肿瘤相关成纤维细胞中G蛋白偶联雌激素受体胞浆转位介导的旁分泌对乳腺癌细胞生长的影响

目的:探讨乳腺癌微环境肿瘤相关成纤维细胞(CAFs)中G蛋白偶联雌激素受体(GPER)活化对成纤维细胞生长因子2(FGF2)的表达调控作用及其诱导的旁分泌对肿瘤细胞生长的影响。方法:免疫荧光法检测单独培养及与ER+乳腺癌MCF-7与ER-乳腺癌MDA-MB-468细胞共培养条件下CAFs和GPER突变型CAFs中GPER的表达定位。用荧光定量PCR法与ELISA法分别检测17-β雌二醇(E2)或GPER特异性激动剂G1处理后单独培养或与乳腺癌细胞共培养的CAFs细胞以及与乳腺癌细胞共培养的GPER突变型CAFs的FGF2mRNA表达和FGF2分泌水平;流式细胞术与CCK-8法检测与CAFs共培养的两种乳腺癌细胞经E2和G1处理后细胞增殖能力的变化,以及FGF2中和抗体的干预作用。结果:单独培养下,GPER定位于CAFs及GPER突变型CAFs的细胞核,与两种乳腺癌细胞共培养后,CAFs细胞中GPER出现明显胞浆转位,而GPER突变型CAFs无此现象。E2和G1处理后,共培养条件下的CAFs中FGF2的mRNA表达及上清液中的FGF2含量均明显增加(均P0.05),但单独培养的CAFs与共培养条件下的GPER突变型CAFs无上述变化(均P0.05)。E2和G1处理后,共培养条件下的两种乳腺癌细胞的增殖能力均明显增强(均P0.05),但该作用均被FGF2中和抗体取消。结论:在乳腺癌微环境下,CAFs中GPER有明显的胞浆转位,这有利于雌激素/GPER/FGF2通路的活化,从而可能促进在乳腺癌的进展。     

抗雄激素条件下癌相关成纤维细胞对前列腺癌LNCaP细胞增殖的影响

目的体外研究抗雄激素条件下癌相关成纤维细胞(cancer associated fibroblasts,CAFs)对雄激素依赖性前列腺癌LNCaP细胞增殖能力的影响。方法体外原代培养源于人前列腺癌基质的CAFs,并用抗雄激素制剂氟他胺(Flutamide)进行处理,制备出条件培养液CAFs-Flu-CM。CAFs原代培养液制备出另一条件培养液CAFs-CM作为对照研究。观察LNCaP细胞在RPMI-1640培养液、CAFs-CM培养液和CAFs-Flu-CM培养液中的生长增殖能力和差异性。结果与RPMI-1640培养液培养的LNCaP细胞相比,10-6mol/L Flutamide可抑制其生长(76.55±7.1 vs 114.51±9.8,P<0.05);浓度为0.75g/L的CAFs-CM却可显著提高LNCaP细胞的增殖能力(237.26±18.3 vs 114.51±9.8,P<0.05)。经10-6mol/L Flutamide处理后,CAFs-Flu-CM刺激LNCaP细胞增殖的能力明显减弱(32.73±5.6 vs 237.26±18.3,P<0.05)。结论源于人前列腺癌基质的CAFs可提高LNCaP细胞的增殖能力,但经抗雄制剂处理后,CAFs功能受到干扰,刺激LNCaP细胞增殖的能力明显减弱。联合靶向CAFs的肿瘤治疗策略可能会提高前列腺癌内分泌治疗的有效性,可作为临床治疗前列腺癌新的靶点。     

胰腺星状细胞通过白细胞介素-22影响胰腺癌侵袭转移及化疗耐药的研究

目的探讨具有不同白细胞介素-22(IL-22)表达水平的胰腺星状细胞(PSC)对胰腺癌细胞侵袭转移及化疗耐药的影响。方法将人原代胰腺癌间质PSC细胞与胰腺癌PANC-1细胞共培养后检测IL-22的表达及胰腺癌细胞的生长增殖能力、迁移和侵袭能力变化;构建稳定敲低或者过表达IL-22的人原代胰腺癌间质PSC细胞并与胰腺癌PANC-1细胞进行体外共培养, 检测PANC-1细胞的生长增殖、迁移和侵袭能力;利用稳定敲低或者过表达IL-22的人原代胰腺癌间质PSC细胞并与胰腺癌PANC-1细胞进行体外共培养后检测E-钙黏蛋白(E-cadherin)和波形蛋白(Vimentin)的表达情况;将含有不同表达水平的胰腺癌间质PSC细胞与胰腺癌PANC-1细胞体外共培养并加入不同浓度梯度的吉他西滨培养72 h后检测细胞存活率, 计算细胞耐受吉他西滨的半数抑制浓度(IC50);将稳定敲低或者过表达IL-22的人原代胰腺癌间质PSC细胞并与胰腺癌PANC-1细胞进行混合后种植于裸鼠皮下, 40 d后处理裸鼠取出肿瘤并绘制肿瘤生长曲线, 比较两组细胞的成瘤能力。采用t检验分析各组间差异的显著性。结果单纯PANC...     

CytoMPS原位疫苗治疗肝癌的抗瘤作用及其免疫效应

目的制作成细胞因子缓释微粒原位瘤苗(CytoMPS-ISV),观测CytoMPS-ISV抗瘤作用及其免疫效应。方法 C57BL/6J小鼠皮下移植性肝癌模型瘤内注射CytoMPS3次制作CytoMPS-ISV,观察其抗肿瘤作用及小鼠生存率;流式细胞仪检测小鼠外周血淋巴细胞变化,免疫组化法检测肿瘤组织的CD4+、CD8+和NK1.1+细胞浸润情况,体外检测小鼠脾CTL和NK细胞的杀瘤活性,检测疫苗诱导脾CTL的抗体阻断作用。结果与PBS注射组和对照组相比,瘤内注射CytoMPS后肿瘤生长受到显著抑制(P0.01)。瘤内注射CytoMPS组血液中及局部浸润的CD4+、CD8+和NK细胞明显高于PBS组和对照组(P0.05);淋巴细胞杀伤试验结果显示瘤内注射CytoMPS组小鼠的脾CTL对靶细胞的杀伤率明显高于PBS组(P0.01)和对照组(P0.05);抗体阻断作用试验显示接种疫苗的小鼠脾CTL的杀瘤活性可被抗CD8+、抗MHC-I单克隆抗体所阻断,但不被抗CD4+、抗MHC-II单克隆抗体所阻断。结论 CytoMPS-ISV可以显著抑制肿瘤生长,能明显增强机体的抗肿瘤免疫作用,其诱导的CTL杀瘤特性是由MHC-I限制的CD8+T细胞所介导。     

猪毛乳头细胞条件培养基的生物学活性研究

目的:研究猪毛乳头细胞条件培养基的生物学活性。方法:通过体外培养低传代猪毛乳头细胞,收集其基础培养基的上清液配制成条件培养基,用于培养高传代人毛乳头细胞,观察细胞形态及细胞生长曲线的变化;同时观察高传代人毛乳头细胞与低传代猪毛乳头细胞共培养情况。结果:条件培养基使高传代人毛乳头细胞出现了凝集生长现象,其生长曲线显著优于基础培养基培养的高传代人毛乳头细胞(P<0.05),其细胞的3H-TdR测定值亦显著升高(P<0.05)。高传代人毛乳头细胞与低传代猪毛乳头细胞共培养后出现凝集性生长趋势。结论:低传代猪毛乳头细胞在体外培养状态下可分泌具有明确生物学活性的物质。     

成年恒河猴骨髓基质干细胞的体外培养

目的温对猴骨髓基质干细胞(BMSCs)进行体外培养及扩增，观察其原代及传代细胞的生长特点及生物学特点。方法 抽取4只成年猴髂骨骨髓，用全骨髓培养法进行体外培养获得BMSCs，胰酶消化传代，用条件培养基培养传代细胞。逐日倒置显微镜观察细胞生长情况，对传代细胞进行HE染色及碱性磷酸酶(ALP)染色。结果 成年雄性恒河猴BMSCs体外培养生长良好，原代细胞10-13d汇成单层，传代后4～7d长满瓶底。HE染色光镜下观察见BMSCs为单核细胞，细胞呈梭形、多角形，传代细胞碱性磷酸酶染色呈强阳性。结论 猴BMSc的体外培养增殖能力强，可诱导为成骨细胞，可作为灵长类动物骨组织工程的种子细胞。     

DC疫苗对荷人前列腺癌免疫重建NOD/SCID小鼠的抑瘤作用

目的 探讨PC-3细胞冻融抗原致敏的树突状细胞(dendritic cells,DC)疫苗(PC-3-DC)对荷人前列腺癌免疫重建NOD/SCID小鼠(hu-PBL-NOD/SCID)的抑瘤作用.方法 采用人外周血淋巴细胞(peripheral blood lymphocytes,PBL)腹腔注射法建立hu-PBL-NOD/SCID小鼠模型,随机分为实验组(PC-3-DC组)和对照组(DC组、PBS组),腹腔分别注射PC-3-DC疫苗、未致敏的DC和PBS.每周1次,共2次,然后接种1×107 PC-3细胞,观察鼠成瘤率、成瘤潜伏期、肿瘤体积以及测定特异性CTL活性.结果 ELISA法可检测到小鼠血清中人IgG水平,hu-PBL-NOD/SCID嵌合模型重建成功,各组小鼠间成瘤率无明显差异,但PC-3-DC组成瘤潜伏期延长,肿瘤生长缓慢,2周后肿瘤体积明显小于DC组和PBS组,差异有统计学意义(P＜0.05),实验组脾淋巴细胞对PC-3细胞有特异性杀伤效应,而对K562细胞则无杀伤活性.结论 负载PC-3冻融抗原的DC疫苗可诱导人T淋巴细胞活化增殖,能有效抑制hu-PBL-NOD/SCID小鼠肿瘤的生长.     

人结肠癌相关成纤维细胞的原代培养及其生物学特性

目的 探讨人结肠癌相关成纤维细胞(CAFs)及结肠正常成纤维细胞(NFs)的培养和鉴定方法,分析两者在生物学特性和蛋白表达方面的差异.方法 采用组织块法和胶原酶消化法进行人结肠CAFs和NFs的原代培养;选择第3代人结肠CAFs及NFs,通过形态学观察和免疫细胞化学染色进行鉴定;细胞计数试剂盒(CCK-8)法检测人结肠CAFs和NFs的增殖活性,并绘制7d生长曲线;酶联免疫吸附试验(ELISA)法测定结肠CAFs和NFs细胞上清液中骨桥蛋白(OPN)、转化生长因子-β(TGF-β)、血管内皮生长因子(VEGF)及基质金属蛋白酶(MMP)-2含量.结果 结肠CAFs胞质突减少,细胞不规则,排列紊乱,可见双核及多核细胞,除细胞角蛋白(CK)外,波形蛋白(vimentin)与α平滑肌肌动蛋白(α-SMA)均为阳性表达;与结肠NFs比较,结肠CAFs增殖活性明显增强;ELISA法测定结肠CAFs和NFs细胞上清液中蛋白含量,其中OPN分别为(2.106±0.137) μg/L和(1.499±0.151) μg/L(P ＜0.01),TGF-β分别为(331.203±12.203) ng/L和(315.391±12.998) ng/L(P＜0.01),VEGF分别为(17.216±0.632) ng/L和(14.887 ±0.661) ng/L(P＜0.01),MMP-2分别为(1.830±0.192) μg/L和(1.436±0.102) μg/L(P ＜0.01).结论 通过组织块法和胶原酶消化法原代培养皆可获得高纯度的人结肠CAFs和NFs;两者在形态结构、增殖活性和蛋白表达等方面差异有统计学意义,这些差异可能是人结肠CAFs发挥其促结肠癌发生发展作用的生物学基础.     

Transwell小室内建立小鼠成骨-破骨细胞共培养体系

目的 :Transwell小室内建立体外小鼠成骨-破骨细胞共培养体系,并检测体系对成骨及破骨细胞活性的影响。方法:体外培育小鼠成骨细胞MC3T3-E1和小鼠单核巨噬细胞RAW264.7,RANKL诱导小鼠单核巨噬细胞RAW264.7分化为成熟破骨细胞后,于Transwell小室内建立成骨-破骨细胞共培养体系。通过CCK-8实验、茜素红染色、TRAP染色检测细胞的成骨、破骨活性。采用PCR、Western Blot方法检测成骨细胞MC3T3-E1中OPG、ALP、RANKL、TGF-b1的基因表达以及RANKL的蛋白表达,检测破骨细胞RANK、NF-κB的基因表达和蛋白表达。结果 :小鼠成骨细胞MC3T3-E1和小鼠破骨细胞可在Transwell小室内建立共培养体系;共培养体系影响小鼠成骨细胞与破骨细胞的分化活性,镜下可见成骨细胞分化增多,破骨细胞分化稍减少。共培养体系中成骨细胞基因OPG(0.65±0.08)、ALP(0.16±0.01)较单独培养OPG(1.00±0.08)、ALP(1.01±0.16)表达下降,而TGF-b1(4.42±0.21)、RANKL(4.12±1.04)较单独培养组TGF-b1(1.00±0.10)、RANKL(1.00±0.09)表达上升;破骨细胞相关RANK(0.63±0.06)、NF-κB(0.64±0.08)基因表达较单独培养组的RANK(1.00±0.08)、NF-κB(1.00±0.09)下降,差异均有统计学意义。同时共培养组的OPG(0.43±0.05)、NF-κB(0.59±0.05)的蛋白表达较单独培养组的OPG(0.84±0.06)、NF-κb(1.13±0.03)减少;共培养组RANKL(0.54±0.03)的蛋白表达则较单独培养组的RANKL(0.31±0.03)增加,差异有统计学意义,均与基因表达变化趋势一致。结论:小鼠成骨细胞MC3T3-E1和小鼠破骨细胞可在Transwell小室内建立共培养体系,共培养体系中成骨细胞活性高于破骨细胞活性。     

肝癌患者来源异种移植瘤模型的建立及药物敏感性研究

目的通过建立肝癌患者来源的异种移植瘤模型(PDX)探究肝癌索拉菲尼敏感性相关的长链非编码RNA(lncRNA)。方法选取上海市第一人民医院2021年5月至2022年10月的17例肝癌标本建立PDX模型。PDX模型传代14 d后通过随机数表法分为对照组与索拉菲尼给药组。给药21 d, 观察7 d后收取肿瘤组织, 给药期间记录小鼠体重。根据公式:肿瘤生长抑制(TGI)指数=ΔT/ΔC, 计算PDX肿瘤组织TGI指数并评价药物敏感性, 通过lncRNA测序筛选肝内胆管癌(ICC)索拉菲尼敏感与耐药的差异lncRNA。在人ICC细胞系HuCCT1中转染小干扰RNA(siRNA)降低lncRNA SCDAL的表达, 根据siRNA序列分组为ctr、si-1、si-2和si-3。采用细胞计数试剂盒(CCK-8)检测细胞增殖能力, 根据给药浓度分组为0 μmol/L(0.1%二甲基亚砜)、4 μmol/L、10 μmol/L。组间比较采用t检验, 多组间两两比较采用单因素方差分析, Turkey事后检验确定组间差异。结果肝细胞肝癌(HCC)及ICC PDX模型鼠索拉非尼给药组与对照组体重差异无统计学...     

A novel xenograft model of human hepatocellular carcinoma in immunocompetent mice based on the microcarrier-6

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignant tumors that threaten human health; thus, the establishment of an animal model with clinical features similar to human hepatocellular carcinoma is of important practical significance.MethodsTaking advantage of the novel microcarrier-6, human HCC cells were injected into immunocompetent mice to establish a novel human HCC patient-derived xenograft (PDX) model. Primary HCC cells were isolated from fresh hepatocellular carcinoma tissues, which were subsequently co-cultured with microcarrier-6 to construct a three-dimensional tumor cell culture model in vitro. The HCC-microcarrier complex was implanted into mice by subcutaneous inoculation, and the tumor formation time, tumor formation rate, and pathological manifestation were recorded. Changes of immune parameters in mice were detected by flow cytometry.ResultsThe success rate was 60% (6/10) in the establishment of hepatocellular carcinoma PDX mouse model, and the total tumor formation rate of the tumor-forming model is 90–100%. H&E staining and immunohistochemical experiments indicate that the model well retained the characteristics of the primary tumor. Interestingly, M2 macrophages in tumor-bearing mice increased significantly, and the levels of CD4⁺ T cells were significantly reduced.ConclusionsThrough the application of the microcarrier-6 in immunocompetent mice, we successfully established a novel human HCC PDX model, which can be used to better study and further elucidate the occurrence and pathogenic mechanism of HCC, improve the predictability of toxicity and drug sensitivity in HCC.     

胃癌细胞-树突状细胞融合疫苗对肿瘤细胞增殖周期影响的研究

目的为胃癌细胞-树突状细胞(DC)融合瘤苗免疫治疗胃癌患者提供实验依据及理论基础。方法采用细胞融合技术制备胃癌细胞-DC融合疫苗,对融合疫苗影响体外培养肿瘤细胞增殖周期及体内种植瘤组织细胞周期的特点进行研究。(1)胃癌患者外周血单个核细胞经粒细胞-巨噬细胞刺激因子(GM-CSF)、白介素(IL)-4、肿瘤坏死因子(TNF)-α诱导分化获取DC；(2)DC与SGC7901细胞经聚乙二醇诱导融合,HAT/HT选择培养获得纯净融合细胞；(3)流式细胞术检测融合疫苗对体外培养胃癌细胞周期及体内种植瘤组织细胞周期的影响。结果(1)外周血单个核细胞诱导分化可获得具备典型特征及细胞表型的DC；(2)DC与SGC7901细胞经PEG诱导融合,HAT/HT选择培养获得纯净融合细胞；(3)体内应用融合疫苗组癌细胞周期比例：G_0/G_1间期(76．77±4．38)%,S期(16．50±2．90)%,G_2/M期(6．73±1．59)%；与对照组比较,P＜0．01,差异有统计学意义。体内应用融合疫苗组种植瘤组织细胞增殖指数为23．34±3．51,与对照组(65．73±4．43)比较,P＜0．05,差异有统计学意义。结论融合细胞疫苗对肿瘤细胞增殖周期可产生显著影响,明显减慢肿瘤生长速度,抑制肿瘤细胞分裂增殖,是融合细胞疫苗发挥更强生物学效应的基础之一。     

树突状细胞肿瘤疫苗诱导抗胃癌作用的实验研究

目的探讨树突状细胞(dendriticcells,DCs)肿瘤疫苗诱导的抗胃癌效应对荷瘤裸小鼠的作用。方法使用胃癌细胞冻融抗原,体外致敏从小鼠骨髓诱导分化来源的DCs成为肿瘤疫苗,用其来刺激脾脏淋巴细胞,得到肿瘤抗原特异的细胞毒性T淋巴细胞(CTL),观察其对荷瘤裸小鼠肿瘤生长的影响以及早期凋亡诱导作用。结果经重组小鼠粒细胞巨噬细胞集落刺激因子(rmGM-CSF)和重组小鼠白细胞介素4(rmIL-4)体外诱导小鼠骨髓细胞,得到大量形态典型、具备强烈刺激增殖能力、高表达CD1a、CD11c、CD40和CD80的DCs。肿瘤抗原致敏DCs刺激脾脏淋巴细胞成为CTL后,可显著抑制裸小鼠皮下移植瘤生长并致瘤细胞早期凋亡增加。结论DCs肿瘤疫苗可通过CTL的作用,抑制荷瘤裸小鼠的胃癌细胞生长及促进胃癌细胞早期凋亡。     

The effectiveness of active specific immunotherapy using interferon-γ-gene-transduced tumor cells in a murine tumor model

Active specific immunotherapy was examined in BALB/c mice using sonicated tumor extract(SE) from plasmacytoma MOPC104E or interferon-γ-(IFN-γ)-genetransduced MOPC104E (Muγ), employing interleukin-1 (IL-1) as an adjuvant. Subcutaneous(s.c.) MOPC104E tumor growth was significantly suppressed in mice given a single preimmunization of IL-1 plus Muγ-SE, 9 days prior to inoculation, whereas the tumor growth in mice similarly pretreated with IL-1 alone or IL-1 plus MOPC104E-SE(MOPC-SE) was not affected; the mean tumor diameters on day 21 being 6.8mm, 15.3mm, and 13.2mm, respectively. Two-dose preimmunization with Muγ-SE alone or IL-1 alone given 10 and 7 days prior to s.c. inoculation also resulted in profound suppression of tumor growth compared to the control. As postsurgical immunization, MOPC104E cells were injected into the foot pads of mice, followed by amputation of the tumor-bearing foot 20 days later, then treatment with IL-1 plus MOPC-SE or IL-1 plus Muγ-SE on days 4, 7, and 10 after the amputation. The mean survival of the mice treated with IL-1 plus Muγ-SE was significantly prolonged compared to that of the mice treated with IL-1 plus MOPC-SE, at 90.3 daysvs 40.9 days, respectively (P<0.05 by the Cox-Mantel test). These results suggest that SE prepared from IFN-γ-genetransduced MOPC104E is more effective for active specific immunotherapy than SE prepared from MOPC104E.     

Combination effects of CDDP and hyperthermia in mouse bladder tumor (MBT-2)

The combination effects of CDDP and hyperthermia in mouse bladder tumor (MBT-2) were investigated both in vivo and in vitro. MBT-2 was transplanted into the hind leg of a C3H/He mouse. Then the leg was dipped in a hot water bath immediately after the intraperitoneal administration of CDDP. The antitumor effects were evaluated from tumor volume. The CDDP plus hyperthermia (43 degrees C) group showed remarkable tumor growth retardation. The in vitro colony forming assay showed that MBT-2 cultured cells treated with CDDP at 42 degrees C exhibited great colony forming inhibition in comparison with the CDDP alone cells. The effects of CDDP plus hyperthermia on the cell cycle progression of MBT-2 cultured cells were studied by using flow cytometry. The results showed that the cells treated with CDDP at 42 degrees C exhibited an accumulation of cells in the C2 phase for many hours as compared with the CDDP alone cells, indicating thermal enhancement of DNA damage in MBT-2 cultured cells treated with CDDP.     

Vitamin D Supplementation is a Promising Therapy for Pancreatic Ductal Adenocarcinoma in Conjunction with Current Chemoradiation Therapy

Background

The cancer-associated fibroblasts (CAFs) in pancreatic ductal adenocarcinoma (PDAC) are well known to play a dominant role in distant metastasis. Nevertheless, the effect on CAFs with current chemoradiation therapies remains uncertain.

Objective

This study aimed to reveal the role of CAFs under current chemoradiation therapy (CRT) and investigate the factors regulating CAFs.

Methods

α-SMA-positive cells in 86 resected PDAC specimens with/without preoperative CRT were evaluated by immunohistochemistry. Various factors, including the plasma levels of vitamin D, were investigated for association with the number of CAFs or distant metastasis-free survival (DMFS). Human pancreatic satellite cells (hPSCs) extracted from clinical specimens were used to validate the factors.

Results

All PDAC samples contained CAFs but the number varied widely. Multivariate analysis for DMFS indicated a larger number of CAFs was a significant risk factor. Univariate analysis for the number of CAFs identified two clinical factors: preoperative CRT and lower plasma levels of vitamin D. In subgroup analysis, the higher plasma level of vitamin D was a dominant factor for longer DMFS in PDAC patients after preoperative CRT. These results were validated by using extracted hPSCs. Irradiation activated stromal cells into CAFs facilitating malignant characteristics of PDAC and the change was inhibited by vitamin D supplementation in vitro.

Conclusion

In conjunction with established current therapies, vitamin D supplementation may be an effective treatment for PDAC patients by inactivating CAFs.

     

Gene therapy-mediated expression by tumor cells of the angiogenesis inhibitor flk-1 results in inhibition of neuroblastoma growth in vivo

BACKGROUND/PURPOSE: Preventing tumors from forming new blood vessels appears to be an effective new anticancer approach. Antiangiogenic therapy usually is cytostatic, however, and, therefore, long-term angiogenesis inhibition is likely to be required. The objective of this study was to determine if sustained gene therapy-mediated expression of these agents from tumor cells could restrict tumor growth in vivo. METHODS: Two replication-defective retroviral vectors were made, one encoding both the soluble, truncated vascular endothelial growth factor receptor (VEGF-R2), flk-1, together with green fluorescent protein (GFP), and the other encoding GFP alone. These vectors were then used to transduce murine neuroblastoma cells (NXS2). Stable, high expression of the flk-1 transgene was confirmed in the former population of cells by Western analysis. Flk-1 protein was isolated from cell culture supernatants and tested in human umbilical vein endothelial cell (HUVEC) proliferation and migration assays to confirm that functional protein was being made. Finally, in vivo activity was assessed by injecting 10(6) tumor cells subcutaneously into SCID mice and monitoring subsequent tumor growth. RESULTS: Purified flk-1 (0.1 micromol/L) was able to inhibit basic fibroblast growth factor (bFGF) stimulated HUVEC proliferation by 44% and VEGF-stimulated migration by 30%. In vitro growth rates for the transduced cell lines were similar to the unmodified cell line. In vivo, however, after 23 days, tumors from flk-1 expressing neuroblastoma cells were less than 33% the average volume of tumors from cells expressing only the GFP transgene (mean volume, 1.9 cm(3) v 5.8 cm(3), P<.001). GFP expression alone had no effect on tumor growth when compared with unmodified tumor cells. CONCLUSIONS: Engineered expression of flk-1, a competitive inhibitor of VEGF, by tumor cells results in the production of an inhibitor of endothelial cell proliferation and migration that greatly restricts the growth of the tumor cells in vivo. Gene therapy-mediated delivery of angiogenesis inhibitors may provide an alternative approach to treating refractory tumors such as neuroblastoma.     

乳腺癌间质成纤维细胞对MDA-MB-231种植肿瘤生长和转移的影响及其机理探讨

目的通过裸鼠接种方式进行体内实验,观察乳腺癌间质成纤维细胞（CAFs）对种植肿瘤生长和转移的影响,并探讨其作用机理。方法选择MDA．MB．231乳腺癌细胞系细胞（简称MDA）、乳腺癌患者的CAFs和癌旁正常乳腺组织的正常成纤维细胞（NFs）,结合不同的试剂,包括生理盐水（NS）、基质细胞衍生因子-1（SDF-1）配体拈抗剂1,4,8,11．四氮杂环十四烷（AMD3100,简称AMD）,组合成以下6种处理：MDA＋NS、NFs＋NS、MDA＋NFs＋NS、MDA＋NFs＋AMD、MDA＋CAFs＋AMD及MDA＋CAFs＋NS。将36只Bal b／c无胸腺裸小鼠按配伍随机方法分成6组,分别接受上述处理。将细胞悬液接种于裸鼠,接种后饲养46d处死,观察种植肿瘤的大小,有无淋巴结、肺及肝脏转移。采用ELISA法检测6组裸鼠的血浆SDF-1浓度,采用real-timePCR法检测6组裸鼠肿瘤组织中的SDF．1mRNA水平,采用Westernblot法检测6组裸鼠肿瘤组织中SDF-1蛋白的表达水平。结果除NFs＋NS组外,其余5组均有种植肿瘤形成。MDA＋CAFs＋NS组裸鼠的种植肿瘤体积[（9．092±2．662）cm3]、血浆SDF．1浓度[（75．25±16．23）ng／L]、肿瘤组织中SDF-1 mRNA（中位数为14．714）及其蛋白（中位数为0．673）的表达水平均最高,与其他5组比较差异均有统计学意义（P〈0．050）。6组裸鼠的肝和肺组织均未见转移灶。但MDA＋CAFs＋NS组有4只、MDA＋NS组有2只裸鼠存在腋窝淋巴结转移,其他组均未发现腋窝淋巴结转移。结论乳腺癌CAFs对乳腺癌的生长起促进作用,且可促进肿瘤淋巴结的转移;其作用可能是通过乳腺癌CAFs分泌SDF-1,与其特定的受体即趋化因子受体4（cxcR4）结合来实现的。     

Targeting of Insulin-like Growth Factor-I Receptor with a Monoclonal Antibody Inhibits Growth of Hepatic Metastases from Human Colon Carcinoma in Mice

Background Colorectal carcinomas (CRC) express high levels of insulin-like growth factor-I/II (IGF-I/II) and the receptor (IGF-IR). We hypothesized that selective inhibition of IGF-IR would inhibit hepatic growth of human CRC in mice. Methods Human CRC cells were treated in vitro with anti-IGF-IR monoclonal antibody (MoAB) with and without oxaliplatin to assess cytotoxicity. The effect of anti-IGF-IR MoAB on IGF-I-induced vascular endothelial growth factor (VEGF) production in human CRC cells was assessed by Northern blot and ELISA. We injected human CRC cells intrahepatically in nude mice, and then administered anti-IGF-IR MoAB with and without oxaliplatin. We delayed treatment in one group until large hepatic tumors were present. We assessed tumors for apoptosis, proliferation, and angiogenesis. Results Anti-IGF-IR MoAB and oxaliplatin inhibited CRC cell growth in vitro and combination treatment was even more effective. IGF-I stimulation of CRC cells resulted in significant upregulation of VEGF and this was completely inhibited by pretreatment with anti-IGF-IR MoAB. Anti-IGF-IR MoAB significantly inhibited hepatic growth of tumors in mice. Anti-IGF-IR MoAB plus oxaliplatin led to a significantly greater inhibition of tumor growth. Anti-IGF-IR MoAB plus oxaliplatin was just as effective at inhibiting growth of larger, more advanced liver tumors. Anti-IGF-IR MoAB, alone and in combination with oxaliplatin, led to a significant increase in tumor cell apoptosis, and a significant inhibition of tumor cell proliferation and angiogenesis. Conclusions These findings suggest that IGF-IR is a potential target for therapy in patients with advanced CRC.     

Use of verapamil to enhance the antiproliferative activity of BCNU in human glioma cells: an in vitro and in vivo study

The authors have evaluated the antiproliferative activity of verapamil, alone or in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in brain-tumor cells. These effects were studied in vitro using four human glioma cell lines and in vivo using glioblastoma multiforme cells transplanted to athymic nude mice. The results showed that verapamil when used alone produced inhibition of tumor growth; however, when verapamil was used in combination with BCNU (in vitro), significant dose-dependent suppression of proliferation occurred in all four cell lines. The in vivo results were far more dramatic. Mice treated with BCNU (25 mg/kg) plus verapamil (50 mg/kg) achieved a 200-fold decrease in tumor growth with a greater than 80% regression in tumor size. Complete cures were achieved in 80% of the mice observed for at least 50 days following the completion of therapy. These findings support the use of verapamil in overcoming drug resistance in malignant brain tumors.