Cisplatin selects for CD133+ cells in lung cancer cells

Objective Platinum-based chemotherapy is the first-line treatment for non-small cell lung cancer,but the chemoresistance of tumor cells continues to be a considerable challenge in the management of NSCLCs,leading to recurrence of most patients.CD133(prominin-1)is a five-transmembrane glycoprotein,and recent evidence suggests that CD133+cells are the cause of drug resistance and tumor recurrence.In this study,the correlation between cisplatin and CD133+cells was investigated systematically.Methods Four lung cancer cell lines,including A549,H460,801D and H1299,were treated with different concentrations of cisplatin.Cell viability was determined by MTT assay.Sphere-forming assay was performed to detect the capability of sphere-forming.CD133+cells was detected by BD FACScaliber flow cytometer.Results The results showed that cisplatin could increase the number of CD133+cells in both time-and dose-dependent manner.The enrichment would weaken but the proportion of CD133+cells was still higher than the basic level as incubation time extended after cisplatin was withdrawn.Compared with adherent culture,the proportion of CD133+cells was higher when the cells were maintained suspension culture.The proportion of CD133+cells significantly increased when cisplatin was provided in suspension culture.Conclusion These results revealed that cisplatin induces the enrichment of CD133+cells and CD133 is a new therapeutic target.Our data partially explained drug resistance to second-line chemotherapy in cisplatin-treated patients with NSCLCs.     

CD133-positive tumor cell content is a predictor of early recurrence in colorectal cancer

Background

The aims of this study were to demonstrate the tumorigenicity of CD133+ colon cancer cells in vitro, analyze the correlations between spheroid formation and clinicopathologic variables, and screen for overexpressed genes in CD133+ colon cancer stem cells. Moreover, the aim of this study was to establish a living tumor tissue bank using surgically resected specimens.

Methods

Using LoVo cell line, we isolated CD133+ cells and performed clonogenic assay and animal experiment to test tumorigenicity of CD133+ cells. Twenty-nine surgical samples were freshly collected from 27 patients who received curative or palliative surgery, and the samples were mechanically and enzymatically dissociated into single cells.

Results

We confirmed the enhanced tumorigenicity of CD133+ cells isolated from LoVo cell line both in vitro and in vivo. Of these 29 samples, 8 (28%) contained >3% CD133+ cells. Sphere formation was significantly higher in samples from patients with lymphatic invasion than in those without lymphatic invasion [54.5% (6/11) vs. 12.5% (2/16); P=0.033] and in samples containing >3% of CD133+ cells than in those containing ≤3% of CD133+ cells [36.4% (4/11) vs. 0% (0/16); P=0.019].

Conclusions

These findings indicate that CD133 is a valid marker for identifying cancer stem cells from fresh surgically resected colorectal cancer tissues. Furthermore, we successfully established a living tumor tissue bank using surgically resected colorectal tissues with a viability of >70%.     

Characterization of the conversion between CD133+ and CD133- cells in colon cancer SW620 cell line

The state of cancer stem cells (CSC) under reversible fluctuations, which has been revealed in breast cancer cells most recently, suggests that subpopulations with distinct phenotypes and functions within cancer cells can undergo inter-conversion. To investigate the possibility in colon cancer cells, we employed CD133 as the CSC marker, and characterized CD133 expression pattern and the biological features of the CD133⁺ and CD133^- subsets. Flow cytometry revealed that CD133 was bimodally expressed in SW620 cells among eight colon cancer cell lines. The CD133⁺ clonal SW620 cells displayed a differential gene expression profile, higher cellular reactive oxygen species (ROS), enhanced tumorigenesis and resistance to 5-fluorouracil. The conversion in term of the CD133 phenotype of the sorted cells was observed in vitro and in vivo. The fraction of the CD133⁺ cells decreased from 99% to 80% in the sorted CD133⁺ population while rising from 5 to 10% in the sorted CD133^- population during the first 20-day cultivation and then stayed almost unchanged. A fraction (about 20%) of the CD133⁺ clonal cells lost their CD133 marker while about 10% of the CD133^- clonal cells acquired the CD133 marker. 5-Azacytidine enhanced the fraction of the CD133⁺ cells in both of the CD133⁺ and CD133^- clonal cells. Our data demonstrate that CD133 expression is dynamic and reversible, and reveal the inter-conversion between the CD133⁺ and the CD133^- SW620 cells, suggesting that the CD133 phenotype of SW620 cell population is retained by the conversion between the two cell subsets.     

CD133肿瘤干细胞标志物在肺癌中的应用进展

CD133作为肿瘤干细胞标志物已被广泛应用于临床,在肺癌干细胞的分选中亦具有重要作用。全文就肿瘤干细胞概念、CD133基因和结构、CD133与肿瘤干细胞的关系、CD133的临床应用以及CD133作为肿瘤干细胞标志物的局限性作一综述。     

肿瘤干细胞标志物CD133、CD44、OCT-4在小细胞肺癌中的表达及其临床意义

目的：探讨肿瘤干细胞标志物CD133、CD44、OCT-4与小细胞肺癌临床病理特征之间的相关性及临床意义。方法应用免疫荧光技术检测小细胞肺癌细胞株NCI-H82中肿瘤干细胞标志物CD133、CD44、OCT-4的表达;同时应用免疫组织化学法检测79例小细胞肺癌组织中CD133、CD44、OCT-4的表达。结果在小细胞肺癌细胞株NCI-H82中,CD133和CD 44的荧光信号为阳性表达,OCT-4的荧光信号为阴性表达。小细胞肺癌组织中CD133和CD44表达与肿瘤直径、淋巴结转移和临床分期相关,差异具有统计学意义（P＜0.05）。小细胞肺癌组织中OCT-4为阴性表达。结论 CD133和CD44可能是小细胞肺癌肿瘤干细胞的标志物,对小细胞肺癌的诊断和治疗有一定的临床意义。     

CD133+ cells contribute to radioresistance via altered regulation of DNA repair genes in human lung cancer cells

Background

Radioresistance in human tumors has been linked in part to a subset of cells termed cancer stem cells (CSCs). The prominin 1 (CD133) cell surface protein is proposed to be a marker enriching for CSCs. We explore the importance of DNA repair in contributing to radioresistance in CD133+ lung cancer cells.

Materials and methods

A549 and H1299 lung cancer cell lines were used. Sorted CD133+ cells were exposed to either single 4 Gy or 8 Gy doses and clonogenic survival measured. ?-H2AX immunofluorescence and quantitative real time PCR was performed on sorted CD133+ cells both in the absence of IR and after two single 4 Gy doses. Lentiviral shRNA was used to silence repair genes.

Results

A549 but not H1299 cells expand their CD133+ population after single 4 Gy exposure, and isolated A549 CD133+ cells demonstrate IR resistance. This resistance corresponded with enhanced repair of DNA double strand breaks (DSBs) and upregulated expression of DSB repair genes in A549 cells. Prior IR exposure of two single 4 Gy doses resulted in acquired DNA repair upregulation and improved repair proficiency in both A549 and H1299. Finally Exo1 and Rad51 silencing in A549 cells abrogated the CD133+ IR expansion phenotype and induced IR sensitivity in sorted CD133+ cells.

Conclusions

CD133 identifies a population of cells within specific tumor types containing altered expression of DNA repair genes that are inducible upon exposure to chemotherapy. This altered gene expression contributes to enhanced DSB resolution and the radioresistance phenotype of these cells. We also identify DNA repair genes which may serve as promising therapeutic targets to confer radiosensitivity to CSCs.     

CD133 negative glioma cells form tumors in nude rats and give rise to CD133 positive cells

CD133 is a cell surface marker expressed on progenitors of haematopoietic and endothelial cell lineages. Moreover, several studies have identified CD133 as a marker of brain tumor-initiating cells. In this study, human glioblastoma multiforme biopsies were engrafted intracerebrally into nude rats. The resulting tumors were serially passaged in vivo, and monitored by magnetic resonance imaging. CD133 expression was analyzed at various passages. Tumors initiated directly from the biopsies expressed little or no CD133, and showed no contrast enhancement suggesting an intact blood-brain barrier. During passaging, the tumors gradually displayed more contrast enhancement, increased angiogenesis and a shorter survival. Real-time qPCR and immunoblots showed that this was accompanied by increased CD133 expression. Primary biopsy spheroids and xenograft tumors were subsequently dissociated and flow sorted into CD133 negative and CD133 positive cell populations. Both populations incorporated BrdU in cell culture, and expressed the neural precursor marker nestin. Notably, CD133 negative cells derived from 6 different patients were tumorgenic when implanted into the rat brains. For 3 of these patients, analysis showed that the resulting tumors contained CD133 positive cells. In conclusion, we show that CD133 negative glioma cells are tumorgenic in nude rats, and that CD133 positive cells can be obtained from these tumors. Upon passaging of the tumors in vivo, CD133 expression is upregulated, coinciding with the onset of angiogenesis and a shorter survival. Thus, our findings do not suggest that CD133 expression is required for brain tumor initiation, but that it may be involved during brain tumor progression.     

Expression of the stem cell marker CD133 in recurrent glioblastoma and its value for prognosis

BACKGROUND:

Experimental data suggest that glioblastoma cells expressing the stem cell marker CD133 play a major role in radiochemoresistance and tumor aggressiveness. To date, however, there is no clinical evidence that the fraction of CD133‐positive cells in glioblastoma that recurs after radiochemotherapy may be relevant for prognosis.

METHODS:

The authors used immunohistochemistry to assess CD133 expression in 37 paired glioblastoma samples, including 1 primary tumor sample and 1 recurrent tumor sample, after patients received adjuvant radiochemotherapy. To assess the actual composition of the CD133‐positive glioblastoma cell population, fluorescence‐associated cell sorting (FACS) analysis was used to sort CD133‐positive/CD45‐negative cells that were assayed for tumor‐specific chromosomal aberrations using interphase fluorescence in situ hybridization. To rule out endothelial precursor cells, CD133‐positive fractions also were assayed with anti‐CD34 by FACS.

RESULTS:

In recurrent glioblastomas, the percentage of CD133‐positive cells was increased by 4.6‐fold compared with the percentage in primary glioblastomas, although, in some tumors, it increased up to 10‐fold and 20‐fold. Unexpectedly, the increase in CD133 expression was associated significantly with longer survival after tumor recurrence. An analysis of tumor‐specific chromosomal aberrations and in vivo studies revealed that the CD133‐positive cell compartment of recurrent glioblastoma was composed of both cancer stem cells and nontumor neural stem cells. The latter cells represented from 20% to 60% of the CD133‐positive cell population, and their relative percentage favorably affected the survival of patients with recurrent glioblastoma. Endothelial CD133‐positive/CD34‐positive precursors did not contribute to the CD133‐positive cell population.

CONCLUSIONS:

The authors hypothesized that, similar to the phenomenon described in glioblastoma models, neural stem/progenitor cells that are recruited by the tumor from surrounding brain may exert an antitumorigenic effect. Cancer 2011. © 2010 American Cancer Society.     

肝细胞性肝癌组织CD133+细胞肿瘤干细胞特性的研究

目的 越来越多的研究表明,肝癌细胞系中能检测到肿瘤干细胞(cancer stem cells,CSCs)的存在.本研究旨在探讨从肝细胞性肝癌(hepatocellular carcinoma,HCC)组织样本中分离获得的CD133+细胞是否具有CSCs特性.方法 将2014-02-01-2015-06-30广西医科大学附属肿瘤医院肝胆外科25例手术获得的新鲜HCC组织和对应癌旁组织,采用酶消化法分别消化成单个肝癌细胞和单个肝细胞,利用流式细胞术检测部分单个肝癌细胞和单个肝细胞CD133的表达率.用剩余的单个肝癌细胞进行原代培养,流式细胞术将培养获得的肝癌细胞分选为CD133+和CD133-细胞,通过平板克隆形成实验、肿瘤球形成实验和裸鼠移植瘤形成实验对比分析这两组细胞的CSCs特性.结果 25例HCC组织中CD133的表达率为3.8％～8.3％,平均值为(5.8±1.6)％,而癌旁组织CD133的表达率为0.1％～0.4％,平均值为(0.2±0.1)％,两者比较差异有统计学意义,t=17.12,P＜0.001.CD133+和CD133-细胞的平均克隆率分别为(25.2±0.8)％和(7.6±0.8)％,两者比较差异有统计学意义,t=81.95,P＜0.001.CD133+和CD133-细胞的平均成球率分别为(20.3±0.6)％和(12.5±1.4)％,两者比较差异有统计学意义,t=68.17,P＜0.001.CD133+细胞的裸鼠移植瘤形成能力明显高于CD133-细胞.结论 从HCC组织样本中分离获得的CD133+细胞具有明显的CSCs特性.     

Clinical relevance of stem cell surface markers CD133, CD24, and CD44 in colorectal cancer

Colon cancer stem cells (CSC) identified by cell surface markers CD133, CD24, and CD44, have been shown to be involved with tumor formation, chemotherapy resistance, and the progression of metastatic disease. Using an in silico translational approach, we hypothesize that a combination of these CSC markers has prognostic value in a large cohort of patients with colorectal cancer. Clinicopathologic and RNA expression data from a total of 594 colorectal cancer (CRC) patients from TCGA were analyzed. The expression of CD133, CD24, and CD44 was individually defined as “high” or “low” based on the median expression. Disease specific survival (DSS) and overall survival (OS) were not associated with tumors that are CD133-high or CD44-high alone. Patients with CD24-high tumors have significantly better DSS (P<0.001) and OS (P = 0.043). CD24-high, CD44-high and CD133-high tumors were associated with significantly greater EGFR, KRAS and Ki67 expression (all P<0.001). CD133, CD24 and CD44-high tumors were independently enriched for conventional stemness-related signaling pathways such as Wnt/β-catenin and Hedgehog signaling pathways. There was no survival difference linked to CD133-high/CD44-low patients, but CD44-high/CD24-low patients have worse DSS (P = 0.005) compared with CD44-low/CD24-high patients. CD133-high/CD24-low tumors show significant negative enrichment of MYC targets, E2F targets, G2M checkpoint and mitotic spindle gene sets, suggesting less cell proliferation in these tumors. Patients with CD133-high/CD24-low tumors have worse DSS (P = 0.004) and OS (P = 0.044), and are more likely to have early and late recurrences. In conclusion, we demonstrated that CD133-high/CD24-low tumors may predict colorectal cancer prognosis.     

Active glycolytic metabolism in CD133(+) hepatocellular cancer stem cells: regulation by MIR-122

Although altered metabolic pathway is an important diagnostic maker and therapeutic target in cancer, it is poorly understood in cancer stem cells (CSCs). Here we show that the CD133 (+) hepatocellular CSCs have distinct metabolic properties, characterized by more active glycolysis over oxidative phosphorylation, compared to the CD133 (−) cells. Inhibition of PDK4 and LDHA markedly suppresses CD133 (+) stemness characteristics and overcome resistance to sorafenib (current chemotherapeutic agent for hepatocellular cancer). Addition of glucose or lactate to CD133 (−) cells promotes CSC phenotypes, as evidenced by increased CD133 (+) cell population, elevated stemness gene expression and enhanced spheroid formation. Furthermore, the liver-specific miRNA, miR-122, inhibits CSC phenotypes by regulating glycolysis through targeting PDK4. Our findings suggest that enhanced glycolysis is associated with CD133 (+) stem-like characteristics and that metabolic reprogramming through miR-122 or PDK4 may represent a novel therapeutic approach for the treatment of hepatocellular cancer.     

CD133和CD45在非小细胞肺癌组织中的表达及临床意义

目的 探讨CD133和CD45在非小细胞肺癌(NSCLC)组织中的表达及临床意义.方法 采用免疫组织化学SABC法检测65例NSCLC组织中CD133和CD45的表达,分析两者与肿瘤大小、组织学类型、TNM分期、组织分化程度及淋巴结转移之间的关系.结果 CD133和CD45在NSCLC组织中的阳性表达率分别为69.2％...     

CD133作为肺癌干细胞标记物的应用及其局限性

肺癌是临床上最常见的恶性肿瘤之一,尚缺乏预后较为理想的治疗方案。肿瘤干细胞学说认为肺癌干细胞是肺癌发生、发展、转移、复发、耐受放化疗以及肺癌细胞具有侵袭性的主要原因。近年来越来越多的机构利用CD133糖基化表位来鉴定、分离、提纯肺癌干细胞。然而,随着研究的深入,CD133作为肺癌干细胞标记物逐渐受到质疑。本文对近年来利用CD133作为肺癌干细胞表面标记物的应用及其局限性做一综述。     

下调CD133表达对肝癌干细胞上皮间质化和侵袭能力影响及机制探讨

目的 肿瘤干细胞是肿瘤复发和转移的原因.探讨CD133基因RNA干扰(RNA interference,RNAi)的重组质粒,对人肝癌细胞株MHCC-H的CD133基因表达、上皮间质化和侵袭能力的影响.方法 通过免疫磁珠分选MHCC-H细胞中的CD133阳性细胞,设计并合成特异性靶向CD133的小分子干扰RNA(small interfering RNA,siRNA)片段,并构建pSuper retro GFP-neo-siRNA-CD133表达质粒,将其转入MHCC-H-CD133+细胞,并设空白对照组、阳性对照组.通过G418筛选出稳定株.通过粘附实验、Boyden小室实验和软琼脂克隆形成实验观察各细胞株侵袭能力的变化.采用明胶酶法测定肝癌细胞的基质金属蛋白酶-2(matrix metalloproteinase-2,MMP 2)和MMP-9的含量,蛋白质印迹法检测上皮间质化相关基因的变化.结果 通过免疫磁珠分选后的MHCC-H细胞中CD133表达率为83.5％.qRT-PCR结果显示,shCD133组相对空白对照组的CD133表达量下降了80％,P＜0.05.蛋白质印迹法检测结果显示,shCD133组的CD133表达明显下降,约为空白对照组的12％和shNC组的10％,均P＜0.05.shCD133组的粘附能力为86.9±12.4,较空白对照组的568.5±53.2和shNC组的538.8±35.6明显下降,P＜0.05.Boyden小室实验发现,shCD133组穿膜细胞数为(80.6±11.4)个,较空白对照组的(228.5±33.2)个和shNC组的(230.8±32.9)个明显减少,P＜0.05.软琼脂克隆形成率的检测发现,shCD133的细胞克隆形成(60±5)个,比空白对照组的(178±23)个和shNC组的(168±25)个明显下降,P＜0.05.明胶酶法检测发现,shCD133组MMP-2和MMP-9相对活性为0.4±0.14和0.6±0.16,较shNC组的1.03±0.19和1.3±0.16明显下降,P＜0.05.蛋白质印迹法检测结果表明,shCD133组N-cadherin蛋白表达为36.3±4.5,Snail为53.6±6.7,Slug为41.63±5.6,Twist为39.4±3.9,均明显低于空白对照组的87.6±8.6、80.6±7.5、81.9±9.2和83.9±9.1,也明显低于shNC组的89.4±9.6、83.5±8.9、85.1±8.7和87.6±9.3,均P＜0.05;而shCD133组E-cadherin表达为88.4±9.2,较空白对照组的57.6±8.7和shNC组的53.9±8.9明显上调,P＜0.05.结论 沉默MHCC-H细胞CD133基因的表达能有效抑制其上皮间质化和侵袭能力,CD133基因可能成为肝癌基因治疗的有效靶点.     

Cancer stem/progenitor cells are highly enriched in CD133+CD44+ population in hepatocellular carcinoma

Both our previous study and other reports have suggested that CD133, originally classified as a hematopoietic stem cell marker, could be used for enrichment of cancer stem cells (CSCs) in human hepatocellular carcinoma (HCC). It was also noted that not all of CD133⁺ cells were representative of CSCs. Further identification and characterization of CSCs or tumor‐initiating cells in HCC are necessary to better understand hepatocarcinogenesis. In present study, we demonstrated that CSC phenotype could be precisely defined by co‐expression of CD133 and CD44 cell surface markers. CD133⁺CD44⁺ HCC cells showed stem cell properties, including extensive proliferation, self‐renewal, and differentiation into the bulk of cancer cells. In vivo xenograft experiments revealed that, actually, the highly tumorigenic capacity of CD133⁺ cells as previously described was primarily attributed to CD133⁺CD44⁺ cell subpopulation, instead of their CD133⁺CD44^? counterparts. Moreover, cells double‐positive for CD133 and CD44 exhibited preferential expression of some stem cell‐associated genes and were more resistant to chemotherapeutic agents due to the upregulation of ATP‐binding cassette (ABC) superfamily transporters, including ABCB1, ABCC1, and ABCG2, further supporting these cells as HCC cell origin. Our findings suggest that CD133⁺CD44⁺ cells might represent true cancer stem/progenitor cells in HCC, which could allow a better understanding of HCC initiation and progression, as well as establish a precise target for the development of more effective therapies.     

Low glucose promotes CD133mAb-elicited cell death via inhibition of autophagy in hepatocarcinoma cells

CD133 on cancer stem cells is a potential therapeutic target. In this study, CD133 antibody (CD133mAb) treatment resulted in cell death in hepatoma LM3, HepG2, Hep3B and Huh-7 cells, especially under low glucose condition. The treatment also inhibited formation of spheroids, colonies, and xenograft tumors. Ectopic CD133 enabled hepatocyte L02 to be suppressed by CD133mAb and increased spheroid formation. CD133mAb caused cell death in primary HCC cells and sensitized them to Doxorubicin and Cisplatin. The antibody effect was attributed to suppressing autophagy and promoting necrotic cell death. Therefore, targeting CD133 under low glucose condition is a potential therapeutic approach for hepatocarcinomas.     

CD24 negative lung cancer cells,possessing partial cancer stem cell properties,cannot be considered as cancer stem cells

Cancer stem cells (CSCs) play vital role in lung cancer progression, resistance, metastasis and relapse. Identifying lung CSCs makers for lung CSCs targeting researches are critical for lung cancer therapy. In this study, utilizing previous identified lung CSCs as model, we compared the expression of CD24, CD133 and CD44 between CSCs and non-stem cancer cells. Increased ratio of CD24- cells were found in CSCs. CD24- cells were then sorted by flow cytometry and their proliferative ability, chemo-resistance property and in vivo tumor formation abilities were detected. A549 CD24- cells formed smaller colonies, slower proliferated in comparison to A549 CD24+ cells. Besides, A549 CD24- exhibited stronger resistance to chemotherapy drug. However, A549 CD24- didn’t exert any stronger tumor formation ability in vivo, which is the gold standard of CSCs. These results showed that CD24- A549 cells showed some properties of CSCs but not actually CSCs. This study provides evidence that CD24 cannot be considered as lung CSCs marker.     

肝癌细胞系Huh-7中CD133阳性细胞亚群的分离及其特性的研究

目的：研究CD133在人肝癌细胞系Huh-7中的表达,初步探讨肝癌中CD133阳性细胞亚群的干细胞特性。方法：流式细胞荧光激活分选技术纯化肝癌细胞系Huh-7中CD133肿瘤细胞,体外培养并观察其增殖、体内成瘤及分化能力。结果i流式细胞仪检测肝癌细胞系Huh-7中有62．3％的细胞CD133呈阳性表达,流式细胞荧光激活分选的CD133肿瘤细胞在无血清培养基中第3、5、7d的吸光度（OD值）分别为0．310、0．362、0．564,均高于相同条件下未分选细胞和CD133阴性细胞;CD133阳性细胞亚群成瘤能力明显强于阴性细胞亚群;CD133阳性细胞亚群在培养体系中的比例逐日下降,至培养的第8天,由第1天的92．3％下降至61．4％。结论：肝癌细胞系Huh-7中CD133阳性细胞亚群比其它细胞亚群具有较强增殖、成瘤和分化能力,是具有肝癌干细胞特性的细胞亚群。     

5-Fluorouracil upregulates the activity of Wnt signaling pathway in CD133-positive colon cancer stem-like cells

Background and Objective:CD133-positive colon cancer stem like cells (CSLCs) are resistant to the conventional cytotoxic drug 5-fluorouracil (5-FU).Wnt signaling pathway plays important roles in colon cancer carcinogenesis and metastasis, and regulates the self-renewal capacity of CSLCs.In the present study, we explored the impact of 5-FU on Wnt signaling pathway of CD133-positive colon CSLCs, and the relation between Wnt signaling pathway and drug resistance of CD133-positive colon CSLCs.Methods:Magnetic a...