Significance of the expression of green fluorescent protein on detection of glioma invasion in vivo

Objective: To investigate the invasion and metastasis of glioma in vivo by xenotransplanted tumor established by implanting C6 glioma cells transfected with green fluorescent protein (GFP) gene in vitro into the brain of SD rats. Methods: C6 cells were transfected with a plasmid vector (pEGEP-N3) containing the GFP gene. Stable GFP-expressing clones were isolated and performed examination by flow cytometry and electron microscope. GFP-expressing cells were stereotactically injected into the brain parenchyma of SD rats to establish xenotransplanted tumor. Four weeks later rats were killed and continuous brain sections respectively were examined by HE staining, immunohistochemistry method and fluorescence microscopy for detection of tumor cell invasion. Xenotransplanted tumor was primarily cultured to determine the storage of exotic GFP gene in vivo. Results: There were not obvious changes in cell cycle and ultrastructure for the cells transfected with GFP gene. C6 cells transfected with GFP gene maintained stable high-level GFP expression in the central nervous system during their growth in vivo. GFP fluorescence clearly demarcated the primary tumor margin and readily allowed for the detection of distant invasion on the single-cell level, which was evidently superior to HE and immunohistochemistry staining. There was not GFP gene loss of transfected cells in vivo. Conclusions: It is suggested that C6 cells transfected with GFP gene can be visualized by fluorescent microscopy after intracranial implantation. This model is an excellent experimental animal model in research on invasion of glioma. Foundation item: This work was supported by a grant from the National Natural science foundation of China (No. 39970752). Biography: LI Xia (1975–), master of neurosurgery, Xijing Hospital, the Fourth Military Medical University, majors in gene diagnosis and gene therapy of brain malignant tumor.     

绿色荧光蛋白转染胶质瘤细胞的体内实验研究

 目的　利用体外转染绿色荧光蛋白 (Enhanced Green Fluorescent Protein,EGFP)基因的胶质瘤细胞建立大鼠移植模型 ,并探索利用该模型研究肿瘤体内侵袭的可行性。方法　携有EGFP基因的 p EGFP- N3质粒体外转染 C6胶质瘤细胞 ,筛选稳定表达 GFP的细胞克隆并以立体定向法植入 SD大鼠脑实质内建立肿瘤移植模型。 4周后处死大鼠并制作连续石蜡切片 ,相邻切片分别作 HE、免疫组化染色和荧光显微镜检测。结果　GFP于体内稳定表达 ,在荧光显微镜下可较容易区分肿瘤细胞和非肿瘤细胞 ,并能检测到侵袭至远端的单个肿瘤细胞 ,其检测效率明显高于HE和免疫组化染色。结论　EGFP基因体外转染 C6胶质瘤细胞并建立大鼠脑内肿瘤移植模型 ,是研究胶质瘤体内侵袭的有效模型和方法。     

绿色荧光蛋白表达对检测胶质细胞瘤体内侵袭的意义

利用体外转染绿色荧光蛋白基因的胶质瘤细胞大鼠移植瘤模型,研究胶质瘤体内侵袭行为。方法：携有增强型绿色荧光蛋白（ＥｎｈａｎｃｅｄＧｒｅｅｎＦｌｕｏｒｅｓｃｅｎｔＰｒｏｔｅｉｎ,ＥＧＦＰ）基因的ｐＥＧＦＰ－Ｎ３质粒体转染Ｃ６胶瘤细胞,筛选稳定表达绿色荧光蛋白的细胞克隆,并作流式细胞仪和电镜检测,以立体定向法植入ＳＤ大鼠脑实质内建立大鼠移植模型。４周后处死大鼠并作鼠脑连续石蜡切片,相邻切片分别作苏木素－     

RI基因真核表达载体的构建及其对C6胶质瘤细胞生长的影响

目的通过构建核糖核酸酶抑制因子(ribonuclease inhibitor, RI)基因的真核表达载体——pLNCX-ri, 并转染C6神经胶质瘤细胞,探讨RI抑制肿瘤生长的作用机制.方法用Nde I / Xho从已构建的pET-ri上切下1.4 kb的RI基因片段,再构建到pLNCX上,获得真核表达载体(pLNCX-ri),采用Lipofect AMINE辅助转染大鼠C6神经胶质瘤细胞,经G418筛选获得稳定转染的细胞克隆,用Western blotting 检测RI基因的表达水平.将转染阳性的C6神经胶质瘤细胞接种于大鼠皮下,观察肿瘤的生长情况.结果在转染的C6神经胶质瘤细胞中,RI基因的表达量明显高于未转染的C6神经胶质瘤细胞,转染阳性的C6神经胶质瘤细胞在大鼠体内的成瘤潜伏期23±5.7天(对照组14±3.5天),瘤组织重量转染组1.35±0.43g比对照组2.40±0.61g(P＜0.01)明显下降,瘤组织的血管密度转染组27.2±4.31比对照组47±6.54(P＜0.01)明显减少.结论RI基因的转染对肿瘤的生长有明显抑制作用,其作用机制可能是抑制肿瘤组织血管的形成.     

IL-24基因对大鼠胶质瘤细胞生长状况的影响

目的　探讨IL 2 4基因对C6大鼠胶质瘤细胞生长状况的影响。方法　应用逆转录病毒载体 ,将IL 2 4基因导入C6细胞 ,经G4 18筛选后获得表达IL 2 4分子的阳性细胞克隆C6 /IL 2 4 ;用RT PCR方法检测目的基因表达 ;四甲基偶氮唑蓝 (MTT)法检测细胞体外增殖状况 ,流式细胞技术检测细胞的增殖活性 ,并制作荷瘤动物模型 ,观察C6 /IL 2 4和C6细胞的体内致瘤性。结果　RT PCR检测表明 ,外源IL 2 4基因于mRNA水平在C6 /IL 2 4细胞已获得稳定表达。C6 /IL 2 4细胞系的体外增殖性较亲代C6细胞明显下降 ,流式细胞术检测其细胞增殖指数 (PI)为 (2 9.71± 0 .89) %。 9只接种C6 /IL 2 4细胞的实验组大鼠中 ,6只颅内成瘤 ,肿瘤体积为 (14 .0 8± 9.81)mm3 ,明显小于接种C6细胞大鼠的肿瘤体积 (P <0 .0 5 )。结论　外源性IL 2 4基因可部分抑制胶质瘤细胞异常增殖的肿瘤特性。     

The brain slice chamber,a novel variation of the Boyden Chamber Assay,allows time-dependent quantification of glioma invasion into mammalian brain <Emphasis Type="Italic">in vitro</Emphasis>

Summary Glioma cell invasion occurs in a complex micromilieu consisting of neural and glial cells, myelinated fiber tracts, blood vessels and extracellular matrix proteins. The present work describes the brain slice chamber (BSC) as a novel experimental model for assessing invasion of glioma cells into adult mammalian white and gray matter on the basis of the well known Boyden chamber system. As a matrix for invasive tumor cells we used freshly prepared brain tissue from adult pigs. The tissue was sectioned into 40 m slices that were mechanically fixed to a millipore filter. The neural structures and the three-dimensional architecture of the slice was preserved as verified by immunohistochemistry, light- and electron microscopy. Human U-373 and U87 astrocytoma cells stably transfected with green fluorescent protein (GFP) were assessed for their invasiveness into the brain-slices during a 24 h period. Invasion of U-87 GFP cells was quantified at different time intervals by confocal laser scanning microscopy showing more intense invasion into white compared to gray matter. Two cytostatics (vincristin and paclitaxel) which both are known to affect the cytoskeleton, inhibited glioma cell invasion in a dose dependent manner, which makes the presented model system suitable for functional experiments. In conclusion, the BSC represents a valid and rapid experimental model that may be used to describe the invasive behavior of glioma cells within the preserved three-dimensional structure of mammalian brain tissue in vitro.     

褪黑激素抗胶质瘤作用的体内试验

目的探讨ＭＬＴ体内抗胶质瘤作用。方法观察荷Ｃ６鼠胶质瘤大鼠接受ＭＬＴ治疗后肿瘤大小,生存期,病理组织学及细胞凋亡的变化。结果ＭＬＴ治疗后Ｃ６胶质瘤生长延缓,荷瘤大鼠生存期延长,诱发肿瘤细胞凋亡。结论ＭＬＴ有一定抑瘤作用,值得进一步临床实验。     

p16基因对脑胶质瘤细胞作用的实验研究

目的 探索ｐ１６基因在脑胶质瘤发生发展过程中的作用及ｐ１６基因在脑胶质瘤临床诊断、治疗中的应用前景。方法 采用脂质体转染的方法,将外源野生型ｐ１６基因导入胶质瘤细胞株Ｕ２５１、Ｃ６,筛选阳怀克隆。同时以空载体质粒ｐＣＤＮＡ３为对照,免疫组化检测ｐ１６基因表达,用ＭＴＴ 测定瘤细胞生长曲线及对转染的瘤细胞在裸鼠体内形成瘤块的变化进行分析。结果 转染ｐ１６基因的Ｕ２５１、Ｃ６细胞有外源ｐ１６基因的整合     

连接蛋白43基因治疗鼠C6脑胶质瘤的体内实验研究

目的 研究连接蛋白（Cx）43基因抑制脑胶质瘤生长的作用。方法 将Cx43基因表达缺失的大鼠C6胶质瘤细胞（对照组）和转染Cx43 cDNA的C6细胞（转染组）种植于SD大鼠右侧尾状核,荷载C6脑胶质瘤鼠用Cx 43 cDNA原位治疗（治疗组）,并以空载体治疗作为对照（空载组）,每组10只,观察各组大鼠一般情况、生存期、MRI动态变化及病理改变,通过原位杂交及免疫组化染色检测肿瘤Cx43 mRNA及蛋白表达,以AgNOR平均计数检测增殖活性,以Tunel法检测细胞凋亡。结果 对照组和空载组大鼠均于3周内死亡;转染组6只大鼠和治疗组8只大鼠观察120 d内无自然死亡。除治疗组1只尚有残瘤外,余瘤体消失,转染组和治疗组肿瘤细胞均有Cx43 mRNA及蛋白表达,且增殖治疗组1只尚有残瘤外,余瘤体消失。转染组和治疗组肿瘤细胞均有Cx43 mRNA及蛋白表达,且增殖活性下降,但凋亡不增加。结论 转染Cx43基因后的C6胶质瘤细胞致瘤性显著降低,且对荷载脑胶质瘤鼠具有明显的治疗作用,有可能成为恶性胶质瘤基因治疗的优先靶的之一。     

单纯疱疹病毒-胸苷激酶基因转染骨髓基质干细胞治疗大鼠脑胶质瘤

目的研究转染单纯疱疹病毒-胸苷激酶(HSV-tk)基因的骨髓基质干细胞(BMSCs)对大鼠脑胶质瘤的治疗作用。方法原代培养BMSCs,AdCMV-tk转染BMSCs。逆转录-多聚酶链反应(RT-PCR)检测BMSCs/tk对tk基因的转录。以BrdU标记BMSCs/tk,观察其趋瘤性。四甲基偶氮唑盐(MTT)法检测其对C6细胞的旁观者效应。将BMSCs/tk注入脑胶质瘤大鼠肿瘤对侧脑组织,原位末端标记(TUNEL)法检测脑内肿瘤细胞凋亡,动态MRI监测肿瘤体积的变化,观察荷瘤鼠生存期。结果全骨髓细胞培养法可获得纯化的BMSCs。感染AdCMV-tk 21 d后,BMSCs仍有明显的tk基因表达。转染tk基因后的BMSCs仍具有明显的趋瘤性。BMSCs/tk不仅在体外可产生明显的旁观者效应,在荷瘤鼠脑内也显示了明显的诱导肿瘤细胞凋亡的旁观者效应,凋亡阳性率为20.38%±2.57%,与BMSCs组(2.56%±0.52%)和对照组(2.74%±0.38%)相比,差异均有统计学意义(P值分别为0.023和0.025)。BMSCs/tk移植3周时,BMSCs/tk组、BMSCs组和对照组肿瘤体积分别为(8.28±2.64)、(134.51±16.37)和(147.22±31.05)mm3,BMSCs/tk组远小于BMSCs组(P=0.001)和对照组(P<0.01),并且部分大鼠脑内肿瘤消失。BMSCs/tk组大鼠生存期为(52.60±13.11)d,与对照组相比明显延长(P=0.01)。结论HSV-tk转染的BMSCs可能成为脑胶质瘤治疗的有效手段。     

IN VIVO GROWTH OF CEREBRAL C6 GLIOMA CELLS TRANSFECTED AND TREATED WITH CX43 GENE

Objective: To study the role of connexin gene (Cx43) on the development of glioma and the feasibility of using Cx43cDNA as a target of gene therapy of gliomas.Methods: Parental rat C6 cells and C6 cells transfected with Cx43cDNA were implanted into right caudate nucleus of SD rats as control and transfected group.Rats bearing cerebral C6 gliomas were treated with Cx43cDNA and empty vector as treated group and empty vector group. The general manifestation, survival time, MRI dynamic scanning and histopathological changes of all rats were observed. In situ hybridization and immunohisto- chemistry were used for examination of Cx43mRNA and its protein in gliomas. Average number of AgNOR staining was used for detection of cell proliferation activity, and TUNEL method for determination of cell apoptosis. Results: All rats in control and empty vector group died of cerebral gliomas within 3 weeks after implantation of C6 cells. Six out of nine rats in the transfected group and eight out of ten rats in treated group kept alive beyond 120 days with totally disappearing of the tumor foci, except one treated rat having a little residue of tumor. In gliomas of transfected and treated groups Cx43 gene expression was upregulated, proliferation activity was lowered,However, the apoptotic cells did not increase.Conclusion: The present study indicates that Cx43 gene is of crucial importance in the development of malignant glioma. It can be an effective target for gene therapy of gliomas.     

表皮生长因子受体反义cDNA对C6鼠脑胶质瘤体内治疗的研究

目的研究表皮生长因子受体（ＥＧＦＲ）反义ｃＤＮＡ对Ｃ６鼠脑胶质瘤的体内治疗效果。方法将野生型及已转染ＥＧＦＲ反义ｃＤＮＡ的Ｃ６鼠胶质瘤细胞接种于鼠脑右侧尾状核（对照组８只,转染组６只）,并对鼠脑内已形成的Ｃ６胶质瘤用脂质体包裹的ＥＧＦＲ反义ｃＤＮＡ瘤区原位注射（治疗组９只）。观察各组大鼠的一般情况、生存期、肿瘤病理学和磁共振成像（ＭＲＩ）动态改变,采用Ａｇ－ＮＯＲ计数、ＴＵＮＥＬ法检测肿瘤细胞增殖活性及凋亡。结果对照组８只大鼠平均生存期为１７．３天,转染组６只及治疗组６只大鼠生存期明显延长,除因病理检查人为处死外,无自然死亡,存活期超过２００天。ＭＲＩ检查对照组大鼠脑内有明显瘤灶,转染组大鼠未形成瘤灶,治疗组大鼠脑内瘤灶治疗后消失,均与病理学检查结果一致。而且治疗组大鼠Ｃ６细胞增殖活性降低,大量细胞凋亡,ＥＧＦＲ表达减少。结论ＥＧＦＲ可望成为基因治疗的优选靶基因     

D-amino acid oxidase gene therapy sensitizes glioma cells to the antiglycolytic effect of 3-bromopyruvate

Glioma tumors are refractory to conventional treatment. Glioblastoma multiforme is the most aggressive type of primary brain tumors in humans. In this study, we introduce oxidative stress-energy depletion (OSED) therapy as a new suggested treatment for glioblastoma. OSED utilizes D-amino acid oxidase (DAO), which is a promising therapeutic protein that induces oxidative stress and apoptosis through generating hydrogen peroxide (H2O2). OSED combines DAO with 3-bromopyruvate (3BP), a hexokinase II (HK II) inhibitor that interferes with Warburg effect, a metabolic alteration of most tumor cells that is characterized by enhanced aerobic glycolysis. Our data revealed that 3BP induced depletion of energetic capabilities of glioma cells. 3BP induced H2O2 production as a novel mechanism of its action. C6 glioma transfected with DAO and treated with D-serine together with 3BP-sensitized glioma cells to 3BP and decreased markedly proliferation, clonogenic power and viability in a three-dimensional tumor model with lesser effect on normal astrocytes. DAO gene therapy using atelocollagen as an in vivo transfection agent proved effective in a glioma tumor model in Sprague-Dawley (SD) rats, especially after combination with 3BP. OSED treatment was safe and tolerable in SD rats. OSED therapy may be a promising therapeutic modality for glioma.     

腺病毒介导的大鼠增殖抑制基因抑制大鼠胶质瘤的生长

目的:探讨腺病毒介导的大鼠增殖抑制基因(rat hyperplasia suppressor gene,rHSG)对大鼠胶质瘤生长的抑制作用.方法:建立大鼠C6胶质瘤动物模型,分别在接种C6胶质瘤细胞后第4和11天,于肿瘤原位注射0.9％氯化钠溶液(对照组)、重组腺病毒Adv-rHSG-GFP和腺病毒Adv-GFP,并于接种C6胶质瘤细胞后第9、15和21天取脑胶质瘤观察肿瘤的生长情况,免疫组织化学法检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)和rHSG蛋白的表达以及微血管密度(microvessel density,MVD),RT-PCR和蛋白质印迹法检测rHSG mRNA及蛋白的表达.结果:Adv-rHSG-GFP组肿瘤体积明显小于对照组和Adv-GFP组(P＜0.01).Adv-rHSG-GFP组PCNA标记指数和MVD均低于对照组和Adv-GFP组.Adv-rHSG-GFP组rHSG mRNA和蛋白的相对表达量均明显高于对照组和Adv-GFP组(P＜0.01).结论:Adv-rHSG-GFP能够抑制胶质瘤血管生成和肿瘤细胞增殖,对恶性胶质瘤的生长具有明显的抑制作用.     

Experimental study on the gene therapy of malignant glioma with antisense VEGF RNA

Objective: To study the effect of antisense VEGF RNA on rat C6 gliomas in vivo and find out the feasibility of antiangiogenesis therapy with antisense VEGF RNA formalignant gliomas. Methods: Parental rat C6 glioma cells and C6 cells transfected with antisense VEGF cDNA were implanted intracerebrally and subcutaneously into SD rats as control and transfected group. Rats bearing cerebral and subcutaneous C6 gliomas were treated with antisense VEGF cDNA as treated group and sense VEGF cDNA and empty vector as control of treated group. The general manifestation, survival time, MRI and histopathological changes of all rats were observed. The volume of subcutaneously implanted tumors was determined regularly. In situ hybridization and immunohistochemical staining were used for detection of VEGF gene expression of gliomas while PCNA immunostaining and TUNEL method for examination of proliferation activity and apoptosis of gliomas, respectively. Results: The survival of the rats in transfected and treated group was prolonged.There were two rats surviving over 90 d in the treated group and their tumors disappeared. The VEGF gene expression, the number of microvessels and the proliferation activity were decreased and a large amount of apoptotic cells could be found in cerebral and subcutaneous gliomas in treated and transfected groups. Conclusion:VEGF is one of the candidate genes for gene therapy of malignant gliomas. Antisense VEGF RNA combined with other therapies should be studied further for enhancing the therapeutic effect of malignant gliomas.     

Migratory patterns of lac-z transfected human glioma cells in the rat brain

Malignant brain tumors are characterized by extensive tumor-cell infiltration into the normal brain tissue. The present work describes the migratory behavior of human glioma cells transplanted into the adult rat brain with the aim of exploiting the extent of active cell migration and passive cell displacement within the central nervous system. To detect every transplanted tumor cell, a stably bacterial β-galactosidase (loc-z) transfected human glioma cell line was used. To distinguish between an active cell migration process and passive cell displacement, rat brains were also implanted with inert fluorescent polystyrene microspheres and the distribution of tumor cells and micro- spheres was studied 1 hr and 3 days after implantation. One hour after implantation the tumor cells were strictly localized at the implantation site. However, 3 days after implantation, both tumor cells and microspheres showed an extensive distribution within the brain. Confirming earlier neuropathological and experimental studies, it is shown that the loc-z-transfected glioma cells had the capacity to move within the Virchow-Robin and subarachnoid spaces. However, since fluorescent micro-spheres were also found in these areas, this spread of tumor cells may be primarily mediated by the extensive cerebrospinal fluid flow that exists within the brain. Three days after implantation, the glioma cells also showed an active migration over the corpus callosum. In comparison, the fluorescent microspheres showed only limited spread along the callosal body. It is concluded that the bacterial lac-z gene can be stably transfected into human glioma cells and, since every tumor cell can be visualized within the brain, this model provides a tool for studying the mechanisms behind tumor-cell invasion of the brain. © 1995 Wiley-Liss, Inc.     

In vivo tumor delivery of the green fluorescent protein gene to report future occurrence of metastasis

The green fluorescent protein (GFP) gene was administered to intraperitoneally (i.p.) growing human stomach cancer in nude mice to visualize future regional and distant metastases. GFP retroviral supernatants were injected i.p. from day 4 to day 10 after i.p. implantation of the cancer cells. Tumor and metastasis fluorescence was visualized every other week with the use of fluorescence optics via a laparotomy on the tumor-bearing animals. At 2 weeks after retroviral GFP delivery, GFP-expressing tumor cells were observed in gonadal fat, greater omentum, and intestine, indicating that these primary i.p. growing tumors were efficiently transduced by the GFP gene and could be visualized by its expression. At the second and third laparotomies, GFP-expressing tumor cells were observed to have spread to lymph nodes in the mesentery and other regional sites. At the fourth laparotomy, widespread tumor growth was visualized by GFP expression, inducing liver metastasis. No normal tissues were found to be transduced by the GFP retrovirus. Thus, reporter gene transduction of the primary tumor enabled detection of its subsequent metastasis. This gene therapy model could be applied to primary tumors before resection or other treatment to have a fluorescent early detection system for metastasis and recurrence.     

绿色荧光蛋白基因标记的胃癌细胞系的建立

目的 建立能连续传代稳定高表达绿色荧光蛋白（GFP）的胃癌细胞株,探讨胃癌的生长与转移特征。方法 利用脂质体将携带GFP-cDNA的真核表达载体质粒转染胃癌细胞株GC9811-P,经过G418筛选和克隆化培养,用荧光显微镜及流式细胞仪检测转染的EGFP基因在癌细胞体内外的表达,MTT比色法观察细胞生长,Western blot检测转染细胞和未转染细胞肿瘤相关抗原的表达。结果 携带EGFP的真核表达载体能有效地转染胃癌细胞GC9811-P,转染的细胞能稳定、高效和持久地表达EGFP,与未转染细胞比较,他们的生物学特性未改变（P〉0. 05）。在裸鼠皮下肿瘤中,EGFP也能稳定、高效的表达。结论 胃癌GC9811P-GFP的建立为研究肿瘤侵袭和转移的发生机制提供了理想的细胞株。     

Orthotopic Transplant Mouse Models with Green Fluorescent Protein-expressing Cancer Cells to Visualize Metastasis and Angiogenesis

There have been major efforts in metastasis research in recent years, especially on the role of angiogenesis in the metastatic process. Much of the information in this area has been obtained from model systems that are not representative of clinical cancer. The technique of surgical orthotopic implantation (SOI) has allowed the development of clinically relevant metastatic models of human cancer in immunodeficient rodents such as the nude and SCID mouse. In order to allow direct visualization of the metastatic process, we took advantage of the green fluorescent protein (GFP) of the jellyfish, Aequorea victoria. A series of cancer cell lines have been stably transfected with vectors containing humanized GFP cDNA. To utilize GFP expression for metastasis studies, fragments of subcutaneously growing tumor, which were comprised of GFP-expressing cells, were implanted by SOI in nude mice. Subsequent metastases were visualized in systemic organs by GFP fluorescence in the lung, liver, bones, brain and other organs down to the single-cell level. With this fluorescent tool, we detected and visualized for the first time tumor cells at the microscopic level in fresh viable tissue in their normal host organs even in the live animal. Angiogenesis is readily visualized in the transplanted GFP-expressing tumors in real time in situ in the live animal using simple laparotomy and fluorescent techniques. The results with the GFP-transfected tumor cells, combined with the use of SOI, demonstrate a fundamental advance to visualize and study cancer metastasis and the role of angiogenesis and other factors in the metastatic process. This revised version was published online in July 2006 with corrections to the Cover Date.     

Lymph node metastasis of oral cancer visualized in live tissue by green fluorescent protein expression

Here, we report the establishment of a stably transfected cell line which expresses high levels of green fluorescent protein (GFP), thus permitting the detection and visualization of developing tumors and lymph node metastases after injection into nude mice. Cells of the human oral squamous carcinoma cell line (SAS-L1) were transfected with an expression vector containing a cDNA encoding humanized GFP and the neomycin resistance gene. A clone with stable high-level expression of GFP was selected in vitro using G418. To study metastasis formation, GFP-expressing cells were injected orthotopically into the tongue of nude mice. The resultant tumor growth in the tongue and micrometastases in the lymph nodes could be visualized by GFP fluorescence. Therefore a useful model has been developed for the study of oral cancer, firstly to understand the metastatic process and secondly for the evaluation of potential treatments.