.009 8)(图2B), 说明悬浮球具有更强的自我更新能力。 2.3 Transwell小室侵袭能力经24 h侵袭，在倒置显微镜下计数ID8细胞组和悬浮球细胞组穿过微孔膜的细胞数分别为(58.0±7.1)，(96.4±18.8)个。结果表明，相对于ID8细胞组，悬浮球细胞组穿过微孔膜细胞数量明显增多(P=0.002 7；图3)，显示出悬浮球细胞具有更强的侵袭能力。 2.4 耐受化疗药物的能力体外顺铂作用48 h后，ID8细胞组的细胞凋亡率为(68.1±5.8)%，而悬浮球细胞组凋亡率为(31.7±3.2)%，悬浮球组细胞的凋亡细胞量明显少于ID8细胞组(P=0.000 7；图4)，显示出悬浮球细胞更强的耐药性。 2.5 肿瘤干性标志ALDH1表达水平定量PCR检测结果发现，在ID8细胞和悬浮球细胞中ALDH1 mRNA的表达水平，差异有显著性意义(P=0.000 4；图5A)。同时，Western blot 检测ALDH1在悬浮球细胞中表达的半定量结果为0.58±0.05，而ID8细胞中ALDH1的表达量仅为0.14±0.04，差异有显著性意义(P=0.003；图5B)。 2.6 小鼠体内致瘤能力小鼠体内成瘤实验显示，与ID8细胞组相比，ID8悬浮球细胞组瘤组织增长速度较快，瘤体积较大(图6A，B)；同时，ID8悬浮球细胞组小鼠的成瘤潜伏期要明显短于ID8细胞组，ID8悬浮球细胞组种瘤后第12-14天在小鼠腹股沟皮下陆续长出肿瘤，而ID8细胞组直到第18天开始才陆续长出瘤体(图6C)。表明悬浮球细胞具有更强的致瘤能力。"

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