Proteomic alteration in gastic adenocarcinomas from Japanese patients

Background

Recently, a small population of cancer stem cells in adult and pediatric brain tumors has been identified. Some evidence has suggested that CD133 is a marker for a subset of leukemia and glioblastoma cancer stem cells. Especially, CD133 positive cells isolated from human glioblastoma may initiate tumors and represent novel targets for therapeutics. The gene expression and the drug resistance property of CD133 positive cancer stem cells, however, are still unknown.

Results

In this study, by FACS analysis we determined the percentage of CD133 positive cells in three primary cultured cell lines established from glioblastoma patients 10.2%, 69.7% and 27.5%, respectively. We also determined the average mRNA levels of markers associated with neural precursors. For example, CD90, CD44, CXCR4, Nestin, Msi1 and MELK mRNA on CD133 positive cells increased to 15.6, 5.7, 337.8, 21.4, 84 and 1351 times, respectively, compared to autologous CD133 negative cells derived from cell line No. 66. Additionally, CD133 positive cells express higher levels of BCRP1 and MGMT mRNA, as well as higher mRNA levels of genes that inhibit apoptosis. Furthermore, CD133 positive cells were significantly resistant to chemotherapeutic agents including temozolomide, carboplatin, paclitaxel (Taxol) and etoposide (VP16) compared to autologous CD133 negative cells. Finally, CD133 expression was significantly higher in recurrent GBM tissue obtained from five patients as compared to their respective newly diagnosed tumors.

Conclusion

Our study for the first time provided evidence that CD133 positive cancer stem cells display strong capability on tumor's resistance to chemotherapy. This resistance is probably contributed by the CD133 positive cell with higher expression of on BCRP1 and MGMT, as well as the anti-apoptosis protein and inhibitors of apoptosis protein families. Future treatment should target this small population of CD133 positive cancer stem cells in tumors to improve the survival of brain tumor patients.     

CD133(+) and CD133(-) glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles

Although glioblastomas show the same histologic phenotype, biological hallmarks such as growth and differentiation properties vary considerably between individual cases. To investigate whether different subtypes of glioblastomas might originate from different cells of origin, we cultured tumor cells from 22 glioblastomas under medium conditions favoring the growth of neural and cancer stem cells (CSC). Secondary glioblastoma (n = 7)-derived cells did not show any growth in the medium used, suggesting the absence of neural stem cell-like tumor cells. In contrast, 11/15 primary glioblastomas contained a significant CD133(+) subpopulation that displayed neurosphere-like, nonadherent growth and asymmetrical cell divisions yielding cells expressing markers characteristic for all three neural lineages. Four of 15 cell lines derived from primary glioblastomas grew adherently in vitro and were driven by CD133(-) tumor cells that fulfilled stem cell criteria. Both subtypes were similarly tumorigenic in nude mice in vivo. Clinically, CD133(-) glioblastomas were characterized by a lower proliferation index, whereas glial fibrillary acidic protein staining was similar. GeneArray analysis revealed 117 genes to be differentially expressed by these two subtypes. Together, our data provide first evidence that CD133(+) CSC maintain only a subset of primary glioblastomas. The remainder stems from previously unknown CD133(-) tumor cells with apparent stem cell-like properties but distinct molecular profiles and growth characteristics in vitro and in vivo.     

CD66c is a novel marker for colorectal cancer stem cell isolation,and its silencing halts tumor growth in vivo

BACKGROUND:

Despite the well recognized expression of the cell surface markers cluster of differentiation 44 (homing cell adhesion molecule) and CD133 (Prominin 1) on human colorectal cancer stem cells (CCSCs), these molecules do not appear to be effective targets for stem cell‐directed therapies. Because the surface marker CD66c (also known as carcinoembryonic antigen‐related cell adhesion molecule 6) has demonstrated promise as a therapeutic target in pancreatic malignancy, the authors evaluated its potential as a target for stem cell‐directed treatment of colorectal cancer.

METHODS:

First, the authors characterized CD66c expression by flow cytometry and immunohistochemistry in colon cancer samples and in normal colon tissues. Then, the coexpression of CD66c and CD133 was evaluated on putative CCSCs. CD66c expression also was measured in stem cell‐enriched colon spheres. Finally, the effects of small‐interfering RNA‐mediated CD66c silencing on the in vitro and in vivo growth of Caco2 colon cancer cells were evaluated.

RESULTS:

CD66c expression was significantly higher in colon cancers than in contiguous normal colon tissues and paralleled cancer stage. CD66c was absent in CD133‐positive cells that were isolated from normal colon, whereas its expression was brightest (CD66c^bright) in CD133‐positive cells from colon cancer samples. In vitro experiments demonstrated that colon spheres were considerably enriched in a CD66c^bright population in a fashion comparable to the enrichment observed in fresh liver metastases. In vitro proliferation and clonogenic potential were hampered when CD66c was silenced in Caco2 cells. Finally, in vivo xenograft experiments demonstrated that CD66c silencing almost completely abrogated the tumorigenic potential of Caco2 cells.

CONCLUSIONS:

CD66c^bright expression was associated with colon cancer stem cells and CD66c silencing blocked tumor growth, thereby opening the way to a potential new treatment for colon cancer. Cancer 2013. © 2012 American Cancer Society.     

Enrichment of CD133‐expressing cells in rectal cancers treated with preoperative radiochemotherapy is an independent marker for metastasis and survival

BACKGROUND:

The transmembrane glycoprotein CD133 (cluster of differentiation 133; also known as Prominin or PROM1) has been described as a potential stem cell marker in colorectal cancer and is associated with higher tumorigenic potential and resistance to radiochemotherapy (RCT). In this study, CD133 expression was evaluated in pre‐RCT tumor biopsies and the corresponding post‐RCT surgical specimens from patients with locally advanced rectal adenocarcinoma, and expression levels were correlated with histopathologic features and clinical follow‐up.

METHODS:

One hundred twenty‐six patients with International Union Against Cancer (UICC) stage II/III rectal cancer who received preoperative 5‐fluorouracil (5‐FU)‐based RCT within the German Rectal Cancer Trials were investigated. Pre‐RCT and post‐RCT CD133 expression levels were determined using immunohistochemistry and were correlated with histopathologic parameters, tumor regression grade, cancer recurrence, and patient survival.

RESULTS:

Compared with pre‐RCT biopsies, significantly higher CD133 expression was observed in tumor specimens (P = .01). However, no correlations were observed for either biopsies or tumor specimens between CD133 expression levels, histopathologic characteristics, or survival. In matched analyses of corresponding biopsy/tumor pairs, patients who had an increased fraction of CD133‐expressing (CD133+) cells after preoperative RCT had significantly higher residual tumor stages (P = .02) and lower histopathologic tumor regression (P < .01). Moreover, these patients had significantly reduced disease‐free survival and cancer‐specific overall survival in univariate analysis (P < .001 and P = .004, respectively) and multivariate analysis (P = .003 and P = .024, respectively).

CONCLUSIONS:

The enrichment of CD133+ cancer cells during preoperative RCT was correlated with minor local tumor response, increased distant cancer recurrence, and decreased survival. The current results indicate that the up‐regulation of intratumoral CD133 expression, in contrast to absolute pre‐RCT and post‐RCT CD133 levels, plays an important role in tumor progression and metastasis in patients with rectal cancer who are receiving neoadjuvant RCT. Cancer 2013. © 2012 American Cancer Society.     

Development of clinically relevant orthotopic xenograft mouse model of metastatic lung cancer and glioblastoma through surgical tumor tissues injection with trocar

Objective

Orthotopic models are important in cancer research. Here we developed orthotopic xenograft mouse model of metastatic lung cancer and glioblastoma with a specially designed system.

Methods

Tiny fragments of surgical tumors were implanted into the mice brain with a trocar system. Immunohistochemistry was performed to detect brain tumor stem cells among glioblastoma tissues, including both the original and resulting ones with monoclonal antibody against CD133.

Results

Besides the constant high take rates in both models; brain transplants perfectly resembled their original tumors in biological behaviors. The brain tumor stem cells, positively stained with CD133 were found, though not frequently, in both original and resulting glioblastoma tissues.

Conclusions

Orthotopic model established with a trocar system is effective and injection of tumor tissues containing stem cells promise the forming of new tumor mass when grafted.     

Modulation of invasive properties of CD133(+) glioblastoma stem cells: A role for MT1‐MMP in bioactive lysophospholipid signaling

Future breakthroughs in cancer therapy must accompany targeted agents that will neutralize cancer stem cells response to circulating growth factors. Since the brain tissue microenvironmental niche is a prerequisite for expression of the stem cell marker CD133 antigen in brain tumors, we investigated the invasion mechanisms specific to CD133(+) U87 glioblastoma cells in response to lysophosphatidic acid (LPA) and sphingosine 1‐phosphate (S1P), two circulating bioactive lysophospholipids and potent inducers of cancer. A CD133(+) U87 glioma cell population was isolated from parental U87 glioblastoma cells using magnetic cell sorting technology. The CD133(+)‐enriched cell population grew as neurospheres and showed enhanced maximal response to both LPA (～5.0‐fold) and S1P (～2.5‐fold) at 1 µM when compared to parental U87 cells. The increased response to LPA in CD133(+) cells, reflected by increased levels of phosphorylated ERK, was found independent of the cooperative functions of the membrane‐type‐1 matrix metalloproteinase (MT1‐MMP), while this cooperativity was essential to the S1P response. Quantitative RT‐PCR was performed and we found higher gene expression levels of the S1P receptors S1P1 and S1P2, and of the LPA receptor LPA1 in CD133(+) cells than in their parental U87 cells. These increased levels reflected those observed from in vivo experimental U87 tumor implants. Our data suggest that the CD133(+) cell subpopulation evokes most of the lysophospholipid response within brain tumors through a combined regulation of S1P/LPA cell surface receptors signaling and by MT1‐MMP. The emergence of lead compounds targeting the stem cell niche and S1P/LPA signaling in CD133(+) cancer cells is warranted. © 2009 Wiley‐Liss, Inc.     

CD133/prominin1 is prognostic for GBM patient's survival, but inversely correlated with cysteine cathepsins' expression in glioblastoma derived spheroids

Introduction

CD133 is a marker for a population of glioblastoma (GBM) and normal neural stem cells (NNSC). We aimed to reveal whether the migratory potential and differentiation of these stem cells is associated with CD133 expression and with cathepsin proteases (Cats).

Materials and methods.

The invasiveness of normal NNSC, GBM/CD133+ cell lines and GBM spheroids was evaluated in 3D collagen, as well as of U87-MG and normal astrocytes (NHA) grown in monolayers in 2D Matrigel. Expression of Cats B, L and S was measured at mRNA and activity levels and their relation to invasiveness, to CD133 mRNA in 26 gliomas, and to the survival of these patients.

Results

The average yield of CD133+ cells from GBM samples was 9.6 %. Survival of patients with higher CD133 mRNA expression was significantly shorter (p< 0.005). Invasion, associated with proteolytic degradation of matrix, was higher in normal stem cells and GBM spheroids and cells than in isolated GBM CD133+ cells. In glioma samples, there was no correlation between CD133 mRNA expression and Cat mRNAs, but there was an inverse correlation with Cat activities.

Conclusions

The study confirms CD133 as a prognostic marker for the survival of GBM patients. We demonstrated that NNSC have higher invasion potential and invade the collagen matrix in a mode different from that of GBM, initiating stem cell spheres. This result could have implications for the design of new therapeutics, including protease inhibitors that specifically target invasive tumour stem cells. Increased activity of cathepsins in CD133– cells suggests their role in the invasive behaviour of GBM.     

HPV‐avertable cancer risks in India: A pooled analysis of 9 observational studies

Recent advances in cancer stem cell biology have shown that cancer stem‐like cells with epithelial–mesenchymal transition (EMT) phenotypes are more aggressive and cause relapse; however, absence of a specific marker to isolate these EMT stem‐like cells hampers research in this direction. Cell surface markers have been identified for isolating cancer stem‐like cells, but none has been identified for isolating cancer stem‐like cells with EMT phenotype. Recently, we discovered that Vimentin, an intracellular EMT tumor cell marker, is present on the surface of colon metastatic tumor nodules in the liver. In our study, we examined the potential of targeting cell surface Vimentin (CSV) to isolate stem‐like cancer cells with EMT phenotype, by using a specific CSV‐binding antibody, 84‐1. Using this antibody, we purified the CSV‐positive, CD133‐negative (csVim⁺CD133⁻) cell population from primary liver tumor cell suspensions and characterized for stem cell properties. The results of sphere assays and staining for the stem cell markers Sox2 and Oct4A demonstrated that csVim⁺CD133⁻ cells have stem‐like properties similar to csVim⁻CD133⁺ population. Our investigation further revealed that the csVim⁺CD133⁻ cells had EMT phenotypes, as evidenced by the presence of Twist and Slug in the nucleus, the absence of EpCAM on the cell surface and basal level of expression of epithelial marker E‐cadherin. The csVimentin‐negative CD133‐positive stem cells do not have any EMT phenotypes. csVim⁺CD133⁻ cells exhibited more aggressively metastatic in livers than csVim⁻CD133⁺ cells. Our findings indicate that csVim⁺CD133⁻ cells are promising targets for treatment and prevention of metastatic hepatocellular carcinoma.     

Correlation between the prognostic value and the expression of the stem cell marker CD133 and isocitrate dehydrogenase1 in glioblastomas

Cancer stem cells are thought to be responsible for tumor recurrence and resistance in glioblastomas. An isocitrate dehydrogenase1 (IDH1) mutation, affecting codon132 of the isocitrate dehydrogenase1 gene, has prognostic significance in glioblastomas. We investigated whether stem cell marker expression [CD133, CD34, and vascular endothelial growth factor (VEGF)] and IDH1 mutation correlate with clinical factors and prognosis in glioblastoma. CD133, CD34, and VEGF expression was evaluated by immunohistochemistry in 67 cases of glioblastoma identified between 2005 and 2012. IDH1 mutation was assessed by immunohistochemistry, peptide-nucleic-acid mediated PCR clamping, and direct gene sequencing. Diffuse CD133 expression was detected in 12 (17.9 %) cases and was associated with poor overall survival (OS) (P = 0.010) and progression-free survival (P = 0.017). CD34 and VEGF expression were not associated with prognosis in these samples. IDH1 mutation was detected in ten (14.9 %) cases. Eight were clinically secondary tumors and two were primary tumors (P < 0.001); the mean age of the secondary tumor patients was significantly younger (P = 0.001, 41.20 vs. 59.14). IDH1-positive patients had longer OS than IDH1-negative patients (25.78 vs. 22.95 months), but this difference was not significant. In addition, IDH1 and CD34 expression showed a negative correlation (P = 0.024). Multivariate analysis showed that age, extent of surgery, and diffuse CD133 expression correlated with OS. CD133 may be a survival marker for glioblastoma. Further characterization of CD133, IDH1, and vascular markers in glioblastoma may help identify new therapeutic targets.     

Impact of CD133 positive stem cell proportion on survival in patients with glioblastoma multiforme

Background

The aim of the study was to assess the impact of CD133-positive (CD133+) cancer stem cell proportions on treatment results of glioblastoma multiforme (GBM) patients.

Patients and methods

Patients with GBM (n = 42) received postoperative radiotherapy (± chemotherapy). Surgically excised GBM tissue sections were immunohistochemically examined for CD133 expression. The proportions of CD133+ GBM cells were determined (%). The proportion of CD133+ GBM stem cells was established by 2 independent researchers whose results were in good accordance (R = 0.8, p < 0.01). Additionally, CD133 expression levels were correlated with patients overall survival.

Results

The proportion of CD133+ cells varied between patients, being from 0.5% to 82%. Mean and median proportions of CD133+ cells of the entire study group were 33% ± 24% (mean ± SD) and 28%, respectively. Clinical data do not support the association between higher proportion of stem cells and the aggressiveness of GBM. Median survival time of the study group was 10.0 months (95% CI 9.0–11.0). The survival time clearly depended on the proportion of CD133+ cells (log rank test, p = 0.02). Median survival times for patients with low (< median) and high (≥ median) proportion of CD133+ cells were 9.0 months (95% CI 7.6–10.5) and 12.0 months (95% CI 9.3–14.7), respectively. In multivariate analysis, the proportion of CD133+ cells emerged as a significant independent predictor for longer overall survival (HR 2.0, 95% CI 1.0–3.8, p = 0.04).

Conclusions

In patients with higher stem cell proportion, significantly longer survival times after postoperative radiotherapy were achieved. Underlying reasons and possible higher sensitivity of GBM stem cells to fractionated radio-therapy should be clarified in further studies.     

CD133~+胶质母细胞瘤细胞的分离与恶性行为特征

背景与目的:在恶性胶质瘤中存在一类恶性度极高的前体细胞,它们控制着肿瘤的生长与恶性演进,也是肿瘤复发的根源。本研究对胶质母细胞瘤进行肿瘤干细胞的分离与恶性行为特征分析,旨在验证胶质瘤干细胞的存在,并为未来该领域研究打下基础。方法:充分消化术中切除的胶质母细胞瘤组织块至单细胞悬液,流式细胞术检测CD133+肿瘤细胞百分率,免疫磁珠分选法分离CD133+和CD133-胶质母细胞瘤细胞;免疫荧光法检测两类细胞中巢蛋白(nestin)、胶质纤维酸性蛋白(GFAP)和神经元特异性烯醇化酶(NSE)的表达水平;Transwell双室培养体系检测两类瘤细胞的侵袭能力,血清依赖实验检测两类瘤细胞在低营养条件下的生存能力,软琼脂克隆形成实验检测瘤细胞的克隆形成能力;去胸腺小鼠腹股沟皮下接种瘤细胞,观察成瘤率。结果:流式细胞术检测CD133+肿瘤细胞百分率为(2.31±0.57)%。CD133+细胞高表达nestin,低表达GFAP和NSE;CD133-细胞则相反。与CD133-细胞比较,CD133+细胞具有更高的细胞侵袭能力、低营养耐受能力和克隆形成能力。CD133+细胞在小鼠体内成瘤率为87%(26/30),CD133-细胞为7%(2/30)。结论:CD133+胶质母细胞瘤细胞具有更高的恶性细胞表型,提示其具有高度研究价值。     

Isolation and characterization of cancer stem like cells in human glioblastoma cell lines

To identify and compare the features of stem like cells in human glioblastoma cell lines U251, U87MG, A172 with primary cultured glioblastoma stem cells, the ratio of CD133⁺ cells, the ability of tumor sphere formation, and self-renewing capacity of U251, U87MG, A172 cells in serum free medium plus EGF, bFGF and B27 supplement were detected. The results suggested that there might be more cancer stem like cells in U251 cells compared with others. CD133⁺ cells enriched in SP cells and in U251 cells cultured with the serum free medium. They expressed the neural stem cell markers CD133 and Nestin, but lacked of neuronal and astrocyte marker MAP2, β-III tubulin and GFAP. They could apparently generate both neurons and glial cells after serum retrieved in vitro. Gli1, Bmi1, Notch2 and PTEN were also found expressed highly in them. Moreover, CD133⁺ cells were more resistant to hypoxia, irradiations and some chemotherapeutics than CD133⁻cells. So we suggested that glioblastoma stem like cells were existed in CD133⁺ cells in U251 cell line with characteristics of self-renew and generation of an unlimited progeny of non-tumorigenic cells. Molecular and functional characterization of such a tumorigenic population may be exploited in the development of novel cancer therapeutic drugs.     

CD133 is indicative for a resistance phenotype but does not represent a prognostic marker for survival of non‐small cell lung cancer patients

Despite advances in anticancer treatment, lung cancer still has poor prognosis. Recently, a cancer stem cell (CSC) hypothesis has emerged describing a small subset of tumor cells with stem cell properties. CSCs found in many solid tumors express CD133 antigen on the cell surface. The presence of CSC is correlated with poor survival of patients with glioblastomas, colon or prostate cancers. In this study, we evaluated whether CD133 expression in non‐small cell lung cancer (NSCLC) has a prognostic value in patients' survival. We also analyzed whether CD133 positivity of NSCLC correlates with the expression of resistance‐related proteins, angiogenic factors, oncogenes, proliferative activity or apoptosis. CD133 expression was retrospectively examined in a total of 88 cases of previously untreated NSCLC by immunohistochemistry. We found no correlation between CD133 positivity or the amount of CD133⁺ cells with NSCLC patients' survival, expression of oncogenes c‐myc, c‐N‐ras, c‐jun, c‐fos, c‐erbB1, c‐erbB2 or p53, angiogenic factors VEGF, VEGFR‐1, FGF, FGFR‐1, tissue factor and with proliferative activity or apoptosis in NSCLC tissues. However, there was a significant association between the expression of resistance‐related proteins glutathione S‐transferase, thymidylate synthase, catalase, O⁶‐methylguanine‐DNA methyltransferase and p170 and CD133. Because CD133 expression is linked to a resistant phenotype, detection of CD133⁺ cells may be useful to predict efficacy of cytotoxic therapy but CD133 is not a strong prognostic parameter for survival of patients with NSCLC.     

Identification and characterization of cancer stem cells in ovarian yolk sac tumors

Recent evidence supports the cancer stem cell theory, that is, that malignant tumors arise from cells termed cancer stem cells or tumor‐initiating cells that have the ability to self‐renew and are responsible for maintaining the tumor. Cells with marked tumor‐initiating capacity have recently been identified in a number of solid tumors. CD133 (PROM1, human prominin‐1) has been used as a marker to detect stem cells (progenitor cells) and cancer stem cells (tumor‐initiating cells) in various tissues. Ovarian yolk sac tumors (YSTs) are rare and highly malignant. The present study was designed to evaluate the tumor‐forming ability of CD133⁺ cells in ovarian YST cell lines and to examine the characteristics of CD133⁺ cells, such as cell growth and invasiveness. Our data suggest ovarian YST to be maintained by a rare fraction of cancer stem‐like cells that express the cell surface marker CD133. (Cancer Sci 2010)     

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。