Comparative Gene Expression Profiling of Primary and Metastatic Renal Cell Carcinoma Stem Cell-Like Cancer Cells.
Comparative Gene Expression Profiling of Primary and Metastatic Renal Cell Carcinoma Stem Cell-Like Cancer Cells.
复制标题
DOI：
10.1371/journal.pone.0165718
复制
发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Szczylik C
中科院分区：
文献类型：
--
作者：
Khan MI;Czarnecka AM;Lewicki S;Helbrecht I;Brodaczewska K;Koch I;Zdanowski R;Król M;Szczylik C
癌症研究的最新进展表明，肿瘤是高度异质性的，并且在单个肿瘤中发现多种表型不同的细胞群。癌症发展和肿瘤生长是由特定类型的细胞-干细胞样癌细胞（SCLCC）驱动的，这也是转移扩散和耐药性的原因。本研究旨在验证肾细胞癌细胞系中SCLCC的存在。随后，我们的目的是表征CD 105+细胞的表型和细胞生物学，之前被定义为肾细胞癌肿瘤起始细胞。该项目的主要目标是描述原发性肿瘤和转移性起源的干细胞样癌细胞的基因表达谱。采用Real-time PCR检测干细胞基因（Oct-4、Nanog和Ncam）和软琼脂集落形成试验检测肾细胞癌（RCC）细胞系的干细胞特性。对RCC细胞进行⑶ 105+和⑶ 133+细胞的FACS分析。验证分离的CD 105+细胞表达间充质标志物-CD 24、CD 146、CD 90、CD 73、CD 44、CD 11b、CD 19、CD 34、CD 45、HLA-DR和碱性磷酸酶。悬滴法检测CD 105+细胞间的粘附力。通过分离的CD 105+形成的自由漂浮的3D球体的分析得到验证，因为球体已被假设含有未分化的多能祖细胞。最后，从原发性（Caki-2）和转移性（ACHN）肾细胞癌细胞系中分选CD 105+细胞。用Agilent的人GE 4x 44 K v2微阵列进行分选的CD 105+细胞的基因表达谱分析。差异表达的基因被进一步归类为典型途径。网络分析和下游分析用IntimidityPathwayAnalysis进行。转移性RCC细胞系（ACHN和Caki-1）与原代RCC细胞系相比表现出更高的集落形成能力。转移性RCC细胞系含有许多CD 105+细胞亚群，并且具有较高的干细胞基因表达（Oct-4和Nanog）。CD 105+细胞在悬滴条件下呈葡萄状漂浮结构。分选的CD 105+细胞对人间充质干细胞（MSC）标志物CD 90、CD 73、CD 44、CD 146和碱性磷酸酶活性呈阳性，但对CD 24和造血谱系标志物CD 34、CD 11b、CD 19、CD 45、CD 11b、CD 11 c、CD 11 d和HLA-DR. 1411基因通常在CD 105+细胞中差异表达（来自原代[Caki-2]和转移性RCC [ACHN]细胞）与健康肾上皮细胞系（ASE-5063）的比较。TGF-β、Wnt/β-catenine、上皮-间质转化（EMT）、Rap 1信号传导、PI 3 K-Akt信号传导和Hippo信号传导途径在CD 105+细胞中失调。TGFB 1、ERBB 2和TNF是这些细胞中激活的最重要的转录调节因子。总之，RCC-CD 105+细胞呈现干细胞样特性。这些干细胞样癌细胞可能代表一种新的治疗靶点。CD 105+细胞独特的基因表达谱可用作后续功能研究和药物设计的初始数据。
Recent advancement in cancer research has shown that tumors are highly heterogeneous, and multiple phenotypically different cell populations are found in a single tumor. Cancer development and tumor growth are driven by specific types of cells—stem cell-like cancer cells (SCLCCs)—which are also responsible for metastatic spread and drug resistance. This research was designed to verify the presence of SCLCCs in renal cell cancer cell lines. Subsequently, we aimed to characterize phenotype and cell biology of CD105+ cells, defined previously as renal cell carcinoma tumor-initiating cells. The main goal of the project was to describe the gene-expression profile of stem cell-like cancer cells of primary tumor and metastatic origin. Real-time PCR analysis of stemness genes (Oct-4, Nanog and Ncam) and soft agar colony formation assay were conducted to check the stemness properties of renal cell carcinoma (RCC) cell lines. FACS analysis of CD105+ and CD133+ cells was performed on RCC cells. Isolated CD105+ cells were verified for expression of mesenchymal markers—CD24, CD146, CD90, CD73, CD44, CD11b, CD19, CD34, CD45, HLA-DR and alkaline phosphatase. Hanging drop assay was used to investigate CD105+ cell-cell cohesion. Analysis of free-floating 3D spheres formed by isolated CD105+ was verified, as spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Finally, CD105+ cells were sorted from primary (Caki-2) and metastatic (ACHN) renal cell cancer cell lines. Gene-expression profiling of sorted CD105+ cells was performed with Agilent’s human GE 4x44K v2 microarrays. Differentially expressed genes were further categorized into canonical pathways. Network analysis and downstream analysis were performed with Ingenuity Pathway Analysis. Metastatic RCC cell lines (ACHN and Caki-1) demonstrated higher colony-forming ability in comparison to primary RCC cell lines. Metastatic RCC cell lines harbor numerous CD105+ cell subpopulations and have higher expression of stemness genes (Oct-4 and Nanog). CD105+ cells adopt 3D grape-like floating structures under handing drop conditions. Sorted CD105+ cells are positive for human mesenchymal stem cell (MSC) markers CD90, CD73, CD44, CD146, and alkaline phosphatase activity, but not for CD24 and hematopoietic lineage markers CD34, CD11b, CD19, CD45, and HLA-DR. 1411 genes are commonly differentially expressed in CD105+ cells (both from primary [Caki-2] and metastatic RCC [ACHN] cells) in comparison to a healthy kidney epithelial cell line (ASE-5063). TGF-β, Wnt/β-catenine, epithelial-mesenchymal transition (EMT), Rap1 signaling, PI3K-Akt signaling, and Hippo signaling pathway are deregulated in CD105+ cells. TGFB1, ERBB2, and TNF are the most significant transcriptional regulators activated in these cells. All together, RCC-CD105+ cells present stemlike properties. These stem cell-like cancer cells may represent a novel target for therapy. A unique gene-expression profile of CD105+ cells could be used as initial data for subsequent functional studies and drug design.
登录
查看更多内容
影响因子：
2.2
作者：
Banerjee, Meenal;Bhonde, Ramesh R.
通讯作者：
Bhonde, Ramesh R.
影响因子：
11.2
作者：
Adorno-Cruz V;Kibria G;Liu X;Doherty M;Junk DJ;Guan D;Hubert C;Venere M;Mulkearns-Hubert E;Sinyuk M;Alvarado A;Caplan AI;Rich J;Gerson SL;Lathia J;Liu H
通讯作者：
Liu H
影响因子：
4.8
作者：
Bussolati, Benedetta;Bruno, Stefania;Camussi, Giovanni
通讯作者：
Camussi, Giovanni
影响因子：
45.3
作者：
Escudier, Bernard;Eisen, Tim;Bukowski, Ronald M.
通讯作者：
Bukowski, Ronald M.
影响因子：
50.3
作者：
Dolado, Ignacio;Swat, Aneta;Nebreda, Angel R.
通讯作者：
Nebreda, Angel R.