Gehlot Prashasnika, Shukla Vivek, Gupta Sanjay, Makidon Paul E
Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA.
TGIB Branch, NCI, Betehsda, MD, USA.
J Transl Med. 2016 Feb 12;14:49. doi: 10.1186/s12967-016-0785-0.
Aldehyde dehydrogenase 1 (ALDH1) activity has been implicated in the therapeutic drug resistance of many malignancies and has been widely used as a marker to identify stem-like cells, including in primary liver cancer. Cancer stem cells (CSCs) are thought to play a crucial role in cancer progression and metastasis. In order to clarify the validity of the rabbit VX2 liver cancer model, we questioned if it expresses ALDH1 as a potential marker of CSCs. Hepatocellular carcinoma is a common malignancy worldwide and has poor prognosis. Most of the animal models used to study hepatocellular carcinoma are rodent models which lack clinical relevance. The rabbit VX2 model is a large animal model useful for preclinical and for developing drugs targeting cancer stem cells.
We used flow cytometry to identify rabbit VX2 liver tumor cells that express ALDH1A1 activity at a high level and confirmed the results with RT-PCR, immunohistochemical and western blot analyses. Further, mRNA and protein expression analysis of tumor samples also express the markers for stemness like klf4, oct3/4, CD44 and nanog as well as the differentiation marker α-fetoprotein.
We used Aldefluor flow cytometry-based assay to identify cells with high ALDH1 activity in the rabbit VX2 liver cancer model. We used the brightest 4.39 % of the total cancer cell population in our study. We performed semi-quantitative as well as real time PCR to characterize the stemness derived from VX2 tumors and tissues from normal rabbit liver. We demonstrated that VX2 tumors have higher expression of cancer stem cell markers such as AlDH1A1 and CD44 in comparison to normal rabbit liver cells. Additionally, real time PCR analysis of the same samples using syber-green demonstrated the significant change (p > 0.05) in the expression of genes. We validated the gene expression of the stemness markers by performing western blot and immunofluorescence. We showed that cancer stem cell markers (AlDH1A1, CD44) and the differentiation marker α-fetoprotein were upregulated in VX2 tumor cells. The same extent of upregulation was observed in stemness markers (klf4, oct3/4 and nanog) in VX2 tumors in comparison to normal rabbit liver.
The overall results of this study indicate that ALDH1 is a valid CSC marker for VX2 cancer. This finding suggests that the rabbit VX2 liver cancer model is useful in studying drug resistance in hepatocellular carcinoma and may be useful for basic and preclinical studies of other types of human cancer.
醛脱氢酶1（ALDH1）活性与多种恶性肿瘤的治疗耐药性有关，并已被广泛用作鉴定干细胞样细胞的标志物，包括在原发性肝癌中。癌症干细胞（CSCs）被认为在癌症进展和转移中起关键作用。为了阐明兔VX2肝癌模型的有效性，我们质疑它是否表达ALDH1作为癌症干细胞的潜在标志物。肝细胞癌是全球常见的恶性肿瘤，预后较差。用于研究肝细胞癌的大多数动物模型是缺乏临床相关性的啮齿动物模型。兔VX2模型是一种大型动物模型，可用于临床前研究以及开发针对癌症干细胞的药物。
我们使用流式细胞术鉴定高表达ALDH1A1活性的兔VX2肝肿瘤细胞，并通过逆转录聚合酶链反应（RT-PCR）、免疫组织化学和蛋白质印迹分析来证实结果。此外，对肿瘤样本的mRNA和蛋白质表达分析还显示其表达干性标志物如klf4、oct3/4、CD44和nanog以及分化标志物甲胎蛋白。
我们使用基于Aldefluor流式细胞术的检测方法在兔VX2肝癌模型中鉴定具有高ALDH1活性的细胞。在我们的研究中，我们使用了癌细胞总数中最亮的4.39%。我们进行了半定量以及实时PCR来表征源自VX2肿瘤和正常兔肝组织的干性。我们证明，与正常兔肝细胞相比，VX2肿瘤中癌症干细胞标志物如AlDH1A1和CD44的表达更高。此外，使用syber-green对相同样本进行的实时PCR分析表明基因表达有显著变化（p>0.05）。我们通过蛋白质印迹和免疫荧光验证了干性标志物的基因表达。我们表明，癌症干细胞标志物（AlDH1A1、CD44）和分化标志物甲胎蛋白在VX2肿瘤细胞中上调。与正常兔肝相比，VX2肿瘤中的干性标志物（klf4、oct3/4和nanog）上调程度相同。
本研究的总体结果表明，ALDH1是VX2癌症的有效癌症干细胞标志物。这一发现表明，兔VX2肝癌模型可用于研究肝细胞癌的耐药性，可能对其他类型人类癌症的基础研究和临床前研究有用。
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