基质金属蛋白酶-2和-9对恶性胶质瘤干细胞侵袭作用的影响
|
摘要
前言与目的
恶性胶质瘤是最常见的原发性中枢神经系统恶性肿瘤,肿瘤细胞迅速增殖、高度侵袭以及新生血管生成是胶质瘤的基本特征。其中,肿瘤细胞高度侵袭是胶质瘤高死亡率的主要原因,同时也是胶质瘤的典型的生物学标志。胶质瘤细胞侵袭的过程主要包括肿瘤细胞粘附到细胞外基质(Extracellular matrix,ECM),ECM成分的降解,以及随后的肿瘤细胞穿透进入邻近的脑组织。围绕着肿瘤侵袭机制的研究一直以来就是胶质瘤研究的重点。目前观点认为,细胞外基质降解酶系统如基质金属蛋白酶家族(Matrix metalloproteinases,MMPs)等参与了ECM的降解过程。MMPs是锌离子依赖的限制性内切酶,MMPs家族在各种生理病理条件下的ECM的降解过程中均发挥了重要的作用。许多研究表明MMPs能够促进肿瘤细胞侵袭入正常组织和促进肿瘤的转移,MMPs表达水平与恶性胶质瘤的侵袭程度密切相关。其中,由于明胶酶(MMP-2,MMP-9)能够裂解Ⅳ型胶原—构成基底膜的主要成分,因此其在胶质瘤侵袭机制中的作用研究日益成为研究的热点。
越来越多的证据表明,在胶质瘤内部存在着一组具有干细胞特性的小的细胞集群-肿瘤干细胞(Cancer Stem Cells,CSCs),胶质瘤CSCs具有自我更新、多向分化以及体内成瘤的能力,可以分化成多种细胞成分,参与肿瘤的发生发展机制,并且与肿瘤的化疗耐药性及放疗抗性密切相关,因此在胶质瘤的发生发展进程中发挥了决定性的作用。但是就胶质瘤CSCs本身的侵袭特性的研究尚未见报道。本研究中我们成功分离并鉴定多形性胶质母细胞瘤(Glioblastoma Multiforme,GBM)细胞系U87、9L、原代培养细胞系GBM1的CSCs;探讨胶质瘤CSCs与非肿瘤干细胞(non-CSCs)的侵袭情况,明确两种细胞亚群的侵袭能力的差异;以及MMP-2、MMP-9表达在GBM的CSCs侵袭机制中的作用,为以CSCs为靶点的胶质瘤治疗方案的筛选提供有力的理论和实验依据。
方法
1实验材料
肿瘤组织取自洛杉矶Cedars-Sinai医学中心神经外科手术标本,经病理诊断证实为GBM。GBM细胞系U87、9L细胞系购自美国ATCC(American Tumor CellCollection)。DMEM/F12培养液、B27(一种干细胞培养补充剂)购自Invitrogen公司,肝素(Heparin)购自Sigma公司,人表皮生长因子(Epidermal Growth Factor,EGF)和碱性成纤维生长因子(basic Fibroblast Growth Factor,bFGF)购自R&DSystems公司。盒式载玻片(Chamber Slides)购自Electron Microscopy Sciences公司。免疫荧光抗体采用:小鼠抗人CD133单抗(1:10,Milteny Biotec),小鼠抗人nestin单抗,小鼠抗人β-mbulinⅢ单抗(1:200,Chemicon),兔抗人GFAP多抗,小鼠抗人myelin/oligodendrocyte单抗(1:1000,Chemicon),Cy3标记的兔抗小鼠或山羊抗兔二抗(1:200,Jackson Immuno Reserech)。细胞侵袭分析试剂盒(ECM550,聚碳酸酯膜,8-μm孔径)购自Chemicon公司(Temecula,CA)。免疫组织化学染色采用:小鼠抗人MMP-2和MMP-9单克隆抗体购自Calbiochem公司(SanDiego,CA)。6-8周龄的免疫缺陷裸鼠、成年雄性F344大鼠(重量200g～220 g),由洛杉矶Cedars-Sinai医学中心实验动物中心提供。
2实验方法
2.1 CSCs培养分离:肿瘤标本经清洗、剪碎、胰酶消化,滤网过滤收集细胞。淋巴细胞分离液除去其中的红细胞,U87、9L细胞系细胞先用含10%胎牛血清(Fetal Bovine Serum,FBS)的DMEM/F12培养液培养扩增,后转至含EGF和bFGF的无血清培养液(DMEM/F12)培养,形成细胞球。
2.2有限稀释法:收集细胞球细胞稀释后培养观察单细胞克隆能力,检测细胞球细胞的自我更新能力。细胞球再经胰酶消化成单细胞,于盒式载玻片中应用含血清培养基进行培养,观察细胞球细胞的多向分化情况。
2.3免疫荧光染色:细胞球细胞及分化的肿瘤细胞固定后,分别滴加干细胞表面特异性标记物,Cy3标记二抗,荧光显微镜下观察细胞分化情况。
2.4细胞颅内种植:应用Stoeling脑立体定向注射仪,将50,000个细胞球细胞或非细胞球细胞定向注入裸鼠右侧纹状体。21天后处死裸鼠,取脑组织观察评价不同亚群肿瘤细胞的颅内成瘤能力。
2.5体外细胞侵袭能力的测定:分别收集细胞球细胞和非细胞球细胞,应用细胞侵袭分析试剂盒,计数侵袭到滤膜背面的穿膜细胞数,以穿膜细胞的数目表示细胞侵袭力的大小。
2.6 H&E染色:如上述方法进行颅内种植,分别于接种后不同时间点14天、21天、28天处死裸鼠,制作16-μm脑组织冰冻切片,观察颅内新生肿瘤的浸润和侵袭性生长情况。
2.7免疫组织化学:冰冻切片,应用MMP-2、MMP-9抗体,采用SABC法检测MMP-2、MMP-9在裸鼠脑组织内的表达情况。
2.8实时定量RT-PCR:采用RNeasy试剂盒(QIAGEN)分别提取细胞及组织的总RNA,逆转录成cDNA后,应用Icycler PCR仪(BIO-RAD,Hercules,CA)行PCR反应。相对mRNA表达水平对比两种细胞亚群的基因表达差异,以及新生脑肿瘤组织中的MMP-2和MMP-9的表达差异。
2.9 Western blot检测:提取细胞及组织的总蛋白,紫外分光光度法蛋白定量。4-20%Tris-甘氨酸SDS-PAGE胶中(Invitrogen)电泳,转印至PVDF膜后,分别加入MMP-2和MMP-9一抗,后加入相应的HRP标记的二抗,显色系统显色。β-actin为内参照。
2.10统计学分析:统计学处理应用SPSS13.0软件。采用双尾Student's t检验,数值以均数±标准差表示。以P＜0.05为有统计学意义。
实验结果
1 CSCs特性分析显示:原代细胞球细胞在有限的传代条件下形成了2代细胞球;表达了神经干细胞(Neural Stem Cells,NSCs)的标记物(CD133和Nestin);这些细胞能够分化成为多种同源子细胞(神经元、星形细胞、少突胶质细胞);经裸鼠颅内种植后形成了新生肿瘤。相反,小于0.5%的非细胞球细胞显示CD133和Nestin的阳性表达,且不具备自我更新、多向分化及颅内成瘤的特性。基于以上结果,我们将这两种细胞亚群定义为:肿瘤干细胞(CSCs)和非肿瘤干细胞(non-CSCs)。
2不同细胞亚群的体外侵袭能力评估显示:CSCs的穿膜细胞数明显多于non-CSCs(GBM1:1106±45,450±12;U87:1194±71,471±22;9L:1270±108,421±13),其差异有统计学意义(P＜0.01)。且前者在穿膜之后再次形成细胞球。
3 MMP-2和MMP-9在不同细胞亚群中的表达
1)实时RT-PCR结果显示,在三个肿瘤细胞组中,CSCs的CD133、Nestin、MMP-2 mRNA的表达水平明显高于non-CSCs(以non-CSCs的值设定为1),CD133(27.6±2.92;23.4±3.57;24.5±1.21);Nestin(22.3±1.12;21.7±1.78;23.8±4.27);MMP-2(297.4±25.3;193.7±31.2;201.6±19.9),但是MMP-9的mRNA表达水平在两个细胞亚群中无明显差异,而且其表达水平显著低于MMP-2 mRNA(5.4±0.47;4.9±0.32;5.1±0.39)。
2) Western blot结果显示,在三组CSCs亚群中,MMP-2表达均为阳性,MMP-9仅在GBM1组的CSCs中有微弱表达;在三组non-CSCs亚群中,MMP-2均为阳性表达且表达强度弱于CSCs组,而MMP-9均为阴性表达。
4不同细胞亚群体内侵袭情况比较
在三个肿瘤细胞组中,经细胞球细胞接种的裸鼠脑组织内均形成高度增殖和侵袭的肿瘤,并伴有广泛的血管生成。相反,在非细胞球细胞接种的动物组中,除在6只9L细胞接种裸鼠中有2只形成了小的颅内肿瘤外,其余实验动物均未形成肿瘤。
5 MMP-2和MMP-9在颅内新生肿瘤组织中的表达
1)免疫组化结果显示,MMP-2阳性表达定位于正常脑组织和肿瘤组织中,尤其特异性表达于肿瘤的中心;相反,MMP-9阳性表达定位在肿瘤的边缘,且在正常脑组织中极少表达。
2)实时RT-PCR结果显示,在正常脑组织中,MMP-2 mRNA有微弱表达(每组中的正常脑组织表达均设为参照值1),而MMP-9 mRNA未见表达;在肿瘤组织中,MMP-2和MMP-9 mRNA表达水平均随肿瘤生长时间明显升高,尤其28天组较14天组其差异有统计学意义(P＜0.0001)(14d/28d:4.96±0.09/27.58±1.07;5.2±0.43/27.09±1.68;3.71±0.56/26.61±1.29),而且MMP-9 mRNA表达水平的升高比例高于MMP-2(MMP-9/MMP-2:2.14±0.25,P＜0.001)。
3) Western blot结果显示,MMP-2在肿瘤组织中表达阳性,在正常脑组织中微弱表达;相反,MMP-9仅在肿瘤组织中阳性表达。与RT-PCR结果一致,两者的表达水平随肿瘤生长而明显升高,特别是将28天组与14天组进行对比,MMP-9的表达变化尤为明显。
结论
1 GBM细胞球细胞具有CSCs的特性,具有自我更新、多向分化以及体内成瘤的能力可以作为理想的CSCs进行深入研究。
2在体内及体外条件下,胶质瘤CSCs表现出较non-CSCs明显增强的侵袭能力。胶质瘤CSCs极大地促进了肿瘤的侵袭能力,至少部分是因为胶质瘤CSCs同时拥有了肿瘤细胞和NSCs的特性,它们在胶质瘤的成瘤过程中发挥了关键的作用,胶质瘤CSCs也许是通过促进肿瘤细胞的侵袭从而促进肿瘤的形成和进展。
3胶质瘤CSCs在体外条件下,主要是通过上调MMP-2的表达促进肿瘤细胞的侵袭和迁移;而在体内条件下,MMP-9对肿瘤的侵袭性生长也许起到了更为重要的作用。因此,胶质瘤CSCs也许是通过明胶酶依赖的机制在不同的条件下促进了肿瘤细胞的侵袭和肿瘤的侵袭性进展。
Introduction
Malignant gliomas are the most common and most aggressive primary brain tumors that respond poorly to current treatment strategies of cytoreductive neurosurgery, involved field radiotherapy,and adjuvant chemotherapy,which are characterized by rapid cell proliferation,a high level of invasiveness into the surrounding brain and increased vascularity.The capability of glioma cells to invade extensively within the central nervous system is a major cause of the high morbidity rate of primary malignant brain tumors.The invasion of tumor cells into brain tissue is a pathologic hallmark of WHO gradesⅡ-Ⅳgliomas and contributes significantly to the failure of current therapeutic treatments.Glioma cell invasion involves the attachment of tumor cells to extracellular matrix(ECM),degradation of ECM components,and subsequent penetration into adjacent brain structures.These processes are accomplished in part by matrix metalloproteinases(MMPs),a family of zinc-dependent proteinases.The members of the MMPs family have the ability to degrade macromolecules of the ECM and are responsible for tumor invasion and infiltration,limiting the effectiveness of the neurosurgical resection of brain tumors.Recent reports have suggested that expressions of gelatinase-A(MMP-2) and gelatinase-B(MMP-9) play an important role in neoplastic tissue invasion or metastasis,since these are critical factors in basement membrane degradation.A better understanding of glioma invasion mechanism will help in developing therapeutic strategies to decrease the invasion of gliomas.
Recently,a large body of evidence has assigned a key role in tumor formation and progression to cancer stem cells(CSCs) by virtue of its ability to self-renew,and differentiate into subpopulations of cells similar to those present in the initial tumor, more importantly,the isolated CSCs are able to form tumors after in vivo implantation. Also glioma CSCs participate in cancer chemoresistance and redioresistance mechanism.To date,invasion has been defined as an important mechanism in glioma tumorigenesis,However,few reports were involved in invasiveness characteristic of glioma CSCs,therefore we isolated and identified glioma CSCs from primary cultured GBM cells-GBM1 and GBM cell lines U87,9L,examined whether glioma CSCs possess potent invasiveness potential and determine tumor progression through promoting tumor invasive processes,and detected the expressions of MMP-2 and -9 in different subpopulations and xenografts in brain tumor models.Here,we reported that malignant glioma CSCs might provide a powerful enhancement for the invasion of glioma through a gelatinases-dependent mechanism.Targeting gelatinases specifically on glioma CSCs may provide a new insight and method for patient therapy by inhibiting glioma invasiveness.
Methods
Cells and culture process
Tumor specimen was obtained from surgical resection of one GBM patient,as approved by the Institute Review Boards at the Neurosurgery Department of Cedars-Sinai Medical Center,LA,CA.Tumor tissue was washed,minced,and enzymatically dissociated.U87 cell line and 9L cell line was purchased from ATCC.Tumor cells were resuspended in DMEM/F12 medium containing 10%fetal bovine serum(FBS) Subsequently monolayer-growing tumor cells were cultured in DMEM/F12 supplemented with epidermal growth factor(EGF),and basic fibroblast growth factor(bFGF)(R&D Systems,Minneapolis,MN).
Subsphere-forming and differentiation assay
The spheres were harvested,dissociated into single cells and were diluted to1-2 cells/well for the subsphere-forming assay Simultaneously,the monolayer cells left in the flasks after harvesting of the sphere were transferred into medium without growth factors but permissive for differentiation.
Immunocytostaining
Spheres and differentiated glioma cells were grown in precoated chamber slides and fixed,and then were stained with the following antibodies:anti-CD133(mouse monoclonal IgG1;1:10;Milteny Biotec),anti-nestin(mouse monoclonal IgG1;1:200; Chemicon),anti-β-tubulinⅢ(mouse monoclonal IgG1;1:200;Chemicon),anti-GFAP (rabbit polyclonal;1:1000;Chemicon),anti-myelin/oligodendrocyte(mouse monoclonal; 1:1000;Chemicon).The primary antibodies were detected with Cy3 or FITC-conjugated anti-mouse or anti-rabbit IgG antibodies(1:200,Jackson Immuno Reserch).
Establishment of in vivo glioma model
Six- to 8-week-old athymic nude mice and adult male F344 rats weighing between 200g and 220 g obtained from Medical Animal Center at Cedars-Sinai Medical Center,LA,CA, stereotactically inoculated with either purified glioma sphere cells or non-sphere cells (50,000/animal) in the right corpus striatum.
Invasion assays
Cell invasion assay kit(ECM 550,polycarbonate membrane,24-well plate with 12 cell culture inserts,8-μm pore size,Chemicon) was used.The number of invasive cells was counted as the indicator of glioma cell invasiveness.
Histological and immunohistochemical analysis of glioma-bearing brain sections
The animals were euthanized 6 weeks after implantation.Brains harvested from tumor cells implanted animals were frozen into sections,which were stained with H&E staining.Also staining were performed using primary antibodies against MMP-2 and MMP-9(1:50,mouse monoclonal IgG,Calbiochem).
Xenografts analysis
Matched sphere cells or non-sphere cells were implanted intracranially with 50,000 cells per animal as described previously.The animals were euthanized on 14,21 and 28 day post-implantation,respectively.
Real-time Quantitative Reverse transcription-PCR
Sphere cells and non-sphere cells from three glioma cell lines,and from xenografts of brain tumor models were respectively subjected to total RNA extraction with RNeasy kit (QIAGEN) and reverse transcripted into cDNA.The real-time PCR for CD 133,Nestin, MMP-2 and MMP-9 was performed.The relative mRNA level of these genes was compared between sphere cells and non-sphere calls,as well gelatinases were detected in normal brain tissues and tumor tissues derived from different time points post-implantation,respectively.
Protein analysis
Western blots contained 20μg protein extracted from glioma cells,and tumor tissues or normal brain tissues derived from xenografts on a 4-20%Tris/glycine SDS-PAGE gel (Invitrogen) and were transferred to 0.45-μm PVDF(Invitrogen).Blots were incubated with antibodies directed against MMP-2 and MMP-9,and in HRP-conjugated IgG.Blots were developed and exposed to film.β-actin as control.
Statistical analysis
Statistical analyses were done using SPSS 13.0.Data graphed with error bars represent mean and SD.A two-sided Student's t test was used to determine the significance of any differences.P＜0.05 as statistical significance.
Results
Isolation and identification of glioma CSCs
Primary cultured sphere glioma cells formed subspheres under limiting passage conditions consistent with self renewal,expressed neural stem cell markets(CD133, nestin),and generated multiple-lineage daughter cells,and formed a neoplasm after intracranial implantation.In contrast,few cells of the non-sphere forming tumor cells in the monolayer culture stained positive for CD133 and nestin.Together these results fulfilled the criteria of two distinct tumor cell subpopulations:CSCs and non-CSCs.
Glioma CSCs presented increased invasive ability combined with elevated levels of MMP-2,but not MMP-9 in vitro
To determine invasiveness capacity for different subpopulations in three glioma cell lines,respectively.The results showed that significantly more sphere cells invaded out of the membrane than non-sphere forming cells(P＜0.01),and single sphere cells formed new subspheres after they passed through ECM gel.
Real-time RT-PCR was used to detect gelatinases mRNA expression.MMP-2 mRNA expression in glioma CSCs ranged from 6.56- to 9.43-fold higher than that in autologous non-CSCs(P＜0.0001),however,only modest differences of MMP-9 mRNA expression between CSCs and non-CSCs subpopulations were observed in three glioma cell lines(P＞0.05),moreover the mRNA expression levels of MMP-9 were lower than those of MMP-2 strikingly,especially in glioma CSCs subpopulation.
Western blot results showed markedly increased secretion of MMP-2 from glioma CSCs versus non-CSCs subpopulation,but except a trace of expression in CSCs subpopulation derived from GBMI cell,MMP-9 expression wasn't detected in other subpopulations.
Tumors derived from sphere glioma cells showed increased tumor invasiveness and proliferation
Regardless of the differing origins of glioma cell lines,the brains implanted with sphere tumor cells presented highly proliferative and invasive neoplasms with extensive vascularity.However,among the animals implanted with non-sphere tumor cells,only 2 of 6 rats implanted with 9L cell line formed small neopalsms,in other brains tumors weren't formed 6 weeks after implantation.
The expression of MMP-2 and MMP-9 were highly up-regulated in brain tumors derived from glioma CSCs
Immunohistochemical results showed,regardless of tumor cell source,MMP-2 was expressed both in normal brain tissues and tumor tissues,and in tumor tissues, MMP-2 was present dominantly in the center of tumor,in contrast,MMP-9 expression was present in very low levels in normal brain tissues,but in the tumors it was greatly restricted to regions at the infiltrating and invading border of the tumor.
Real-time RT-PCR results revealed that the mRNA expression levels of MMP-9 were very low and only a trace of expression of MMP-2 mRNA was detected in normal brain tissues;the expression of MMP-2 and MMP-9 mRNA were markedly elevated in tumor tissues at 3 time points post-implantation with the growth of malignant glioma. The mRNA expression levels of both MMP-2 and MMP-9 at 28 days were striking increased compared with that post-implantation for 14 days.However,the growth rate of MMP-9 mRNA level along with the time after implantation was higher than that of MMP-2,especially on 28 days versus on 14 days post-implantation.
Western blot assay showed that MMP-2 was detected at 72 kDa in both tumor tissues and normal brain tissues,but the expression in tumor tissues was much higher than that in normal brain tissues.MMP-9 was detected at 92 kDa in tumor tissues,but not in normal brain tissues except modest expression in those derived from U87 sphere cells.In addition,western blot results showed increased secretion of MMP-2 and MMP-9 along with the time post sphere tumor cell implantation.Both MMP-2 and MMP-9 expressions 28 days post-implantation were evidently increased compared to those from 14 days,particularly for MMP-9.
Conclusions
1 In our study,we confirmed cultured sphere glioma cells from glioma cell lines formed new spheres with the capacity for self renewal,expressed gene associated with neural stem cells,generated daughter cells of different phenotypes from one mother cell, and formed tumors after intracranial implantation both in mice and rats.These sphere cells could be used for further study as optimal CSCs.
2 Our findings above suggest the fact that glioma CSCs enhance the invasiveness potential of malignant glioma,at least in part,is because the small subpopulation shares both characteristics of glioma tumor cells and NSCs simultaneously.Our results of CSCs inducing the increased invasiveness in cell invasion assay and in xenograft evaluations extend these findings of a key role of CSCs in glioma invasive tumorigenesis.Glioma CSCs maybe contribute to tumor progression through promoting tumor invasion.
3 Together these results suggest that glioma CSCs can up-regulate the expression of MMP-2 that promote their invasion and migration in vitro.Also of interest was our finding that MMP-9 might exhibit stronger invasion and infiltration capability in glioma tumorigenesis than MMP-2 in vivo.Glioma CSCs may possess increased invasiveness ability and enhance tumor invasive progression through gelatinases-related mechanism under different circumstances.
恶性胶质瘤是最常见的原发性中枢神经系统恶性肿瘤,肿瘤细胞迅速增殖、高度侵袭以及新生血管生成是胶质瘤的基本特征。其中,肿瘤细胞高度侵袭是胶质瘤高死亡率的主要原因,同时也是胶质瘤的典型的生物学标志。胶质瘤细胞侵袭的过程主要包括肿瘤细胞粘附到细胞外基质(Extracellular matrix,ECM),ECM成分的降解,以及随后的肿瘤细胞穿透进入邻近的脑组织。围绕着肿瘤侵袭机制的研究一直以来就是胶质瘤研究的重点。目前观点认为,细胞外基质降解酶系统如基质金属蛋白酶家族(Matrix metalloproteinases,MMPs)等参与了ECM的降解过程。MMPs是锌离子依赖的限制性内切酶,MMPs家族在各种生理病理条件下的ECM的降解过程中均发挥了重要的作用。许多研究表明MMPs能够促进肿瘤细胞侵袭入正常组织和促进肿瘤的转移,MMPs表达水平与恶性胶质瘤的侵袭程度密切相关。其中,由于明胶酶(MMP-2,MMP-9)能够裂解Ⅳ型胶原—构成基底膜的主要成分,因此其在胶质瘤侵袭机制中的作用研究日益成为研究的热点。
越来越多的证据表明,在胶质瘤内部存在着一组具有干细胞特性的小的细胞集群-肿瘤干细胞(Cancer Stem Cells,CSCs),胶质瘤CSCs具有自我更新、多向分化以及体内成瘤的能力,可以分化成多种细胞成分,参与肿瘤的发生发展机制,并且与肿瘤的化疗耐药性及放疗抗性密切相关,因此在胶质瘤的发生发展进程中发挥了决定性的作用。但是就胶质瘤CSCs本身的侵袭特性的研究尚未见报道。本研究中我们成功分离并鉴定多形性胶质母细胞瘤(Glioblastoma Multiforme,GBM)细胞系U87、9L、原代培养细胞系GBM1的CSCs;探讨胶质瘤CSCs与非肿瘤干细胞(non-CSCs)的侵袭情况,明确两种细胞亚群的侵袭能力的差异;以及MMP-2、MMP-9表达在GBM的CSCs侵袭机制中的作用,为以CSCs为靶点的胶质瘤治疗方案的筛选提供有力的理论和实验依据。
方法
1实验材料
肿瘤组织取自洛杉矶Cedars-Sinai医学中心神经外科手术标本,经病理诊断证实为GBM。GBM细胞系U87、9L细胞系购自美国ATCC(American Tumor CellCollection)。DMEM/F12培养液、B27(一种干细胞培养补充剂)购自Invitrogen公司,肝素(Heparin)购自Sigma公司,人表皮生长因子(Epidermal Growth Factor,EGF)和碱性成纤维生长因子(basic Fibroblast Growth Factor,bFGF)购自R&DSystems公司。盒式载玻片(Chamber Slides)购自Electron Microscopy Sciences公司。免疫荧光抗体采用:小鼠抗人CD133单抗(1:10,Milteny Biotec),小鼠抗人nestin单抗,小鼠抗人β-mbulinⅢ单抗(1:200,Chemicon),兔抗人GFAP多抗,小鼠抗人myelin/oligodendrocyte单抗(1:1000,Chemicon),Cy3标记的兔抗小鼠或山羊抗兔二抗(1:200,Jackson Immuno Reserech)。细胞侵袭分析试剂盒(ECM550,聚碳酸酯膜,8-μm孔径)购自Chemicon公司(Temecula,CA)。免疫组织化学染色采用:小鼠抗人MMP-2和MMP-9单克隆抗体购自Calbiochem公司(SanDiego,CA)。6-8周龄的免疫缺陷裸鼠、成年雄性F344大鼠(重量200g～220 g),由洛杉矶Cedars-Sinai医学中心实验动物中心提供。
2实验方法
2.1 CSCs培养分离:肿瘤标本经清洗、剪碎、胰酶消化,滤网过滤收集细胞。淋巴细胞分离液除去其中的红细胞,U87、9L细胞系细胞先用含10%胎牛血清(Fetal Bovine Serum,FBS)的DMEM/F12培养液培养扩增,后转至含EGF和bFGF的无血清培养液(DMEM/F12)培养,形成细胞球。
2.2有限稀释法:收集细胞球细胞稀释后培养观察单细胞克隆能力,检测细胞球细胞的自我更新能力。细胞球再经胰酶消化成单细胞,于盒式载玻片中应用含血清培养基进行培养,观察细胞球细胞的多向分化情况。
2.3免疫荧光染色:细胞球细胞及分化的肿瘤细胞固定后,分别滴加干细胞表面特异性标记物,Cy3标记二抗,荧光显微镜下观察细胞分化情况。
2.4细胞颅内种植:应用Stoeling脑立体定向注射仪,将50,000个细胞球细胞或非细胞球细胞定向注入裸鼠右侧纹状体。21天后处死裸鼠,取脑组织观察评价不同亚群肿瘤细胞的颅内成瘤能力。
2.5体外细胞侵袭能力的测定:分别收集细胞球细胞和非细胞球细胞,应用细胞侵袭分析试剂盒,计数侵袭到滤膜背面的穿膜细胞数,以穿膜细胞的数目表示细胞侵袭力的大小。
2.6 H&E染色:如上述方法进行颅内种植,分别于接种后不同时间点14天、21天、28天处死裸鼠,制作16-μm脑组织冰冻切片,观察颅内新生肿瘤的浸润和侵袭性生长情况。
2.7免疫组织化学:冰冻切片,应用MMP-2、MMP-9抗体,采用SABC法检测MMP-2、MMP-9在裸鼠脑组织内的表达情况。
2.8实时定量RT-PCR:采用RNeasy试剂盒(QIAGEN)分别提取细胞及组织的总RNA,逆转录成cDNA后,应用Icycler PCR仪(BIO-RAD,Hercules,CA)行PCR反应。相对mRNA表达水平对比两种细胞亚群的基因表达差异,以及新生脑肿瘤组织中的MMP-2和MMP-9的表达差异。
2.9 Western blot检测:提取细胞及组织的总蛋白,紫外分光光度法蛋白定量。4-20%Tris-甘氨酸SDS-PAGE胶中(Invitrogen)电泳,转印至PVDF膜后,分别加入MMP-2和MMP-9一抗,后加入相应的HRP标记的二抗,显色系统显色。β-actin为内参照。
2.10统计学分析:统计学处理应用SPSS13.0软件。采用双尾Student's t检验,数值以均数±标准差表示。以P＜0.05为有统计学意义。
实验结果
1 CSCs特性分析显示:原代细胞球细胞在有限的传代条件下形成了2代细胞球;表达了神经干细胞(Neural Stem Cells,NSCs)的标记物(CD133和Nestin);这些细胞能够分化成为多种同源子细胞(神经元、星形细胞、少突胶质细胞);经裸鼠颅内种植后形成了新生肿瘤。相反,小于0.5%的非细胞球细胞显示CD133和Nestin的阳性表达,且不具备自我更新、多向分化及颅内成瘤的特性。基于以上结果,我们将这两种细胞亚群定义为:肿瘤干细胞(CSCs)和非肿瘤干细胞(non-CSCs)。
2不同细胞亚群的体外侵袭能力评估显示:CSCs的穿膜细胞数明显多于non-CSCs(GBM1:1106±45,450±12;U87:1194±71,471±22;9L:1270±108,421±13),其差异有统计学意义(P＜0.01)。且前者在穿膜之后再次形成细胞球。
3 MMP-2和MMP-9在不同细胞亚群中的表达
1)实时RT-PCR结果显示,在三个肿瘤细胞组中,CSCs的CD133、Nestin、MMP-2 mRNA的表达水平明显高于non-CSCs(以non-CSCs的值设定为1),CD133(27.6±2.92;23.4±3.57;24.5±1.21);Nestin(22.3±1.12;21.7±1.78;23.8±4.27);MMP-2(297.4±25.3;193.7±31.2;201.6±19.9),但是MMP-9的mRNA表达水平在两个细胞亚群中无明显差异,而且其表达水平显著低于MMP-2 mRNA(5.4±0.47;4.9±0.32;5.1±0.39)。
2) Western blot结果显示,在三组CSCs亚群中,MMP-2表达均为阳性,MMP-9仅在GBM1组的CSCs中有微弱表达;在三组non-CSCs亚群中,MMP-2均为阳性表达且表达强度弱于CSCs组,而MMP-9均为阴性表达。
4不同细胞亚群体内侵袭情况比较
在三个肿瘤细胞组中,经细胞球细胞接种的裸鼠脑组织内均形成高度增殖和侵袭的肿瘤,并伴有广泛的血管生成。相反,在非细胞球细胞接种的动物组中,除在6只9L细胞接种裸鼠中有2只形成了小的颅内肿瘤外,其余实验动物均未形成肿瘤。
5 MMP-2和MMP-9在颅内新生肿瘤组织中的表达
1)免疫组化结果显示,MMP-2阳性表达定位于正常脑组织和肿瘤组织中,尤其特异性表达于肿瘤的中心;相反,MMP-9阳性表达定位在肿瘤的边缘,且在正常脑组织中极少表达。
2)实时RT-PCR结果显示,在正常脑组织中,MMP-2 mRNA有微弱表达(每组中的正常脑组织表达均设为参照值1),而MMP-9 mRNA未见表达;在肿瘤组织中,MMP-2和MMP-9 mRNA表达水平均随肿瘤生长时间明显升高,尤其28天组较14天组其差异有统计学意义(P＜0.0001)(14d/28d:4.96±0.09/27.58±1.07;5.2±0.43/27.09±1.68;3.71±0.56/26.61±1.29),而且MMP-9 mRNA表达水平的升高比例高于MMP-2(MMP-9/MMP-2:2.14±0.25,P＜0.001)。
3) Western blot结果显示,MMP-2在肿瘤组织中表达阳性,在正常脑组织中微弱表达;相反,MMP-9仅在肿瘤组织中阳性表达。与RT-PCR结果一致,两者的表达水平随肿瘤生长而明显升高,特别是将28天组与14天组进行对比,MMP-9的表达变化尤为明显。
结论
1 GBM细胞球细胞具有CSCs的特性,具有自我更新、多向分化以及体内成瘤的能力可以作为理想的CSCs进行深入研究。
2在体内及体外条件下,胶质瘤CSCs表现出较non-CSCs明显增强的侵袭能力。胶质瘤CSCs极大地促进了肿瘤的侵袭能力,至少部分是因为胶质瘤CSCs同时拥有了肿瘤细胞和NSCs的特性,它们在胶质瘤的成瘤过程中发挥了关键的作用,胶质瘤CSCs也许是通过促进肿瘤细胞的侵袭从而促进肿瘤的形成和进展。
3胶质瘤CSCs在体外条件下,主要是通过上调MMP-2的表达促进肿瘤细胞的侵袭和迁移;而在体内条件下,MMP-9对肿瘤的侵袭性生长也许起到了更为重要的作用。因此,胶质瘤CSCs也许是通过明胶酶依赖的机制在不同的条件下促进了肿瘤细胞的侵袭和肿瘤的侵袭性进展。
Introduction
Malignant gliomas are the most common and most aggressive primary brain tumors that respond poorly to current treatment strategies of cytoreductive neurosurgery, involved field radiotherapy,and adjuvant chemotherapy,which are characterized by rapid cell proliferation,a high level of invasiveness into the surrounding brain and increased vascularity.The capability of glioma cells to invade extensively within the central nervous system is a major cause of the high morbidity rate of primary malignant brain tumors.The invasion of tumor cells into brain tissue is a pathologic hallmark of WHO gradesⅡ-Ⅳgliomas and contributes significantly to the failure of current therapeutic treatments.Glioma cell invasion involves the attachment of tumor cells to extracellular matrix(ECM),degradation of ECM components,and subsequent penetration into adjacent brain structures.These processes are accomplished in part by matrix metalloproteinases(MMPs),a family of zinc-dependent proteinases.The members of the MMPs family have the ability to degrade macromolecules of the ECM and are responsible for tumor invasion and infiltration,limiting the effectiveness of the neurosurgical resection of brain tumors.Recent reports have suggested that expressions of gelatinase-A(MMP-2) and gelatinase-B(MMP-9) play an important role in neoplastic tissue invasion or metastasis,since these are critical factors in basement membrane degradation.A better understanding of glioma invasion mechanism will help in developing therapeutic strategies to decrease the invasion of gliomas.
Recently,a large body of evidence has assigned a key role in tumor formation and progression to cancer stem cells(CSCs) by virtue of its ability to self-renew,and differentiate into subpopulations of cells similar to those present in the initial tumor, more importantly,the isolated CSCs are able to form tumors after in vivo implantation. Also glioma CSCs participate in cancer chemoresistance and redioresistance mechanism.To date,invasion has been defined as an important mechanism in glioma tumorigenesis,However,few reports were involved in invasiveness characteristic of glioma CSCs,therefore we isolated and identified glioma CSCs from primary cultured GBM cells-GBM1 and GBM cell lines U87,9L,examined whether glioma CSCs possess potent invasiveness potential and determine tumor progression through promoting tumor invasive processes,and detected the expressions of MMP-2 and -9 in different subpopulations and xenografts in brain tumor models.Here,we reported that malignant glioma CSCs might provide a powerful enhancement for the invasion of glioma through a gelatinases-dependent mechanism.Targeting gelatinases specifically on glioma CSCs may provide a new insight and method for patient therapy by inhibiting glioma invasiveness.
Methods
Cells and culture process
Tumor specimen was obtained from surgical resection of one GBM patient,as approved by the Institute Review Boards at the Neurosurgery Department of Cedars-Sinai Medical Center,LA,CA.Tumor tissue was washed,minced,and enzymatically dissociated.U87 cell line and 9L cell line was purchased from ATCC.Tumor cells were resuspended in DMEM/F12 medium containing 10%fetal bovine serum(FBS) Subsequently monolayer-growing tumor cells were cultured in DMEM/F12 supplemented with epidermal growth factor(EGF),and basic fibroblast growth factor(bFGF)(R&D Systems,Minneapolis,MN).
Subsphere-forming and differentiation assay
The spheres were harvested,dissociated into single cells and were diluted to1-2 cells/well for the subsphere-forming assay Simultaneously,the monolayer cells left in the flasks after harvesting of the sphere were transferred into medium without growth factors but permissive for differentiation.
Immunocytostaining
Spheres and differentiated glioma cells were grown in precoated chamber slides and fixed,and then were stained with the following antibodies:anti-CD133(mouse monoclonal IgG1;1:10;Milteny Biotec),anti-nestin(mouse monoclonal IgG1;1:200; Chemicon),anti-β-tubulinⅢ(mouse monoclonal IgG1;1:200;Chemicon),anti-GFAP (rabbit polyclonal;1:1000;Chemicon),anti-myelin/oligodendrocyte(mouse monoclonal; 1:1000;Chemicon).The primary antibodies were detected with Cy3 or FITC-conjugated anti-mouse or anti-rabbit IgG antibodies(1:200,Jackson Immuno Reserch).
Establishment of in vivo glioma model
Six- to 8-week-old athymic nude mice and adult male F344 rats weighing between 200g and 220 g obtained from Medical Animal Center at Cedars-Sinai Medical Center,LA,CA, stereotactically inoculated with either purified glioma sphere cells or non-sphere cells (50,000/animal) in the right corpus striatum.
Invasion assays
Cell invasion assay kit(ECM 550,polycarbonate membrane,24-well plate with 12 cell culture inserts,8-μm pore size,Chemicon) was used.The number of invasive cells was counted as the indicator of glioma cell invasiveness.
Histological and immunohistochemical analysis of glioma-bearing brain sections
The animals were euthanized 6 weeks after implantation.Brains harvested from tumor cells implanted animals were frozen into sections,which were stained with H&E staining.Also staining were performed using primary antibodies against MMP-2 and MMP-9(1:50,mouse monoclonal IgG,Calbiochem).
Xenografts analysis
Matched sphere cells or non-sphere cells were implanted intracranially with 50,000 cells per animal as described previously.The animals were euthanized on 14,21 and 28 day post-implantation,respectively.
Real-time Quantitative Reverse transcription-PCR
Sphere cells and non-sphere cells from three glioma cell lines,and from xenografts of brain tumor models were respectively subjected to total RNA extraction with RNeasy kit (QIAGEN) and reverse transcripted into cDNA.The real-time PCR for CD 133,Nestin, MMP-2 and MMP-9 was performed.The relative mRNA level of these genes was compared between sphere cells and non-sphere calls,as well gelatinases were detected in normal brain tissues and tumor tissues derived from different time points post-implantation,respectively.
Protein analysis
Western blots contained 20μg protein extracted from glioma cells,and tumor tissues or normal brain tissues derived from xenografts on a 4-20%Tris/glycine SDS-PAGE gel (Invitrogen) and were transferred to 0.45-μm PVDF(Invitrogen).Blots were incubated with antibodies directed against MMP-2 and MMP-9,and in HRP-conjugated IgG.Blots were developed and exposed to film.β-actin as control.
Statistical analysis
Statistical analyses were done using SPSS 13.0.Data graphed with error bars represent mean and SD.A two-sided Student's t test was used to determine the significance of any differences.P＜0.05 as statistical significance.
Results
Isolation and identification of glioma CSCs
Primary cultured sphere glioma cells formed subspheres under limiting passage conditions consistent with self renewal,expressed neural stem cell markets(CD133, nestin),and generated multiple-lineage daughter cells,and formed a neoplasm after intracranial implantation.In contrast,few cells of the non-sphere forming tumor cells in the monolayer culture stained positive for CD133 and nestin.Together these results fulfilled the criteria of two distinct tumor cell subpopulations:CSCs and non-CSCs.
Glioma CSCs presented increased invasive ability combined with elevated levels of MMP-2,but not MMP-9 in vitro
To determine invasiveness capacity for different subpopulations in three glioma cell lines,respectively.The results showed that significantly more sphere cells invaded out of the membrane than non-sphere forming cells(P＜0.01),and single sphere cells formed new subspheres after they passed through ECM gel.
Real-time RT-PCR was used to detect gelatinases mRNA expression.MMP-2 mRNA expression in glioma CSCs ranged from 6.56- to 9.43-fold higher than that in autologous non-CSCs(P＜0.0001),however,only modest differences of MMP-9 mRNA expression between CSCs and non-CSCs subpopulations were observed in three glioma cell lines(P＞0.05),moreover the mRNA expression levels of MMP-9 were lower than those of MMP-2 strikingly,especially in glioma CSCs subpopulation.
Western blot results showed markedly increased secretion of MMP-2 from glioma CSCs versus non-CSCs subpopulation,but except a trace of expression in CSCs subpopulation derived from GBMI cell,MMP-9 expression wasn't detected in other subpopulations.
Tumors derived from sphere glioma cells showed increased tumor invasiveness and proliferation
Regardless of the differing origins of glioma cell lines,the brains implanted with sphere tumor cells presented highly proliferative and invasive neoplasms with extensive vascularity.However,among the animals implanted with non-sphere tumor cells,only 2 of 6 rats implanted with 9L cell line formed small neopalsms,in other brains tumors weren't formed 6 weeks after implantation.
The expression of MMP-2 and MMP-9 were highly up-regulated in brain tumors derived from glioma CSCs
Immunohistochemical results showed,regardless of tumor cell source,MMP-2 was expressed both in normal brain tissues and tumor tissues,and in tumor tissues, MMP-2 was present dominantly in the center of tumor,in contrast,MMP-9 expression was present in very low levels in normal brain tissues,but in the tumors it was greatly restricted to regions at the infiltrating and invading border of the tumor.
Real-time RT-PCR results revealed that the mRNA expression levels of MMP-9 were very low and only a trace of expression of MMP-2 mRNA was detected in normal brain tissues;the expression of MMP-2 and MMP-9 mRNA were markedly elevated in tumor tissues at 3 time points post-implantation with the growth of malignant glioma. The mRNA expression levels of both MMP-2 and MMP-9 at 28 days were striking increased compared with that post-implantation for 14 days.However,the growth rate of MMP-9 mRNA level along with the time after implantation was higher than that of MMP-2,especially on 28 days versus on 14 days post-implantation.
Western blot assay showed that MMP-2 was detected at 72 kDa in both tumor tissues and normal brain tissues,but the expression in tumor tissues was much higher than that in normal brain tissues.MMP-9 was detected at 92 kDa in tumor tissues,but not in normal brain tissues except modest expression in those derived from U87 sphere cells.In addition,western blot results showed increased secretion of MMP-2 and MMP-9 along with the time post sphere tumor cell implantation.Both MMP-2 and MMP-9 expressions 28 days post-implantation were evidently increased compared to those from 14 days,particularly for MMP-9.
Conclusions
1 In our study,we confirmed cultured sphere glioma cells from glioma cell lines formed new spheres with the capacity for self renewal,expressed gene associated with neural stem cells,generated daughter cells of different phenotypes from one mother cell, and formed tumors after intracranial implantation both in mice and rats.These sphere cells could be used for further study as optimal CSCs.
2 Our findings above suggest the fact that glioma CSCs enhance the invasiveness potential of malignant glioma,at least in part,is because the small subpopulation shares both characteristics of glioma tumor cells and NSCs simultaneously.Our results of CSCs inducing the increased invasiveness in cell invasion assay and in xenograft evaluations extend these findings of a key role of CSCs in glioma invasive tumorigenesis.Glioma CSCs maybe contribute to tumor progression through promoting tumor invasion.
3 Together these results suggest that glioma CSCs can up-regulate the expression of MMP-2 that promote their invasion and migration in vitro.Also of interest was our finding that MMP-9 might exhibit stronger invasion and infiltration capability in glioma tumorigenesis than MMP-2 in vivo.Glioma CSCs may possess increased invasiveness ability and enhance tumor invasive progression through gelatinases-related mechanism under different circumstances.
引文
1.Jeremic B,Milicic B,Grujicic D,et al.Clinical prognostic factors in patients with malignant glioma treated with combined modality approach.Am Clin Oncol,2004,27:195-204.
2.魏宇佳,赵洪洋,姜晓兵,等.免疫磁珠法分离、培养人脑胶质瘤干细胞.中华神经外科疾病研究杂志.2008:7(5):426-430.
3.Kong DS,Kim MH,Park WY,et al.The progression of gliomas is associated with cancer stem cell phenotype.Oncol Rep.2008;19(3):639-643.
4.Oka N,Soeda A,Inagaki A,et al.VEGF promotes tumorigenesis and angiogenesis of human glioblastoma stem cells.Biochem Biophys Res Commun.2007;360(3):553-559.
5.Ehtesham M,Mapara KY,Stevenson CB,et al.CXCR4 mediates the proliferation of glioblastoma progenitor cells.Cancer Lett.2009;274(2):305-312.
6.Bao S,Wu Q,Sathornsumetee S,et al.Stem Cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor.Cancer Res.2006;66:7843-7848.
7.Fan X,Matsui W,Khaki L,et al.Notch pathway inhibition depletes stem-like cells and blocks engraftment in embryonal brain tumors.Cancer Res.2006;66:7445-7452.
8.Johannessen TC,Bjerkvig R,Tysnes BB.DNA repair and cancer stem-like cells--potential partners in glioma drug resistance? Cancer Treat Rev.2008;34(6):558-567.
9.Sanchez-Martin M.Brain tumour stem cells:implications for cancer therapy and regenerative medicine.Curr Stem Cell Res Ther.2008;3(3):197-207.
10.Godlewski J,Nowicki MO,Bronisz A,et al.Targeting of the Bmi-1 oncogene/stem cell renewal factor by microRNA-128 inhibits glioma proliferation and self-renewal.Cancer Res.2008;68(22):9125-9130.
11.胡福军,凌志强.肿瘤干细胞介导脑肿瘤化放疗抗拒性研究进展.中华肿瘤防治杂志.2005;15(18):1425-1428.
12.Liu G,Yuan X,Zeng Z,et al.Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma.Mol Cancer.2006;5:67.
13.Bao S,Wu Q,McLendon RE,et al.Glioma stem cells promote radioresistance by preferential activation of the DNA damage response.Nature.2006;444:756-760.
14.P.Kleihues and W.K.Cavenee,Pathology and Genetics:Tumors of the Nervous System.IARC,Lyon(2000).
15.Ogden AT,Waziri AE,Lochhead RA,et al.Identification of A2B5+CD133-tumor-initiating cells in adult human gliomas.Neurosurgery.2008;62(2):505-14;discussion 514-5.
1.Kargiotis O,Chetty C,Gondi CS,et al.Adenovirus-mediated transfer of siRNA against MMP-2 mRNA results in impaired invasion and tumor-induced angiogenesis,induces apoptosis in vitro and inhibits tumor growth in vivo in glioblastoma.Oncogene.2008;27(35):4830-4840.
2.de Groot J,Piao Y,Lu L.AMPA receptors promote perivascular glioma invasion via {beta} 1 integrin-dependent adhesion to the extracellular matrix.Neuro Oncol.2008 Oct 28.[Epub ahead of print]
3.Singh SK,Hawkins C,Dirks PB.Identification of human brain tumour initiating cells.Nature,2004,432:396-401.
4.Yuan X,Curtin J,Yu JS.Isolation of cancer stem cells from adult glioblastoma multiforme.Oncogene,2004,23:9392-9400.
5.Galli R,Binda E,Orfanelli U,et al.Isolation and characterization of tumorigenic,stem-like neural precursors from human glioblastoma.Cancer Res,2004,64:7011-7021.
6.Nakada M,Nakada S,Demuth T,et al.Molecular targets of glioma invasion.Cell Mol Life Sci,2007,64:458-478.
7.Sliwa M,Markovic D,Gabrusiewicz K,et al.The invasion promoting effect of microglia on glioblastoma cells inhibited by cyclosporine A.Brain.2007;130:47-89.
8.Nalabothula N,Lakka SS,Dinh DH,et al.Sense p16 and antisense uPAR bicistronic construct inhibits angiogenesis and induces glioma cell death.Int J Oncol.2007;30:669-678.
9.McDonough WS,Savitch BA,Fortin SP,et al.Increase Fibroblast Growth Factor-Inducible 14 expression levels promote glioma cell invasion via Rac 1 and Neuclear Factor-(kappa)B and correlate with poor patient outcome.Cancer Res.2006;66:9535-9542.
10.Levicar N,Nuttall RK,Lah TT.Proteases in brain tumour progression.Acta Neurochir(Wien).2003;145:825-838.
11.Rao JS.Molecular mechanisms of glioma invasiveness:the role of proteases.Nat Rev Cancer.2003;3:489-501.
12.Yong VW.Metalloproteinases:mediators of pathology and regeneration in the CNS.Nat Rev Neurosci.2005;6:931-944.
13.Chi A,Norden AD,Wen PY.Inhibition of angiogenesis and invasion in malignant gliomas.Expert Rev Anticancer Ther.2007;7(11):1537-1560.
14.Nishida Y,Miyamori H,Thompson EW,et al.Activation of matrix metalloproteinase-2(MMP-2)by membrane type 1 matrix metalloproteinase through an artificial receptor for proMMP-2 generates active MMP-2.Cancer Res.2008;68(21):9096-9104.
15.Neth P,Ries C,Karow M,et al.The Wnt signal transduction pathway in stem cells and cancer cells:influence on cellular invasion.Stem Cell Rev.2007;3(1):18-29.
16.Chen RS,Song YM,Zhou ZY,et al.Disruption of xCT inhibits cancer cell metastasis via the caveolin-1/beta-cateninpathway.Oncogene.2009;28(4):599-609.
17.Kong DS,Kim MH,Park WY,et al.The progression of gliomas is associated with cancer stem cell phenotype.Oncol Rep.2008;19(3):639-643.
18.Yu SC,Ping YF,Yi L,et al.Isolation and characterization of cancer stem cells from a human glioblastoma cell line U87.Cancer Lett.2008;265(1):124-34.
19.Ghods AJ,Irvin D,Liu G,et al.Spheres isolated from 9L gliosarcoma rat cell line possess chemoresistant and aggressive cancer stem-like cells.Stem Cells.2007;25(7):1645-53.
20.Silber J,Lim DA,Petritsch C,et al.miR-124 and miR-137 inhibit proliferation of glioblastoma multiforme cells and induce differentiation of brain tumor stem cells.BMC Med.2008;6:14.
21.Oka N,Soeda A,Inagaki A,et al.VEGF promotes tumorigenesis and angiogenesis of human glioblastoma stem cells.Biochem Biophys Res Commun.2007;360(3):553-559.
22.Ehtesham M,Mapara KY,Stevenson CB,et al.CXCR4 mediates the proliferation of glioblastoma progenitor cells.Cancer Lett.2009;274(2):305-312.
23.Bao S,Wu Q,Sathomsumetee S,et al.Stem Cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor.Cancer Res.2006;66:7843-7848.
24.Fan X,Matsui W,Khaki L,et al.Notch pathway inhibition depletes stem-like cells and blocks engraftment in embryonal brain tumors.Cancer Res.2006;66:7445-7452.
25.Johannessen TC,Bjerkvig R,Tysnes BB.DNA repair and cancer stem-like cells-potential partners in glioma drug resistance? Cancer Treat Rev.2008;34(6):558-567.
26.Sanchez-Martin M.Brain tumour stem cells:implications for cancer therapy and regenerative medicine.Curr Stem Cell Res Ther.2008;3(3):197-207.
27.Godlewski J,Nowicki MO,Bronisz A,et al.Targeting of the Bmi-1 oncogene/stem cell renewal factor by microRNA-128 inhibits glioma proliferation and self-renewal.Cancer Res.2008;68(22):9125-9130.
28.Liu G,Yuan X,Zeng Z,et al.Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma.Mol Cancer.2006;5:67.
29.Bao S,Wu Q,McLendon RE,et al.Glioma stem cells promote radioresistance by preferential activation of the DNA damage response.Nature.2006;444:756-760.
30.Pisati F,Belicchi M,Acerbi F,et al.Effect of human skin-derived stem cells on vessel architecture,tumor growth,and tumor invasion in brain tumor animal models.Cancer Res.2007;67:3054-3063.
31.Munaut C,Noel A,Hougrand O,et al.Vascular endothelial growth factor expression correlates with matrix metalloprotinases MT1-MMP,MMP-2 and MMP-9 in human glioblastomas.Int J Cancer.2003;106:848-855.
32.Kunishio K,Okada M,Matsumoto Y,et al.Matrix metalloproteinase-2 and-9 expression in astrocytic tumors.Brain Tumor Pathol.2003;20:39-45.
33.Nakada M,Okada Y,Yamashita J.The role of matrix metalloproteinases in glioma invasion.Front Biosci.2003;8:e261-269.
34.Komatsu K,Nakanishi Y,Nemoto N,et al.Expression and quantitative analysis of matrix metalloproteinase-2 and-9 in human gliomas.Brain Tumor Pathol.2004;21:105-112.
35.Ries C,Egea V,Karow M,et al.MMP-2,MT1-MMP,and TIMP-2 are essential for the invasive capacity of human mesenchymal stem cells:differential regulation by inflammatory cytokines.Blood.2007;109(9):4055-4063.
36.Baumann F,Leukel P,Doerfelt A,et al.Lactate promotes glioma migration by TGF-beta2 dependent regulation of matrix metalloproteinase-2.Neuro Oncol.2008 Dec 5.[Epub ahead of print]
37.Huang HC,Huang CY,Lin-Shiau SY,et al.Ursolic acid inhibits IL-lbeta or TNF-alphainduced C6 glioma invasion through suppressing the association ZIP/p62 with PKC-zeta and downregulating the MMP-9 expression.Mol Carcinog.2008 Oct 30.[Epub ahead of print]
38.Katoh M,Katoh M.WNT signaling pathway and stem cell signaling network.Clin Cancer Res.2007;13(14):4042-4045.
39.Katoh M.WNT signaling in stem cell biology and regenerative medicine.Curr Drug Targets.2008;9(7):565-570.
40.Annabi B,Laflamme C,Sina A,et al.A MTl-MMP/NF-kappaB signaling axis as a checkpoint controller of COX-2 expression in CD133+ U87 glioblastoma cells.J Neuroinflammation.2009;6:8.
41.Bleau AM,Hambardzumyan D,Ozawa T,et al.PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells.Cell Stem Cell.2009;4(3):226-35.
1.Jeremic B,Milicic B,Grujicic D,et al.Clinical prognostic factors in patients with malignant glioma treated with combined modality approach.Am Clin Oncol,2004;27:195-204.
2.Gondi CS,Lakka SS,Dinh Dh,et al.Downregulation of uPA,uPAR and MMP-9 using small interfering,hairpin RNA(si RNA)inhibits glioma cell invasion,angiogenesis and tumor growth.Neuron Glia Biol,2004;1:165-76.
3.Sliwa M,Markovic D,Gabrusiewicz K,et al.The invasion promoting effect of microglia on glioblastoma cells inhibited by cyclosporine A.Brain,2007;130:47-89.
4.Chen DL,Ping YF,Yu SC,et al.Downregulating FPR restrains xenograft tumors by impairing the angiogenic potential and invasive capability of malignant glioma cells.Biochem Biophys Res Commun.2009;381(3):448-52.
5.Rorive S,Berton A,DTiaene N,et al.Matrix metalloproteinase-9 interplays with the IGFBP2-IGFⅡ complex to promote cell growth and motility in astrocytomas.Glia.2008;56(15):1679-90.
6.Du R,Petritsch C,Lu K,et al.Matrix metalloproteinase-2 regulates vascular patterning and growth affecting tumor cell survival and invasion in GBM.Neuro Oncol.2008;10(3):254-64.
7.Bl(?)zquez C,Salazar M,Carracedo A,et al.Cannabinoids inhibit glioma cell invasion by down-regulating matrix metalloproteinase-2 expression.Cancer Res.2008;68(6):1945-52.
8.Wong ET,Alsop D,Lee D,et al.Cerebrospinal fluid matrix metalloproteinase-9 increases during treatment of recurrent malignant gliomas.Cerebrospinal Fluid Res.2008;5:1.
9.Vlashi E,Kim K,Lagadec C,et al.In vivo imaging,tracking,and targeting of cancer stem cells.J Natl Cancer Inst.2009;101(5):350-9.
10.Pallini R,Ricci-Vitiani L,Banna GL,et al.Cancer stem cell analysis and clinical outcome in patients with glioblastoma multiforme.Clin Cancer Res.2008;14(24):8205-12.
11.Loja T,Chlapek P,Kuglik P,et al.Characterization of a GM7 glioblastoma cell line showing CD133 positivity and both cytoplasmic and nuclear localization of nestin.Oncol Rep.2009;21(1):119-27.
12.Harris MA,Yang H,Low BE,et al.Cancer stem cells are enriched in the side population cells in a mouse model of glioma.Cancer Res.2008;68(24):10051-9.
13.Kong DS,Kim MH,Park WY,et al.The progression of gliomas is associated with cancer stem cell phenotype.Oncol Rep.2008;19(3):639-643.
14.Oka N,Soeda A,Inagaki A,et al.VEGF promotes tumorigenesis and angiogenesis of human glioblastoma stem cells.Biochem Biophys Res Commun.2007;360(3):553-559.
15.Ehtesham M,Mapara KY,Stevenson CB,et al.CXCR4 mediates the proliferation of glioblastoma progenitor cells.Cancer Lett.2009;274(2):305-312.
16.Johannessen TC,Bjerkvig R,Tysnes BB.DNA repair and cancer stem-like cells-potential partners in glioma drug resistance? Cancer Treat Rev.2008;34(6):558-567.
17.Sanchez-Martin M.Brain tumour stem cells:implications for cancer therapy and regenerative medicine.Curr Stem Cell Res Then 2008;3(3):197-207.
18.Godlewski J,Nowicki MO,Bronisz A,et al.Targeting of the Bmi-1 oncogene/stem cell renewal factor by microRNA-128 inhibits glioma proliferation and self-renewal.Cancer Res.2008;68(22):9125-9130.
19.Nakada M,Nakada S,Demuth T,et al.Molecular targets of glioma invasion.Cell Mol Life Sci.2007;64:458-478.
20.Nalabothula N,Lakka SS,Dinh DH,Gujrati M,Olivero WC,Rao JS:Sense p16 and antisense uPAR bicistronic construct inhibits angiogenesis and induces glioma cell death.Int J Oncol.2007;30:669-678.
21.McDonough WS,Savitch BA,Fortin SP,et al.Increase Fibroblast Growth Factor-Inducible 14 expression levels promote glioma cell invasion via Rac 1 and Neuclear Factor-(kappa)B and correlate with poor patient outcome.Cancer Res.2006;66:9535-9542.
22.Levicar N,Nuttall RK,Lah TT.Proteases in brain tumour progression.Acta Neurochir(Wien)2003;145:825-838.
23.Rao JS:Molecular mechanisms of glioma invasiveness:the role of proteases.Nat Rev Cancer.2003;3:489-501.
24.Yong VW.Metalloproteinases:mediators of pathology and regeneration in the CNS.Nat Rev Neurosci 2005;6:931-944.
25.Visse R,NagaseH.Matrixmetalloproteinases and tissue inhibitors of metalloproteinases structure,function and biochemistry.Circ Res,2003,92(8):827-839.
26.Rome C,Arsaut J,Taris C,Couillaud F,Loiseau H:MMP-7(matrilysin)expression in human brain tumors.Mol Carcinog.2007;46:446-452.
27.Sarkar S,Nuttall RK,Liu S,et al.Tenascin-C stimulates glioma cell invasion through matrix metalloproteinase-12.Cancer Res,2006;66(24):11771-11780.
28.Yeh WL,Lu DY,Lee MJ,et al.Leptin induces migration and invasion of glioma cells through MMP-13 production.Glia.2009;57(4):454-64.
29.Iwanami H,Ishizaki M,Fukuda Y,et al.Expression of matrix metalloproteinases(MMP)-12 by myofibroblasts during alkali-burned corneal wound healing.Curr Eye Res.2009;34(3):207-14.
30.Dive V,Bregant S,Huillet C,et al.Detection of matrix metalloproteinase active forms in complex proteomes:evaluation of photoaffinity versus affinity capture.J Proteome Res.2009 Mar 9.[Epub ahead of print]
31.Ries C,Egea V,Karow M,et al.MMP-2,MTl-MMP,and TIMP-2 are essential for the invasive capacity of human mesenchymal stem cells:differential regulation by inflammatory cytokines.Blood.2007;109(9):4055-4063.
32.Baumann F,Leukel P,Doerfelt A,et al.Lactate promotes glioma migration by TGF-beta2 dependent regulation of matrix metalloproteinase-2.Neuro Oncol.2008 Dec 5.[Epub ahead of print]
33.Huang HC,Huang CY,Lin-Shiau SY,et al.Ursolic acid inhibits IL-lbeta or TNF-alphainduced C6 glioma invasion through suppressing the association ZIP/p62 with PKC-zeta and downregulating the MMP-9 expression.Mol Carcinog.2008 Oct 30.[Epub ahead of print]
34.Nishida Y,Miyamori H,Thompson EW,et al.Activation of matrix metalloproteinase-2(MMP-2)by membrane type 1 matrix metalloproteinase through an artificial receptor for proMMP-2 generates active MMP-2.Cancer Res.2008;68(21):9096-104.
35.McClung HM,Thomas SL,Osenkowski P,et al.SPARC upregulates MTl-MMP expression,MMP-2 activation,and the secretion and cleavage of galectin-3 in U87MG glioma cells.Neurosci Lett.2007;419(2):172-7.
36.Munaut C,Noel A,Hougrand O,et al.Vascular endothelial growth factor expression correlates with matrix metalloprotinases MT1-MMP,MMP-2 and MMP-9 in human glioblastomas.Int J Cancer.2003;106:848-855.
37.Lu KV,Jong KA,Rajasekaran AK,et al.Upregulation of tissue inhibitor of metalloproteinases(TIMP)-2 promotes matrix metalloproteinase(MMP)-2 activation and cell invasion in a human glioblastoma cell line.Lab Invest.2004;84:8-20.
38.Nyormoi O,Mills L,Bar-Eli M.An MMP-2/MMP-9 inhibitor,5a,enhances apoptosis induced by ligands of the TNF receptor superfamily in cancer cells.Cell Death Differ.2003;10:558-569.
39.Groves MD,Puduvalli VK,Hess KR,et al.Phase Ⅱ trial of temozolomide plus the matrix metalloproteinase inhibitor,marimastat,in recurrent and progressive glioblastoma multiforme.J Clin Oncol.2002;20:1383-1388.
1.Blum B,Benvenisty N.The tumorigenicity of human embryonic stem cells.Adv Cancer Res.2008;100:133-58.
2.Tian X,Kaufman DS.Differentiation of embryonic stem cells towards hematopoietic cells:progress and pitfalls.Curr Opin Hematol.2008;15(4):312-8.
3.Chidgey AP,Layton D,Trounson A,et al.Tolerance strategies for stem-cell-based therapies.Nature.2008;453(7193):330-7.
4.Taupin P.Adult neurogenesis,neuroinflammation and therapeutic potential of adult neural stem cells.Int J Med Sci.2008;5(3):127-32.
5.Newman MB,Bakay RA.Therapeutic potentials of human embryonic stem cells in Parkinson's disease.Neurotherapeutics.2008;5(2):237-51.
6.Spitkovsky D,Hescheler J.Adult mesenchymal stromal stem cells for therapeutic applications.Minim Invasive Ther Allied Technol.2008;17(2):79-90.
7.Lapidot T,Sirard C,Vormoor J,et al.A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.Nature.1994;367:645-648.
8.Larochelle A,Vormoor J,Hanenberg H,et al.Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow:implications for gene therapy.Nat Med.1996;2:1329-1337.
9.Bonnet D,Dick JE.Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.Nat Med.1997;3:730-737.
10.Al-Hajj M,Wicha MS,Benito-Hernandez A,et al.Prospective identi-fication of tumorigenic breast cancer cells.Proc Natl Acad Sci USA.2003;100:3983-3988.
11.Ignatova TN,Kukekov VG,Laywell ED,et al.Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro.Glia.2002;39:193-206.
12.Singh SK,Clarke ID,Terasaki M,et al.Identification of a cancer stem cell in human brain tumors.Cancer Res.2003;63:5821-5828.
13.Hemmati HD,Nakano I,Lazareff JA,et al.Cancerous stem cells can arise from pediatric brain tumors.Proc Natl Acad Sci USA.2003;100:15178-15183.
14.Singh SK,Hawkins C,Clarke ID,et al.Identification of human brain tumour initiating cells.Nature.2004;432:396-401.
15.Yuan X,Curtin J,Xiong Y,et al.Isolation of cancer stem cells from adult glioblastoma multiforme.Oncogene.2004;23:9392-9400.
16.Galli R,Binda E,Orfanelli U,et al.Isolation and characterization of tumorigenic,stem-like neural precursors from human glioblastoma.Cancer Res.2004;64:7011-7021.
17.Blum B,Benvenisty N.The tumorigenicity of human embryonic stem cells.Adv Cancer Res.2008;100:133-58.
18.Zhu R,Bhattacharya C,Matin A.The role of dead-end in germ-cell tumor development.Ann NY Acad Sci.2007;1120:181-6.
19.Wilck MB,Baden LR.Vaccination after stem cell transplant:a review of recent developments and implications for current practice.Curr Opin Infect Dis.2008;21(4):399-408.
20.Bamabe-Heider F,Frisen J.Stem cells for spinal cord repair.Cell Stem Cell.2008;3(1):16-24.
21.Taupin P.Adult neurogenesis,neuroinflammation and therapeutic potential of adult neural stem cells.Int J Med Sci.2008;5(3):127-32.
22.Screening for prostate cancer:recommendation and rationale2.Ann Intern Med.2002;137:915-916.
23.Bardeesy N,DePinho RA.Pancreatic cancer biology and genetics.Nat Rev Cancer.2002;2:897-909.
24.Troyer DA,Mubiru J,Leach RJ,et al.Promise and challenge:Markers of prostate cancer detection,diagnosis and prognosis.Dis Markers.2004;20:117-128.
25.Garber ME,Troyanskaya OG,Schluens K,et al.Diversity of gene expression in adenocarcinoma of the lung.Proc Natl Acad Sci USA.2001;98:13784-13789.
26.Perou CM,Jeffrey SS,van de Rijn M,et al.Distinctive gene expression patterns in human mammary epithelial cells and breast cancers.Proc Natl Acad Sci USA.1999;96:9212-9217.
27.Perou CM,Sorlie T,Eisen MB,et al.Molecular portraits of human breast tumours.Nature.2000;406:747-752.
28.Futreal PA,Coin L,Marshall M,et al.A census of human cancer genes.Nat Rev Cancer.2004;4:177-183.
29.Hanahan D,Weinberg RA.The hallmarks of cancer.Cell.2000;100:57-70.
30.Clarke MF.Self-renewal and solid-tumor stem cells.Biol Blood Marrow Transplant.2005;11(2 Suppl 2):14-6.
31.Al-Hajj M,Clarke MF.Self-renewal and solid tumor stem cells.Oncogene.2004;23(43):7274-82.
32.Al-Hajj M,Becker MW,Wicha M,et al.Therapeutic implications of cancer stem cells.Curr Opin Genet Dev.2004;14(1):43-7.
33.Jeremic B,Milicic B,Grujicic D,et al.Clinical prognostic factors in patients with malignant glioma treated with combined modality approach.Am J Clin Oncol.2004;27(2):195-204.
34.Yu SC,Ping YF,Yi L,et al.Isolation and characterization of cancer stem cells from a human glioblastoma cell line U87.Cancer Lett.2008;265(1):124-34.
35.Shen G,Shen F,Shi Z,et al.Identification of cancer stem-like cells in the C6 glioma cell line and the limitation of current identification methods.In Vitro Cell Dev Biol Anim.2008 Jul 2.
36.Zheng X,Shen G,Yang X,et al.Most C6 cells are cancer stem cells:evidence from clonal and population analyses.Cancer Res.2007;67(8):3691-7.
37.Ghods AJ,Irvin D,Liu G,et al.Spheres isolated from 9L gliosarcoma rat cell line possess chemoresistant and aggressive cancer stem-like cells.Stem Cells.2007;25(7):1645-53.
38.Wu A,Oh S,Wiesner SM,et al.Persistence of CD133+ cells in human and mouse glioma cell lines:detailed characterization of GL261 glioma cells with cancer stem cell-like properties.Stem Cells Dev.2008;17(1):173-84.
39.Mao Y,Zhou L,Zhu W,et al.Proliferative status of tumor stem cells may be correlated with malignancy grade of human astrocytomas.Front Biosci.2007;12:2252-2259.
40.Gondi CS,Lakka SS,Dinh Dh,et al.Downregulation of uPA,uPAR and MMP-9 using small interfering,hairpin RNA(si RNA)inhibits glioma cell invasion,angiogenesis and tumor growth.Neuron Glia Biol.2004;1(2):165-76.3
41.Yoshizawa T,Hammaker D,Sweeney SE,et al.Synoviocyte innate immune responses:I.Differential regulation of interferon responses and the JNK pathway by MAPK kinases.J Immunol.2008;181(5):3252-8.
42.McKay MM,Morrison DK.Integrating signals from RTKs to ERK/MAPK.Oncogene.2007;26(22):3113-21.
43.Raman M,Chen W,Cobb MH.Differential regulation and properties of MAPKs.Oncogene.2007;26(22):3100-12.
44.Sliwa M,Markovic D,Gabrusiewicz K,et al.The invasion promoting effect of microglia on glioblastoma cells inhibited by cyclosporine A.Brain.2007;130(Pt 2):47-89.
45.Neth P,Ries C,Karow M,et al.The Wnt signal transduction pathway in stem cells and cancer cells:influence on cellular invasion.Stem Cell Rev.2007;3(1):18-29.
46.Neth P,Cicarella M,Egea V,et al.Wnt signaling regulates the invasion capacity of human mesenchymal stem cells.Stem Cells.2006;24(8):1892-903.
47.Katoh M,Katoh M.WNT signaling pathway and stem cell signaling network.Clin Cancer Res.2007;13(14):4042-5.
48.Woll PS,Morris JK,Painschab MS,et al.Wnt signaling promotes hematoendothelial cell development from human embryonic stem cells.Blood.2008;111(1):122-31.
49.Nikolova T,Wu M,Brumbarov K,et al.WNT-conditioned media differentially affect the proliferation and differentiation of cord blood-derived CD 133+ cells in vitro.Differentiation.2007;75(2):100-11.
50.Visse R,NagaseH.Matrixmetalloproteinases and tissue inhibitors of metalloproteinases structure,function and biochemistry.Circ Res,2003,92(8):827-839.
51.Rome C,Arsaut J,Taris C,Couillaud F,Loiseau H:MMP-7(matrilysin)expression in human brain tumors.Mol Carcinog.2007,46:446-452.
52.Sarkar S,Nuttall RK,Liu S,et al.Tenascin-C stimulates glioma cell invasion through matrix metalloproteinase-12.Cancer Res,2006,15;66(24):11771-11780.
53.Yeh WL,Lu DY,Lee MJ,et al.Leptin induces migration and invasion of glioma cells through MMP-13 production.Glia.2009;57(4):454-64.
54.Iwanami H,Ishizaki M,Fukuda Y,et al.Expression of matrix metalloproteinases(MMP)-12 by myofibroblasts during alkali-burned corneal wound healing.Curr Eye Res.2009;34(3):207-14.
55.Dive V,Bregant S,Huillet C,et al.Detection of matrix metalloproteinase active forms in complex proteomes:evaluation of photoaffinity versus affinity caprure.J Proteome Res.2009 Mar 9..[Epub ahead of print]
56.Ries C,Egea V,Karow M,et al.MMP-2,MT1-MMP,and TIMP-2 are essential for the invasive capacity of human mesenchymal stem cells:differential regulation by inflammatory cytokines.Blood.2007;109(9):4055-4063.
57.Baumann F,Leukel P,Doerfelt A,et al.Lactate promotes glioma migration by TGF-beta2 dependent regulation of matrix metalloproteinase-2.Neuro Oncol.2008 Dec 5.[Epub ahead of print]
58.Huang HC,Huang CY,Lin-Shiau SY,et al.Ursolic acid inhibits IL-lbeta or TNF-alphainduced C6 glioma invasion through suppressing the association ZIP/p62 with PKC-zeta and downregulating the MMP-9 expression.Mol Carcinog.2008 Oct 30.[Epub ahead of print]
59.Nishida Y,Miyamori H,Thompson EW,et al.Activation of matrix metalloproteinase-2(MMP-2)by membrane type 1 matrix metalloproteinase through an artificial receptor for proMMP-2 generates active MMP-2.Cancer Res.2008;68(21):9096-104.
60.McClung HM,Thomas SL,Osenkowski P,et al.SPARC upregulates MT1-MMP expression,MMP-2 activation,and the secretion and cleavage of galectin-3 in U87MG glioma cells.Neurosci Lett.2007;419(2):172-7.
61.Munaut C,Noel A,Hougrand O,et al.Vascular endothelial growth factor expression correlates with matrix metalloprotinases MT1-MMP,MMP-2 and MMP-9 in human glioblastomas.Int J Cancer.2003;106:848-855.
62.Lu KV,Jong KA,Rajasekaran AK,et al.Upregulation of tissue inhibitor of metalloproteinases(TIMP)-2 promotes matrix metalloproteinase(MMP)-2 activation and cell invasion in a human glioblastoma cell line.Lab Invest.2004;84:8-20.
63.Chen DL,Ping YF,Yu SC,et al.Downregulating FPR restrains xenograft tumors by impairing the angiogenic potential and invasive capability of malignant glioma cells.Biochem Biophys Res Commun.2009;381(3):448-52.
64.Rorive S,Berton A,Dliaene N,et al.Matrix metalloproteinase-9 interplays with the IGFBP2-IGFⅡ complex to promote cell growth and motility in astrocytomas.Glia.2008;56(15):1679-90.
65.Du R,Petritsch C,Lu K,et al.Matrix metalloproteinase-2 regulates vascular patterning and growth affecting tumor cell survival and invasion in GBM.Neuro Oncol.2008;10(3):254-64.
66.Blazquez C,Salazar M,Carracedo A,et al.Cannabinoids inhibit glioma cell invasion by down-regulating matrix metalloproteinase-2 expression.Cancer Res.2008;68(6):1945-52.
67.Wong ET,Alsop D,Lee D,et al.Cerebrospinal fluid matrix metalloproteinase-9 increases during treatment of recurrent malignant gliomas.Cerebrospinal Fluid Res.2008;5:1.
68.Fan X,Matsui W,Khaki L,et al.Notch pathway inhibition depletes stem-like cells and blocks engraftment in embryonal brain tumors.Cancer Res.2006;66(15):7445-52.
69.Fan X,Mikolaenko I,Elhassan I,et al.Notchl and notch2 have opposite effects on embryonal brain tumor growth.Cancer Res.2004;64(21):7787-93.
70.Clement V,Sanchez P,de Tribolet N,et al.HEDGEHOG-GLI1 signaling regulates human glioma growth,cancer stem cell self-renewal,and tumorigenicity.Curr Biol.2007;17(2):165-72.
71.Massard C,Deutsch E,Soria JC.Tumour stem cell-targeted treatment:elimination or differentiation.Ann Oncol.2006;17(11):1620-1624.
72.Liu G,Yuan X,Yu JS.Analysis of gene expression and chemoresistance of CD 133+ cancer stem cells in glioblastoma.Mol Cancer.2006;5:67.
73.Bao S,Wu Q,McLendon RE,et al.Glioma stem cells promote radioresistance by preferential activation of the DNA damage response.Nature.2006;444(7120):756-760.
2.魏宇佳,赵洪洋,姜晓兵,等.免疫磁珠法分离、培养人脑胶质瘤干细胞.中华神经外科疾病研究杂志.2008:7(5):426-430.
3.Kong DS,Kim MH,Park WY,et al.The progression of gliomas is associated with cancer stem cell phenotype.Oncol Rep.2008;19(3):639-643.
4.Oka N,Soeda A,Inagaki A,et al.VEGF promotes tumorigenesis and angiogenesis of human glioblastoma stem cells.Biochem Biophys Res Commun.2007;360(3):553-559.
5.Ehtesham M,Mapara KY,Stevenson CB,et al.CXCR4 mediates the proliferation of glioblastoma progenitor cells.Cancer Lett.2009;274(2):305-312.
6.Bao S,Wu Q,Sathornsumetee S,et al.Stem Cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor.Cancer Res.2006;66:7843-7848.
7.Fan X,Matsui W,Khaki L,et al.Notch pathway inhibition depletes stem-like cells and blocks engraftment in embryonal brain tumors.Cancer Res.2006;66:7445-7452.
8.Johannessen TC,Bjerkvig R,Tysnes BB.DNA repair and cancer stem-like cells--potential partners in glioma drug resistance? Cancer Treat Rev.2008;34(6):558-567.
9.Sanchez-Martin M.Brain tumour stem cells:implications for cancer therapy and regenerative medicine.Curr Stem Cell Res Ther.2008;3(3):197-207.
10.Godlewski J,Nowicki MO,Bronisz A,et al.Targeting of the Bmi-1 oncogene/stem cell renewal factor by microRNA-128 inhibits glioma proliferation and self-renewal.Cancer Res.2008;68(22):9125-9130.
11.胡福军,凌志强.肿瘤干细胞介导脑肿瘤化放疗抗拒性研究进展.中华肿瘤防治杂志.2005;15(18):1425-1428.
12.Liu G,Yuan X,Zeng Z,et al.Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma.Mol Cancer.2006;5:67.
13.Bao S,Wu Q,McLendon RE,et al.Glioma stem cells promote radioresistance by preferential activation of the DNA damage response.Nature.2006;444:756-760.
14.P.Kleihues and W.K.Cavenee,Pathology and Genetics:Tumors of the Nervous System.IARC,Lyon(2000).
15.Ogden AT,Waziri AE,Lochhead RA,et al.Identification of A2B5+CD133-tumor-initiating cells in adult human gliomas.Neurosurgery.2008;62(2):505-14;discussion 514-5.
1.Kargiotis O,Chetty C,Gondi CS,et al.Adenovirus-mediated transfer of siRNA against MMP-2 mRNA results in impaired invasion and tumor-induced angiogenesis,induces apoptosis in vitro and inhibits tumor growth in vivo in glioblastoma.Oncogene.2008;27(35):4830-4840.
2.de Groot J,Piao Y,Lu L.AMPA receptors promote perivascular glioma invasion via {beta} 1 integrin-dependent adhesion to the extracellular matrix.Neuro Oncol.2008 Oct 28.[Epub ahead of print]
3.Singh SK,Hawkins C,Dirks PB.Identification of human brain tumour initiating cells.Nature,2004,432:396-401.
4.Yuan X,Curtin J,Yu JS.Isolation of cancer stem cells from adult glioblastoma multiforme.Oncogene,2004,23:9392-9400.
5.Galli R,Binda E,Orfanelli U,et al.Isolation and characterization of tumorigenic,stem-like neural precursors from human glioblastoma.Cancer Res,2004,64:7011-7021.
6.Nakada M,Nakada S,Demuth T,et al.Molecular targets of glioma invasion.Cell Mol Life Sci,2007,64:458-478.
7.Sliwa M,Markovic D,Gabrusiewicz K,et al.The invasion promoting effect of microglia on glioblastoma cells inhibited by cyclosporine A.Brain.2007;130:47-89.
8.Nalabothula N,Lakka SS,Dinh DH,et al.Sense p16 and antisense uPAR bicistronic construct inhibits angiogenesis and induces glioma cell death.Int J Oncol.2007;30:669-678.
9.McDonough WS,Savitch BA,Fortin SP,et al.Increase Fibroblast Growth Factor-Inducible 14 expression levels promote glioma cell invasion via Rac 1 and Neuclear Factor-(kappa)B and correlate with poor patient outcome.Cancer Res.2006;66:9535-9542.
10.Levicar N,Nuttall RK,Lah TT.Proteases in brain tumour progression.Acta Neurochir(Wien).2003;145:825-838.
11.Rao JS.Molecular mechanisms of glioma invasiveness:the role of proteases.Nat Rev Cancer.2003;3:489-501.
12.Yong VW.Metalloproteinases:mediators of pathology and regeneration in the CNS.Nat Rev Neurosci.2005;6:931-944.
13.Chi A,Norden AD,Wen PY.Inhibition of angiogenesis and invasion in malignant gliomas.Expert Rev Anticancer Ther.2007;7(11):1537-1560.
14.Nishida Y,Miyamori H,Thompson EW,et al.Activation of matrix metalloproteinase-2(MMP-2)by membrane type 1 matrix metalloproteinase through an artificial receptor for proMMP-2 generates active MMP-2.Cancer Res.2008;68(21):9096-9104.
15.Neth P,Ries C,Karow M,et al.The Wnt signal transduction pathway in stem cells and cancer cells:influence on cellular invasion.Stem Cell Rev.2007;3(1):18-29.
16.Chen RS,Song YM,Zhou ZY,et al.Disruption of xCT inhibits cancer cell metastasis via the caveolin-1/beta-cateninpathway.Oncogene.2009;28(4):599-609.
17.Kong DS,Kim MH,Park WY,et al.The progression of gliomas is associated with cancer stem cell phenotype.Oncol Rep.2008;19(3):639-643.
18.Yu SC,Ping YF,Yi L,et al.Isolation and characterization of cancer stem cells from a human glioblastoma cell line U87.Cancer Lett.2008;265(1):124-34.
19.Ghods AJ,Irvin D,Liu G,et al.Spheres isolated from 9L gliosarcoma rat cell line possess chemoresistant and aggressive cancer stem-like cells.Stem Cells.2007;25(7):1645-53.
20.Silber J,Lim DA,Petritsch C,et al.miR-124 and miR-137 inhibit proliferation of glioblastoma multiforme cells and induce differentiation of brain tumor stem cells.BMC Med.2008;6:14.
21.Oka N,Soeda A,Inagaki A,et al.VEGF promotes tumorigenesis and angiogenesis of human glioblastoma stem cells.Biochem Biophys Res Commun.2007;360(3):553-559.
22.Ehtesham M,Mapara KY,Stevenson CB,et al.CXCR4 mediates the proliferation of glioblastoma progenitor cells.Cancer Lett.2009;274(2):305-312.
23.Bao S,Wu Q,Sathomsumetee S,et al.Stem Cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor.Cancer Res.2006;66:7843-7848.
24.Fan X,Matsui W,Khaki L,et al.Notch pathway inhibition depletes stem-like cells and blocks engraftment in embryonal brain tumors.Cancer Res.2006;66:7445-7452.
25.Johannessen TC,Bjerkvig R,Tysnes BB.DNA repair and cancer stem-like cells-potential partners in glioma drug resistance? Cancer Treat Rev.2008;34(6):558-567.
26.Sanchez-Martin M.Brain tumour stem cells:implications for cancer therapy and regenerative medicine.Curr Stem Cell Res Ther.2008;3(3):197-207.
27.Godlewski J,Nowicki MO,Bronisz A,et al.Targeting of the Bmi-1 oncogene/stem cell renewal factor by microRNA-128 inhibits glioma proliferation and self-renewal.Cancer Res.2008;68(22):9125-9130.
28.Liu G,Yuan X,Zeng Z,et al.Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma.Mol Cancer.2006;5:67.
29.Bao S,Wu Q,McLendon RE,et al.Glioma stem cells promote radioresistance by preferential activation of the DNA damage response.Nature.2006;444:756-760.
30.Pisati F,Belicchi M,Acerbi F,et al.Effect of human skin-derived stem cells on vessel architecture,tumor growth,and tumor invasion in brain tumor animal models.Cancer Res.2007;67:3054-3063.
31.Munaut C,Noel A,Hougrand O,et al.Vascular endothelial growth factor expression correlates with matrix metalloprotinases MT1-MMP,MMP-2 and MMP-9 in human glioblastomas.Int J Cancer.2003;106:848-855.
32.Kunishio K,Okada M,Matsumoto Y,et al.Matrix metalloproteinase-2 and-9 expression in astrocytic tumors.Brain Tumor Pathol.2003;20:39-45.
33.Nakada M,Okada Y,Yamashita J.The role of matrix metalloproteinases in glioma invasion.Front Biosci.2003;8:e261-269.
34.Komatsu K,Nakanishi Y,Nemoto N,et al.Expression and quantitative analysis of matrix metalloproteinase-2 and-9 in human gliomas.Brain Tumor Pathol.2004;21:105-112.
35.Ries C,Egea V,Karow M,et al.MMP-2,MT1-MMP,and TIMP-2 are essential for the invasive capacity of human mesenchymal stem cells:differential regulation by inflammatory cytokines.Blood.2007;109(9):4055-4063.
36.Baumann F,Leukel P,Doerfelt A,et al.Lactate promotes glioma migration by TGF-beta2 dependent regulation of matrix metalloproteinase-2.Neuro Oncol.2008 Dec 5.[Epub ahead of print]
37.Huang HC,Huang CY,Lin-Shiau SY,et al.Ursolic acid inhibits IL-lbeta or TNF-alphainduced C6 glioma invasion through suppressing the association ZIP/p62 with PKC-zeta and downregulating the MMP-9 expression.Mol Carcinog.2008 Oct 30.[Epub ahead of print]
38.Katoh M,Katoh M.WNT signaling pathway and stem cell signaling network.Clin Cancer Res.2007;13(14):4042-4045.
39.Katoh M.WNT signaling in stem cell biology and regenerative medicine.Curr Drug Targets.2008;9(7):565-570.
40.Annabi B,Laflamme C,Sina A,et al.A MTl-MMP/NF-kappaB signaling axis as a checkpoint controller of COX-2 expression in CD133+ U87 glioblastoma cells.J Neuroinflammation.2009;6:8.
41.Bleau AM,Hambardzumyan D,Ozawa T,et al.PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells.Cell Stem Cell.2009;4(3):226-35.
1.Jeremic B,Milicic B,Grujicic D,et al.Clinical prognostic factors in patients with malignant glioma treated with combined modality approach.Am Clin Oncol,2004;27:195-204.
2.Gondi CS,Lakka SS,Dinh Dh,et al.Downregulation of uPA,uPAR and MMP-9 using small interfering,hairpin RNA(si RNA)inhibits glioma cell invasion,angiogenesis and tumor growth.Neuron Glia Biol,2004;1:165-76.
3.Sliwa M,Markovic D,Gabrusiewicz K,et al.The invasion promoting effect of microglia on glioblastoma cells inhibited by cyclosporine A.Brain,2007;130:47-89.
4.Chen DL,Ping YF,Yu SC,et al.Downregulating FPR restrains xenograft tumors by impairing the angiogenic potential and invasive capability of malignant glioma cells.Biochem Biophys Res Commun.2009;381(3):448-52.
5.Rorive S,Berton A,DTiaene N,et al.Matrix metalloproteinase-9 interplays with the IGFBP2-IGFⅡ complex to promote cell growth and motility in astrocytomas.Glia.2008;56(15):1679-90.
6.Du R,Petritsch C,Lu K,et al.Matrix metalloproteinase-2 regulates vascular patterning and growth affecting tumor cell survival and invasion in GBM.Neuro Oncol.2008;10(3):254-64.
7.Bl(?)zquez C,Salazar M,Carracedo A,et al.Cannabinoids inhibit glioma cell invasion by down-regulating matrix metalloproteinase-2 expression.Cancer Res.2008;68(6):1945-52.
8.Wong ET,Alsop D,Lee D,et al.Cerebrospinal fluid matrix metalloproteinase-9 increases during treatment of recurrent malignant gliomas.Cerebrospinal Fluid Res.2008;5:1.
9.Vlashi E,Kim K,Lagadec C,et al.In vivo imaging,tracking,and targeting of cancer stem cells.J Natl Cancer Inst.2009;101(5):350-9.
10.Pallini R,Ricci-Vitiani L,Banna GL,et al.Cancer stem cell analysis and clinical outcome in patients with glioblastoma multiforme.Clin Cancer Res.2008;14(24):8205-12.
11.Loja T,Chlapek P,Kuglik P,et al.Characterization of a GM7 glioblastoma cell line showing CD133 positivity and both cytoplasmic and nuclear localization of nestin.Oncol Rep.2009;21(1):119-27.
12.Harris MA,Yang H,Low BE,et al.Cancer stem cells are enriched in the side population cells in a mouse model of glioma.Cancer Res.2008;68(24):10051-9.
13.Kong DS,Kim MH,Park WY,et al.The progression of gliomas is associated with cancer stem cell phenotype.Oncol Rep.2008;19(3):639-643.
14.Oka N,Soeda A,Inagaki A,et al.VEGF promotes tumorigenesis and angiogenesis of human glioblastoma stem cells.Biochem Biophys Res Commun.2007;360(3):553-559.
15.Ehtesham M,Mapara KY,Stevenson CB,et al.CXCR4 mediates the proliferation of glioblastoma progenitor cells.Cancer Lett.2009;274(2):305-312.
16.Johannessen TC,Bjerkvig R,Tysnes BB.DNA repair and cancer stem-like cells-potential partners in glioma drug resistance? Cancer Treat Rev.2008;34(6):558-567.
17.Sanchez-Martin M.Brain tumour stem cells:implications for cancer therapy and regenerative medicine.Curr Stem Cell Res Then 2008;3(3):197-207.
18.Godlewski J,Nowicki MO,Bronisz A,et al.Targeting of the Bmi-1 oncogene/stem cell renewal factor by microRNA-128 inhibits glioma proliferation and self-renewal.Cancer Res.2008;68(22):9125-9130.
19.Nakada M,Nakada S,Demuth T,et al.Molecular targets of glioma invasion.Cell Mol Life Sci.2007;64:458-478.
20.Nalabothula N,Lakka SS,Dinh DH,Gujrati M,Olivero WC,Rao JS:Sense p16 and antisense uPAR bicistronic construct inhibits angiogenesis and induces glioma cell death.Int J Oncol.2007;30:669-678.
21.McDonough WS,Savitch BA,Fortin SP,et al.Increase Fibroblast Growth Factor-Inducible 14 expression levels promote glioma cell invasion via Rac 1 and Neuclear Factor-(kappa)B and correlate with poor patient outcome.Cancer Res.2006;66:9535-9542.
22.Levicar N,Nuttall RK,Lah TT.Proteases in brain tumour progression.Acta Neurochir(Wien)2003;145:825-838.
23.Rao JS:Molecular mechanisms of glioma invasiveness:the role of proteases.Nat Rev Cancer.2003;3:489-501.
24.Yong VW.Metalloproteinases:mediators of pathology and regeneration in the CNS.Nat Rev Neurosci 2005;6:931-944.
25.Visse R,NagaseH.Matrixmetalloproteinases and tissue inhibitors of metalloproteinases structure,function and biochemistry.Circ Res,2003,92(8):827-839.
26.Rome C,Arsaut J,Taris C,Couillaud F,Loiseau H:MMP-7(matrilysin)expression in human brain tumors.Mol Carcinog.2007;46:446-452.
27.Sarkar S,Nuttall RK,Liu S,et al.Tenascin-C stimulates glioma cell invasion through matrix metalloproteinase-12.Cancer Res,2006;66(24):11771-11780.
28.Yeh WL,Lu DY,Lee MJ,et al.Leptin induces migration and invasion of glioma cells through MMP-13 production.Glia.2009;57(4):454-64.
29.Iwanami H,Ishizaki M,Fukuda Y,et al.Expression of matrix metalloproteinases(MMP)-12 by myofibroblasts during alkali-burned corneal wound healing.Curr Eye Res.2009;34(3):207-14.
30.Dive V,Bregant S,Huillet C,et al.Detection of matrix metalloproteinase active forms in complex proteomes:evaluation of photoaffinity versus affinity capture.J Proteome Res.2009 Mar 9.[Epub ahead of print]
31.Ries C,Egea V,Karow M,et al.MMP-2,MTl-MMP,and TIMP-2 are essential for the invasive capacity of human mesenchymal stem cells:differential regulation by inflammatory cytokines.Blood.2007;109(9):4055-4063.
32.Baumann F,Leukel P,Doerfelt A,et al.Lactate promotes glioma migration by TGF-beta2 dependent regulation of matrix metalloproteinase-2.Neuro Oncol.2008 Dec 5.[Epub ahead of print]
33.Huang HC,Huang CY,Lin-Shiau SY,et al.Ursolic acid inhibits IL-lbeta or TNF-alphainduced C6 glioma invasion through suppressing the association ZIP/p62 with PKC-zeta and downregulating the MMP-9 expression.Mol Carcinog.2008 Oct 30.[Epub ahead of print]
34.Nishida Y,Miyamori H,Thompson EW,et al.Activation of matrix metalloproteinase-2(MMP-2)by membrane type 1 matrix metalloproteinase through an artificial receptor for proMMP-2 generates active MMP-2.Cancer Res.2008;68(21):9096-104.
35.McClung HM,Thomas SL,Osenkowski P,et al.SPARC upregulates MTl-MMP expression,MMP-2 activation,and the secretion and cleavage of galectin-3 in U87MG glioma cells.Neurosci Lett.2007;419(2):172-7.
36.Munaut C,Noel A,Hougrand O,et al.Vascular endothelial growth factor expression correlates with matrix metalloprotinases MT1-MMP,MMP-2 and MMP-9 in human glioblastomas.Int J Cancer.2003;106:848-855.
37.Lu KV,Jong KA,Rajasekaran AK,et al.Upregulation of tissue inhibitor of metalloproteinases(TIMP)-2 promotes matrix metalloproteinase(MMP)-2 activation and cell invasion in a human glioblastoma cell line.Lab Invest.2004;84:8-20.
38.Nyormoi O,Mills L,Bar-Eli M.An MMP-2/MMP-9 inhibitor,5a,enhances apoptosis induced by ligands of the TNF receptor superfamily in cancer cells.Cell Death Differ.2003;10:558-569.
39.Groves MD,Puduvalli VK,Hess KR,et al.Phase Ⅱ trial of temozolomide plus the matrix metalloproteinase inhibitor,marimastat,in recurrent and progressive glioblastoma multiforme.J Clin Oncol.2002;20:1383-1388.
1.Blum B,Benvenisty N.The tumorigenicity of human embryonic stem cells.Adv Cancer Res.2008;100:133-58.
2.Tian X,Kaufman DS.Differentiation of embryonic stem cells towards hematopoietic cells:progress and pitfalls.Curr Opin Hematol.2008;15(4):312-8.
3.Chidgey AP,Layton D,Trounson A,et al.Tolerance strategies for stem-cell-based therapies.Nature.2008;453(7193):330-7.
4.Taupin P.Adult neurogenesis,neuroinflammation and therapeutic potential of adult neural stem cells.Int J Med Sci.2008;5(3):127-32.
5.Newman MB,Bakay RA.Therapeutic potentials of human embryonic stem cells in Parkinson's disease.Neurotherapeutics.2008;5(2):237-51.
6.Spitkovsky D,Hescheler J.Adult mesenchymal stromal stem cells for therapeutic applications.Minim Invasive Ther Allied Technol.2008;17(2):79-90.
7.Lapidot T,Sirard C,Vormoor J,et al.A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.Nature.1994;367:645-648.
8.Larochelle A,Vormoor J,Hanenberg H,et al.Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow:implications for gene therapy.Nat Med.1996;2:1329-1337.
9.Bonnet D,Dick JE.Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.Nat Med.1997;3:730-737.
10.Al-Hajj M,Wicha MS,Benito-Hernandez A,et al.Prospective identi-fication of tumorigenic breast cancer cells.Proc Natl Acad Sci USA.2003;100:3983-3988.
11.Ignatova TN,Kukekov VG,Laywell ED,et al.Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro.Glia.2002;39:193-206.
12.Singh SK,Clarke ID,Terasaki M,et al.Identification of a cancer stem cell in human brain tumors.Cancer Res.2003;63:5821-5828.
13.Hemmati HD,Nakano I,Lazareff JA,et al.Cancerous stem cells can arise from pediatric brain tumors.Proc Natl Acad Sci USA.2003;100:15178-15183.
14.Singh SK,Hawkins C,Clarke ID,et al.Identification of human brain tumour initiating cells.Nature.2004;432:396-401.
15.Yuan X,Curtin J,Xiong Y,et al.Isolation of cancer stem cells from adult glioblastoma multiforme.Oncogene.2004;23:9392-9400.
16.Galli R,Binda E,Orfanelli U,et al.Isolation and characterization of tumorigenic,stem-like neural precursors from human glioblastoma.Cancer Res.2004;64:7011-7021.
17.Blum B,Benvenisty N.The tumorigenicity of human embryonic stem cells.Adv Cancer Res.2008;100:133-58.
18.Zhu R,Bhattacharya C,Matin A.The role of dead-end in germ-cell tumor development.Ann NY Acad Sci.2007;1120:181-6.
19.Wilck MB,Baden LR.Vaccination after stem cell transplant:a review of recent developments and implications for current practice.Curr Opin Infect Dis.2008;21(4):399-408.
20.Bamabe-Heider F,Frisen J.Stem cells for spinal cord repair.Cell Stem Cell.2008;3(1):16-24.
21.Taupin P.Adult neurogenesis,neuroinflammation and therapeutic potential of adult neural stem cells.Int J Med Sci.2008;5(3):127-32.
22.Screening for prostate cancer:recommendation and rationale2.Ann Intern Med.2002;137:915-916.
23.Bardeesy N,DePinho RA.Pancreatic cancer biology and genetics.Nat Rev Cancer.2002;2:897-909.
24.Troyer DA,Mubiru J,Leach RJ,et al.Promise and challenge:Markers of prostate cancer detection,diagnosis and prognosis.Dis Markers.2004;20:117-128.
25.Garber ME,Troyanskaya OG,Schluens K,et al.Diversity of gene expression in adenocarcinoma of the lung.Proc Natl Acad Sci USA.2001;98:13784-13789.
26.Perou CM,Jeffrey SS,van de Rijn M,et al.Distinctive gene expression patterns in human mammary epithelial cells and breast cancers.Proc Natl Acad Sci USA.1999;96:9212-9217.
27.Perou CM,Sorlie T,Eisen MB,et al.Molecular portraits of human breast tumours.Nature.2000;406:747-752.
28.Futreal PA,Coin L,Marshall M,et al.A census of human cancer genes.Nat Rev Cancer.2004;4:177-183.
29.Hanahan D,Weinberg RA.The hallmarks of cancer.Cell.2000;100:57-70.
30.Clarke MF.Self-renewal and solid-tumor stem cells.Biol Blood Marrow Transplant.2005;11(2 Suppl 2):14-6.
31.Al-Hajj M,Clarke MF.Self-renewal and solid tumor stem cells.Oncogene.2004;23(43):7274-82.
32.Al-Hajj M,Becker MW,Wicha M,et al.Therapeutic implications of cancer stem cells.Curr Opin Genet Dev.2004;14(1):43-7.
33.Jeremic B,Milicic B,Grujicic D,et al.Clinical prognostic factors in patients with malignant glioma treated with combined modality approach.Am J Clin Oncol.2004;27(2):195-204.
34.Yu SC,Ping YF,Yi L,et al.Isolation and characterization of cancer stem cells from a human glioblastoma cell line U87.Cancer Lett.2008;265(1):124-34.
35.Shen G,Shen F,Shi Z,et al.Identification of cancer stem-like cells in the C6 glioma cell line and the limitation of current identification methods.In Vitro Cell Dev Biol Anim.2008 Jul 2.
36.Zheng X,Shen G,Yang X,et al.Most C6 cells are cancer stem cells:evidence from clonal and population analyses.Cancer Res.2007;67(8):3691-7.
37.Ghods AJ,Irvin D,Liu G,et al.Spheres isolated from 9L gliosarcoma rat cell line possess chemoresistant and aggressive cancer stem-like cells.Stem Cells.2007;25(7):1645-53.
38.Wu A,Oh S,Wiesner SM,et al.Persistence of CD133+ cells in human and mouse glioma cell lines:detailed characterization of GL261 glioma cells with cancer stem cell-like properties.Stem Cells Dev.2008;17(1):173-84.
39.Mao Y,Zhou L,Zhu W,et al.Proliferative status of tumor stem cells may be correlated with malignancy grade of human astrocytomas.Front Biosci.2007;12:2252-2259.
40.Gondi CS,Lakka SS,Dinh Dh,et al.Downregulation of uPA,uPAR and MMP-9 using small interfering,hairpin RNA(si RNA)inhibits glioma cell invasion,angiogenesis and tumor growth.Neuron Glia Biol.2004;1(2):165-76.3
41.Yoshizawa T,Hammaker D,Sweeney SE,et al.Synoviocyte innate immune responses:I.Differential regulation of interferon responses and the JNK pathway by MAPK kinases.J Immunol.2008;181(5):3252-8.
42.McKay MM,Morrison DK.Integrating signals from RTKs to ERK/MAPK.Oncogene.2007;26(22):3113-21.
43.Raman M,Chen W,Cobb MH.Differential regulation and properties of MAPKs.Oncogene.2007;26(22):3100-12.
44.Sliwa M,Markovic D,Gabrusiewicz K,et al.The invasion promoting effect of microglia on glioblastoma cells inhibited by cyclosporine A.Brain.2007;130(Pt 2):47-89.
45.Neth P,Ries C,Karow M,et al.The Wnt signal transduction pathway in stem cells and cancer cells:influence on cellular invasion.Stem Cell Rev.2007;3(1):18-29.
46.Neth P,Cicarella M,Egea V,et al.Wnt signaling regulates the invasion capacity of human mesenchymal stem cells.Stem Cells.2006;24(8):1892-903.
47.Katoh M,Katoh M.WNT signaling pathway and stem cell signaling network.Clin Cancer Res.2007;13(14):4042-5.
48.Woll PS,Morris JK,Painschab MS,et al.Wnt signaling promotes hematoendothelial cell development from human embryonic stem cells.Blood.2008;111(1):122-31.
49.Nikolova T,Wu M,Brumbarov K,et al.WNT-conditioned media differentially affect the proliferation and differentiation of cord blood-derived CD 133+ cells in vitro.Differentiation.2007;75(2):100-11.
50.Visse R,NagaseH.Matrixmetalloproteinases and tissue inhibitors of metalloproteinases structure,function and biochemistry.Circ Res,2003,92(8):827-839.
51.Rome C,Arsaut J,Taris C,Couillaud F,Loiseau H:MMP-7(matrilysin)expression in human brain tumors.Mol Carcinog.2007,46:446-452.
52.Sarkar S,Nuttall RK,Liu S,et al.Tenascin-C stimulates glioma cell invasion through matrix metalloproteinase-12.Cancer Res,2006,15;66(24):11771-11780.
53.Yeh WL,Lu DY,Lee MJ,et al.Leptin induces migration and invasion of glioma cells through MMP-13 production.Glia.2009;57(4):454-64.
54.Iwanami H,Ishizaki M,Fukuda Y,et al.Expression of matrix metalloproteinases(MMP)-12 by myofibroblasts during alkali-burned corneal wound healing.Curr Eye Res.2009;34(3):207-14.
55.Dive V,Bregant S,Huillet C,et al.Detection of matrix metalloproteinase active forms in complex proteomes:evaluation of photoaffinity versus affinity caprure.J Proteome Res.2009 Mar 9..[Epub ahead of print]
56.Ries C,Egea V,Karow M,et al.MMP-2,MT1-MMP,and TIMP-2 are essential for the invasive capacity of human mesenchymal stem cells:differential regulation by inflammatory cytokines.Blood.2007;109(9):4055-4063.
57.Baumann F,Leukel P,Doerfelt A,et al.Lactate promotes glioma migration by TGF-beta2 dependent regulation of matrix metalloproteinase-2.Neuro Oncol.2008 Dec 5.[Epub ahead of print]
58.Huang HC,Huang CY,Lin-Shiau SY,et al.Ursolic acid inhibits IL-lbeta or TNF-alphainduced C6 glioma invasion through suppressing the association ZIP/p62 with PKC-zeta and downregulating the MMP-9 expression.Mol Carcinog.2008 Oct 30.[Epub ahead of print]
59.Nishida Y,Miyamori H,Thompson EW,et al.Activation of matrix metalloproteinase-2(MMP-2)by membrane type 1 matrix metalloproteinase through an artificial receptor for proMMP-2 generates active MMP-2.Cancer Res.2008;68(21):9096-104.
60.McClung HM,Thomas SL,Osenkowski P,et al.SPARC upregulates MT1-MMP expression,MMP-2 activation,and the secretion and cleavage of galectin-3 in U87MG glioma cells.Neurosci Lett.2007;419(2):172-7.
61.Munaut C,Noel A,Hougrand O,et al.Vascular endothelial growth factor expression correlates with matrix metalloprotinases MT1-MMP,MMP-2 and MMP-9 in human glioblastomas.Int J Cancer.2003;106:848-855.
62.Lu KV,Jong KA,Rajasekaran AK,et al.Upregulation of tissue inhibitor of metalloproteinases(TIMP)-2 promotes matrix metalloproteinase(MMP)-2 activation and cell invasion in a human glioblastoma cell line.Lab Invest.2004;84:8-20.
63.Chen DL,Ping YF,Yu SC,et al.Downregulating FPR restrains xenograft tumors by impairing the angiogenic potential and invasive capability of malignant glioma cells.Biochem Biophys Res Commun.2009;381(3):448-52.
64.Rorive S,Berton A,Dliaene N,et al.Matrix metalloproteinase-9 interplays with the IGFBP2-IGFⅡ complex to promote cell growth and motility in astrocytomas.Glia.2008;56(15):1679-90.
65.Du R,Petritsch C,Lu K,et al.Matrix metalloproteinase-2 regulates vascular patterning and growth affecting tumor cell survival and invasion in GBM.Neuro Oncol.2008;10(3):254-64.
66.Blazquez C,Salazar M,Carracedo A,et al.Cannabinoids inhibit glioma cell invasion by down-regulating matrix metalloproteinase-2 expression.Cancer Res.2008;68(6):1945-52.
67.Wong ET,Alsop D,Lee D,et al.Cerebrospinal fluid matrix metalloproteinase-9 increases during treatment of recurrent malignant gliomas.Cerebrospinal Fluid Res.2008;5:1.
68.Fan X,Matsui W,Khaki L,et al.Notch pathway inhibition depletes stem-like cells and blocks engraftment in embryonal brain tumors.Cancer Res.2006;66(15):7445-52.
69.Fan X,Mikolaenko I,Elhassan I,et al.Notchl and notch2 have opposite effects on embryonal brain tumor growth.Cancer Res.2004;64(21):7787-93.
70.Clement V,Sanchez P,de Tribolet N,et al.HEDGEHOG-GLI1 signaling regulates human glioma growth,cancer stem cell self-renewal,and tumorigenicity.Curr Biol.2007;17(2):165-72.
71.Massard C,Deutsch E,Soria JC.Tumour stem cell-targeted treatment:elimination or differentiation.Ann Oncol.2006;17(11):1620-1624.
72.Liu G,Yuan X,Yu JS.Analysis of gene expression and chemoresistance of CD 133+ cancer stem cells in glioblastoma.Mol Cancer.2006;5:67.
73.Bao S,Wu Q,McLendon RE,et al.Glioma stem cells promote radioresistance by preferential activation of the DNA damage response.Nature.2006;444(7120):756-760.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |