摘要
目的“血管生成拟态(VM)”是高侵袭恶性肿瘤与经典“肿瘤血管生成”完全不同、不依赖血管内皮细胞的全新肿瘤血管供血模式。胆囊癌作为手术切除率低、易复发转移、预后差的高恶性肿瘤，是否也存在VM，迄今未见报道。本研究通过人胆囊癌石蜡标本、体外细胞三维培养及其干预实验，初探胆囊癌是否存在VM，以及其形态学特征、分子机制、临床意义和NCTD对胆囊癌VM的影响和机制。
方法①收集手术切除经病理确诊原发性胆囊癌、胆囊腺瘤、慢性胆囊炎石蜡标本74例、10例、10例和相关临床病理参数；应用HE染色、CD_(31)和PAS双重染色，观察胆囊癌是否存在VM，并作临床病理参数相关分析、Cox分析、Kaplan-Meier曲线绘制。②通过体外细胞二维培养、Transwell小室侵袭实验、胶原收缩实验、细胞毒实验及CD_(31)免疫荧光检测，明确GBC-SD、SGC-996细胞侵袭、迁移潜能，NCTD IC_(50)及HUVEC细胞鉴定。在此基础上，通过鼠尾Ⅰ型胶原三维培养进行胆囊癌VM体外模拟实验和药物干预实验，实验分GBC-SD细胞对照组、SGC-996细胞对照组、TIMP_1组、TIMP_2和NCTD组，应用HE和PAS染色、光学和电子显微镜观察三维培养组织切片体外模拟VM形态学。③应用SABC、Envision法免疫组化和ELISA放免方法测定人胆囊癌石蜡切片、三维培养组织切片和二、三维培养上清液中VM相关分子MMP_1、MMP_2、MMP_9、MT_1-MMP、VEGF、VE-cad和Ⅳ胶原蛋白表达，RT-PCR测定胆囊癌细胞表达内皮细胞相关基因。
结果①在74例人胆囊癌中发现13.5％(10／74)存在VM，胆囊癌VM与病人性别、年龄、肿瘤部位、瘤体大小、分化程度、Nevin分期、浸润深度、局部淋巴转移无关；但与组织类型(χ~2=10.241，P=0.017)，生存期(χ~2=5.7221，P=0.0168)有关，可能由于VM(+)阳性标本少，本组胆囊癌VM和肝脏转移无显著相关。②GBC-SD和SGC-996细胞的NCTD IC_(50)分别为56.18μg·ml~(-1)和22.66μg·ml~(-1)，GBC-SD细胞体外穿过Transwell小室侵袭能力(P=0.0013)和收缩迁移能力(P＜0.001)明显高于SGC-996细胞，故GBC-SD、SGC-996细胞分属高、低侵袭潜能细胞株。经HE和PAS染色，高倍、倒置光相差显微镜观察，鼠尾Ⅰ型胶原和Matrigel胶三维培养GBC-SD细胞密集如癌巢，48h即形成以肿瘤细胞为内衬的基质型单环或多环中央空洞网络状管道结构，14d管腔成熟；而低侵袭性SGC-996细胞始终未形成模拟VM的管道状结构。③相关分子表达，对胆囊癌石蜡切片检测显示：VM(+)、VM(-)胆囊癌细胞MMP_1、MMP_2、MMP_9、MT1-MMP、VEGF表达明显高于胆囊腺瘤、胆囊炎(P＜0.0001)，VE-Cad、Ⅳ型胶原明显低于胆囊腺瘤、胆囊炎(P＜0.0001)；VM(+)胆囊癌MMP_2(F=5.74，P=0.0187)、MT1-MMP(F=8.78，P=0.0039)表达明显高，而VEGF(F=5.78，P=0.0182)、Ⅳ型胶原(F=5.80，P=0.0181)明显低于VM(-)胆囊癌，MMP_2、MT1-MMP与胆囊癌VM呈正相关，VEGF、Ⅳ型胶原与胆囊癌VM呈负相关；VM(+)胆囊癌MMP_1(F=0.27，P=0.6082)、MMP_9(F=2.80，P=0.0976)、VE-Cad(F=1.37，P=0.2450)表达与VM(-)胆囊癌无差别。对三维培养组织切片检测显示：GBC-SD细胞MMP_1、MMP_2、MMP_9、MT1-MMP、VEGF表达明显高于SGC-996细胞。MMP-1在VM(-)组表达与分化程度、Nevin分期、淋巴结转移、肝脏转移密切相关，并随分化程度越低、Nevin分期越晚(S3～S5)、淋巴结及肝脏转移表达量增加，有显著差异(P＜0.05)，在VM(+)组与临床病理指标无关(P＞0.05)，同为肝脏转移组，VM(+)组MMP-1表达显著高于VM(-)组(P＜0.05)。MMP-2在VM(-)组表达与浸润深度、分化程度、Nevin分期、淋巴结转移、肝脏转移密切相关，并随侵及浆膜、分化程度越低、Nevin分期越晚(S3～S5)、淋巴结及肝脏转移表达量增加，有显著差异(P＜0.05)，在VM(+)组与临床病理类型腺癌有关(P＜0.05)，同为淋巴结、肝脏转移组、中低分化腺癌、浸润深度，Nevin分期(S3～S5)，VM(+)组MMP-2表达均显著高于VM(-)组(P＜0.05)。MMP-9在VM(-)组表达与浸润深度、Nevin分期、淋巴结转移、肝脏转移密切相关，并随侵及浆膜、分化程度越低、Nevin分期越晚(S3～S5)、肝脏转移表达量增加，有显著差异(P＜0.05)，在VM(+)组与浸润深度、Nevin分期、肝脏转移有关，并随侵及浆膜、Nevin分期越晚(S3～S5)、肝脏转移表达量增加，有显著差异(P＜0.05)，同为侵及浆膜组，腺癌，Nevin分期(S3～S5)、肝脏转移，VM(+)组MMP-9表达均显著高于VM(-)组(P＜0．05)。MMP-14在无论VM(-)组和VM(+)组其表达与浸润深度、Nevin分期、淋巴结转移、肝脏转移密切相关，并随侵及浆膜、Nevin分期越晚(S3～S5)、肝脏转移表达量增加，有显著差异(P＜0.05)，同为侵及浆膜组，Nevin分期(S3～S5)、淋巴结及肝脏转移，VM(+)组MMP-14表达均显著高于VM(-)组(P＜0.05)。VEGF在VM(-)组表达与浸润深度、Nevin分期、淋巴结转移、肝脏转移密切相关，并随侵及浆膜、Nevin分期越晚(S3～S5)、肝脏转移表达量增加，有显著差异(P＜0.05)，在VM(+)组仅肝脏转移组表达明显高于无肝脏转移，有显著差异(P＜0.05)。同一病理指标下，VM(-)组和VM(+)组表达VEGF无差异(P＞0.05)，VE-cad在VM(-)组表达与浸润深度、Nevin分期、淋巴结转移、分化程度相关，并随侵及浆膜、Nevin分期越晚(S3～S5)、淋巴结转移、分化差表达量减少，有显著差异(P＜0.05)，在VM(+)组与临床病理指标无关(P＞0.05)，同一病理指标下，VM(-)组和Ⅷ(+)组表达VE-cad无差异(P＞0.05)，Ⅳ胶原在VM(-)组表达与浸润深度、Nevin分期、分化程度、肝脏转移相关，并随侵及浆膜、Nevin分期越晚(S3～S5)、肝脏转移、分化差表达量减少，有显著差异(P＜0.05)，在VM(+)组Ⅳ胶原表达与浸润深度、分化程度相关，并随侵及浆膜，分化差表达量减少，有显著差异(P＜0.05)，同为淋巴结肝脏转移，VM(+)组Ⅳ胶原表达均显著低于VM(-)组(P＜0.05)。二、三维培养上清液ELISA检测显示：三维培养GBC-SD细胞MMP2蛋白表达较三维SGC-996细胞、二维GBC-SD明显增高(P＜0.05)，而三维培养GBC-SD细胞MMP_9蛋白表达较三维SGC-996细胞、二维GBC-SD增高不显(P＞0.05)，RT-PCR显示具有Ⅷ能力的GBC-SD可以强表达内皮细胞相关基因，而SGC-996仅弱或不表达④应用NCTD后，鼠尾Ⅰ型胶原三维培养48h，GBC-SD细胞即丧失形成单环或多环网络样结构能力，细胞稀疏、浮起、固缩，或聚集、核固缩碎裂，细胞凋亡、坏死；TIMP_2则起这种作用的时间明显延迟，TIMP_1无此作用。表明，NCTD有抑制体外模拟胆囊癌VM能力，TIMP_2，TIMP_1无阻止体外模拟胆囊癌VM能力。通过三维培养组织切片和上清液检测，1／2CI_(50)NCTD不仅使MMP_2、MT1-MMP，而且使MMP_1、MMP_9、VEGF阳性的GBC-SD细胞减少、染色变浅，蛋白表达下降；且随时间延长，GBC-SD细胞MMP_2、MMP_9蛋白表达明显受抑制(P＜0.05)，表明，NCTD不仅有效抑制GBC-SD细胞MMP_2、MT1-MMP，而且抑制MMP_1、MMP_9、VEGF、RT-PCR显示NCTD可以有效抑制ANG-1、ANG-2、EPHA2、VEGF表达，从而通过多靶点作用抑制和破坏体外模拟胆囊癌VM的形成和成熟。
结论人原发性胆囊癌存在VM；胆囊癌VM与组织学类型、生存期相关。高侵袭潜能GBC-SD细胞在三维培养中具有形成以癌细胞为内衬的单环或多环网络结构的体外模拟VM能力。其分子机制涉及MMP_2、MT1-MMP，VEGF、EPHA2、Ⅳ型胶原等相关分子。NCTD可有效抑制体外模拟胆囊癌VM；其机制可能与NCTD通过多靶点抑制上述相关分子表达，从而发挥阻遏、破坏形成体外模拟胆囊癌VM的作用。
Objective Vasculogenic mimicry (VM) is a new vessel mode and extravascular fluid pathway for blood supply of aggressive malignant tumor, with its no-reliance on endothelium cells and its difference from classical angiogenesis. It is not known if there is VM in human gallbladder carcinomas, a highly lethal and aggressive malignant tumor with unresectable, early metastasis or invasion and poor prognosis. Based on this viewpoint, we studied the VM in human gallbladder carcinomas, and its morphological character, molecular mechanisms and clinical significance, and the effect of NCTD on VM of gallbladder carcinomas and its mechanisms in vitro.
Methods①74 carcinomas, 10 adenomas and 10 chronic inflammatory lesions of the gallbladder underwent operation and confirmed histopathologically, and their clinical- pathlogical dates were studied. VM in human gallbladder carcinomas and its correlation with clinical-pathlogical dates was observed and analyzed by HE dye, CD_(31) and PAS dyes and light microscope. Cox multiple factor and postoperative 5-year survival rate(Kaplan-Meier survival curve) evalution.②The invasiveness, movement and NCTD' IC_(50) of GBC-SD cells and SGC-996 cells were studied by two-dimensional cultures, Transwell invasive experiment, collagen gel contraction experiment and cell toxic test in vitro. HUVEC was identified by CD_(31) immunofluorescence. The VM models of gallbladder carcinomas established by rat-tail collagen I three-dimensional cultures of GBC-SD cells were used to evaluate the therapy effect of NCTD for VM of gallbladder carcinomas in vitro. The expriments were randomly divided into the control groups of GBC-SD cells and SGC-996 cells, TIMP_1 group, TIMP_2 group and NCTD group, and were taken different treatment. The morphological changes of the VM models of gallbladder carcinomas in vitro were observed by HE and PAS dyes, light and electron microscope.③Expression of the VM related molecules such as MMP_1, MMP_2, MMP_9, MT_1-MMP, VEGF, VE-cad and collagen IV on sections from formalin-fixed paraffin-embedded blocks of human gallbladder carcinomas in vivo, on sections and in supernates from the two and three-dimensional culture tissues of GBC-SD cells and GBC-SD cells in vitro was determined by streptavidin-biotin complex method(SABC), Envision method and ELISA method. RT-PCR was performed in the regions of all tumor cells to observe the gene expression level of endothelial cells.
Results①13.5% (10/74) of human gallbladder carcinomas were found to contain vasculogenic mimicry (VM), namely intratumoral, tumor cell-lined extracellular matrix (ECM)-rich, PAS-positive and vasculogenic-like network patterns that is associated with histological type (χ~2=10.241, P=0.017) and poor overall survival (χ~2=5.7221, P=0.016). VM is also not correlated with sex、age、location and diameter of carcinoma、differentiation degree、Nevin stage、serosal invasion、lymph node metastatis. because the sample number with VM(+) was not sufficient, VM is not associated with liver metastatis.②IC_(50) of NCTD for GBC-SD cells and SGC-996 cells was 56.18μg·ml~(-1) and 22.66μg·ml~(-1) respectively. The potential capacity of Transwell invasiveness (P=0.0013) and collagen gel contraction (P＜0.001) in GBC-SD cells was increased significantly than those of SGC-996 cells in vitro. GBC-SD cells and SGC-996 cells were so called as high- and low-invasive cell lines. Nest-like tumor cells and the network patterns composed of tumor cell-lined, patterned matrix type, loop or back-to-back morn loops network were detected in three-dimensional cultures of GBC-SD cells on matrigel and collagenⅠmatrix by HE and PAS dyes, light and electron microscope. These network patterns were initially formed at 48h, maturated at 14 days. But, SGC-996 cells were found to be incapable of forming throughout the vasculogenic-like network in three-dimensional cultures.③Expression of MMP_1, MMP_2, MMP_9, MT_1-MMP and VEGF was increased significantly in carcinomas with or without VM than adenomas and inflammatory lesions of the gallbladder (P＜0.0001) in vivo. Moreover, expression of VE-cad and collagenⅣwas decreased significantly in carcinomas with or without VM than adenomas and inflammatory lesions of the gallbladder (P＜0.0001). Expression of MMP_2 (F=5.74, P=0.0187) and MT_1-MMP (F=8.78, P=0.0039) in the gallbladder carcinomas with VM was increased significantly than that of the gallbladder carcinomas without VM, that is showed positive correlation between expression of MMP_2, MT_1-MMP and VM of gallbladder carcinomas. Expression of VEGF (F=5.78, P=0.0182) and collagenⅣ(F=5.80, P=0.0181) in the gallbladder carcinomas with VM was decreased significantly than that of the gallbladder carcinomas without VM, that is cued negative correlation between expression of VEGF, collagenⅣand VM of gallbladder carcinomas. Moreover, there is no correlation on expression of MMP_1 (F=0.27, P=0.6082), MMP_9 (F=2.80, P=0.0976) and VE-Cad (F=1.37, P=0.2450) between the gallbladder carcinomas with VM and the gallbladder carcinomas without VM. In the examinations on sections from the three-dimensional culture tissues in vitro, expression of MMP_1、MMP_2、MMP_9、 MT_1-MMP was increased significantly in GBC-SD cells than SGC-996 cells. Moreover, expression of VEGF was also increased significantly in GBC-SD cells than SGC-996 cells, the expression of MMP-1 was closely correlated with cell differention、Nevin stage、lymph node metastases、liver metastatis in case of gallbladder carcinomas without VM. it's expression was significantly higher than that without lymph node and liver metastatis、high differention、Nevin stage(S1～S2) (P＜0.05). there was no correlation between MMP-1 protein expression and pathologic index in case of gallbladder carcinomas with VM(p＞0.05). In the same condition of liver metastatis, MMP-1 protein expression with VM was higher than that of without VM (P＜0.05). the expression of MMP-2 was markedly correlated with cell differention、Nevin stage、lymph node metastases、liver metastatis、serosal invasion in case of gallbladder carcinomas without VM. Increased expression of MMP-2 was observed in the case of lower differention、Nevin stage(S3～S5)、lymph node metastases、liver metastatis、serosal invasion (P＜0.05). In the same condition of liver metastatis, cell differention, Nevin stage(S3～S5)、lymph node metastases、lower differention MMP-2 protein expression with VM was higher than that of without VM (P＜0.05). a significant relationship was observed between MMP-9 expression without VM and Nevin stage、lymph node metastases、liver metastatis、serosal invasion. Increased expression of MMP-9 was observed in the case of lower differention、Nevin stage(S3～S5)、lymph node metastases、liver metastatis、serosal invasion (P＜0.05). the expression of MMP-9 was also markedly correlated with Nevin stage、liver metastatis、serosal invasion in case of gallbladder carcinomas with VM (P＜0.05). In the same condition of liver metastatis、Nevin stage(S3～S5)、serosal invasion, histological type MMP-9 protein expression with VM was higher than that of without VM ( P＜0.05 ). the expression of MMP-14 was significantly related to the Nevin stage、liver metastatis、serosal invasion、lymph node metastases whether VM or not. Increased expression of MMP-14 was observed in the case of Nevin stage(S3～S5)、lymph node metastases、liver metastatis、serosal invasion (P＜0.05). In the same condition of liver metastatis、Nevin stage(S3～S5)、serosal invasion, lymph node metastases MMP-9 protein expression with VM was higher than that of without VM (P＜0.05). VEGF was closely correlated with Nevin stage、lymph node metastases、liver metastatis、serosal invasion in case of gallbladder carcinomas without VM. Increased expression of VEGF was observed in the case of Nevin stage(S3～S5)、liver metastatis、serosal invasion (P＜0.05). the expression of VEGF was also markedly correlated with liver metastatis in case of gallbladder carcinomas with VM (P＜0.05). there was not any different expression of VEGF between VM and without VM according to the same condition, a significant relationship was observed between VE-cad expression without VM and Nevin stage、lymph node metastases、liver metastatis、serosal invasion、differentiation degree, decreased expression of VE-cad was observed in the case of Nevin stage(S3～S5)、lymph node metastases、lower differentiation、serosal invasion (P＜0.05). there was no correlation between VE-cad protein expression and pathologic index in case of gallbladder carcinomas with VM(p＞0.05). there was not any different expression of VE-cad between VM and without VM according to the same condition. There was correlation betweenⅣcollagen and Nevin stage、liver metastatis、serosal invasion、or differentiation degree. decreased expression ofⅣcollagen was observed in the case of Nevin stage(S3～S5)、lower differentiation、serosal invasion、liver metastatis(P＜0.05) without VM. the expression ofⅣcollagen was also markedly correlated with serosal invasion、differentiation degree in case of gallbladder carcinomas with VM (P＜0.05). In the same condition of liver metastatis、lymph node metastasesⅣcollagen expression without VM was higher than that of with VM (P＜0.05), examinations on supernates from the cell cultures in vitro, expression of MMP_2 in GBC-SD cells cultured by collagen 1 matrix was increased significantly, it's expression was significantly higher than that of three--dimensional cultures of SGC-996 cells and the two-dimensional cultures of GBC-SD cells(P＜0.05), expression of MMP_9 in GBC-SD cells cultured by collagen 1 matrix was not increased significantly, it's expression was not significantly higher than that of three--dimensional cultures of SGC-996 cells and the two-dimensional cultures of GBC-SD cells(P＞0.05), gene expression of endothelial cells were strongly observed in three--dimensional cultures of GBC-SD cells, negative or slight exoression were observed in three--dimensional cultures of SGC-996 cells④After treatment with NCTD, GBC-SD cells in three-dimensional cultures on rat-tail collagenⅠat 48h wasn't able completely to form loop or loops vasculogenic-like network; sparseness, floating, shrinkage and aggregation of cell, nuclear shrinkage, chromosome condensation, karyorrhexis, apoptosis, necrosis in some GBC-SD cells were observed by light and electric microscope. Moreover, TIMP_2 exert the inhibitory effect on GBC-SD cells lingeringly when being compared with NCTD (5d vs. 48h), TIMP_1 hasn't the effect. It is shown that inhibitory effect of NCTD for the simulated VM of gallbladder carcinomas is more powerful and earlier than TIMP_2. TIMP1 and TIMP2 couldan't prevent the formation of vasculogenic mimicry. The reduced cell amounts, the shallow dye and the decreased expression of not only MMP_(2-), MT1-MMP-positive GBC-SD cells but also MMP_(1-), MMP_(9-), VEGF-positive GBC-SD cells were observed by examination for sections and supernates from the cell culture tissues after treatment with 1/2CI_(50) NCTD in vitro. As the effect time of NCTD prolonged, the expression of MMP_2 and MMP_9 proteins of GBC-SD cells was inhibited significantly, which is also showed obvious difference on expression of MMP_2 and MMP_9 between the three-dimensional cultures of GBC-SD cells and the two-, three-dimensional cultures of SGC-996 cells and the two-dimensional cultures of GBC-SD cells, which isn't able to form the simulated VM of gallbladder carcinomas. It has been demonstrated that NCTD inhibit effectively not only MMP_2 and MT1-MMP of GBC-SD cells, but also MMP_1, MMP_9, VEGF. further pullback and destroy the form and mature of the simulated VM of gallbladder carcinomas by the more target-point effects. NCTD could effiectively prevent the expression of ANG-1, ANG-2, EPHA2, VEGE
Conclusions Human gallbladder carcinomas were found to have vasculogenic mimicry that is associated with histological type, liver metastasis and poor overall survival. High-invasive potential GBC-SD cells were able to form tumor cell-lined, loop or more loops and simulated VM network of gallbladder carcinomas in three-dimensional cultures of collagenⅠmatrix. Its mechanisms correlate with the expression change of related-molecules such as MMP_2 and MT1-MMP, might also correlate with the expression change of MMP_1, MMP_9, VEGF, EPHA2 and collageⅣ. NCTD can inhibit effectively the simulated VM of gallbladder carcinomas in vitro. Its effect mechanisms was found to associate whith the inhibition of above-mentioned related-molucule expressions by more target-point effects, consequently exert the therapitic effect in the form and mature of the simulated VM of gallbladder carcinomas.
引文
[1] Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis[J]. N Engl J Med, 1995, 333(26):1757-1763
[2] Risau W. Mechanisms of angiogenesis[J]. Nature,1997,386(6626):671-674
[3] Weidner N, Folkman J.Tumoral vascularity as a prognostic factor in cancer[J]. Important Adv Oncol,1996,184:167-190.
[4] Teo NB, Shoker BS, Martin L, et al.Angiogenesis in pre-invasive cancers[J]. Anticancer Res, 2002, 22(4):2061-2072.
[5] Thompson WD, Li WW, Maragoudakis M ,et al. The clinical manipulation of angiogenesis: pathology, side-effects, surprises, and opportunities with novel human therapies[J]. J Pathol,2000,190(3):330-337.
[6] Maniotis AJ, Folberg R, Hess A, et al. Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry[J]. Am J Patho1,1999,155(3):739-752.
[7] Folberg R, Hendrix MJ, Maniotis A J, et al. Vasculogenic mimicry and tumor angiogenesis[J]. Am J Pathol. 2000;156(2):361-381.
[8] Bissell MJ. Tumor plasticity allows vasculogenic mimicry, a novel form of angiogenic switch. A rose by any other name[J]? .Am J Pathol,1999,155(3):675-679.
[9] McDonald DM, Munn L, Jain RK, et al.Vasculogenic mimicry: how convincing, how novel, and how significant[J]? Am J Pathol,2000,156(2):383-388.
[10] Shubik P, Warren BA. Additional literature on "vasculogenic mimicry" not cited [J].Am J Pathol, 2000,156(2):736
[11] Kakinuma T, Nakamura K, Wakugawa M, et al. IL-4, but not IL-13, modulates TARC (thymus and activation-regulated chemokine)/CCL17 and IP-10 (interferon-induced protein of 10kDA)/CXCL10 release by TNF-alpha and IFN-gamma in HaCaT cell line[J]. Cytokine, 2002,20(1):1-6.
[12] Barbetakis N, Antoniadis T, Kaplanis K, et al. Metastatic squamous cell vulvar carcinoma of the lung:a case report and review of the literature[J]. Eur J Gynaecol Oncol,2002,23(5):475-476.
[13] Lee YJ, Nagai N, Siar CH, et al. Angioarchitecture of primary oral malignant melanomas[J]. J Histochem Cytochem ,2002, 50(11):1555-1562.
[14] Sood AK, Seftor EA, Fletcher MS, et al. Molecular determinants of ovarian cancer plasticity. [J]. Am J Pathol, 2001,158(4):1279-1288.
[15] Hendrix MJ, Seftor EA, Kirschmann DA, et al. Molecular biology of breast cancer metastasis. Molecular expression of vascular markers by aggressive breast cancer cells[J]. Breast Cancer Res ,2002,2(6):417-422.
[16] Shirakawa K, Wakasugi H, Heike Y, et al. Vasculogenic mimicry and pseudo-comedo formation in breast cancer[J]. Int J Cancer, 2002, 99(6):821-828.
[17] Shirakawa K, Kobayashi H, Sobajima J, et al. Inflammatory breast cancer: vasculogenic mimicry and its hemodynamics of an inflammatory breast cancer xenograft model[J]. Breast Cancer Res, 2003, 5(3):136-139.
[18] Cai Xuansong, Jia Yongwei, Mei Jiong, et aL Tumor blood vessels formation in osteosarcoma: vasculogenesis mimicry[J]. Chin J Med,2004,117(1):94-98.
[19] Shanna N, Seftor RE, Seftor EA, et al. Prostatic tumor cell plasticity involves cooperative interactions of distinct phenotypic subpopulations: role in vasculogenic mimicry[J]. Prostate ,2002,50(3):189-201.
[20] Liu C, Huang H, Donate F, et al. Prostate-specific membrane antigen directed selective thrombotic infarction of tumors[J]. Cancer Res, 2002, 62(19):5470-5475.
[21] Hao X, Sun B, Zhang S, et al. Microarray study of vasculogenic mimicry in bi-directional differentiation malignant tumor[J] .Zhonghua Yi Xue Za Zhi, 2002 ,82(19):1298-1302.
[22] Buijs JT, Cleton AM, Smit VT, et al. Prognostic significance of periodic acid-Schiff-positive patterns in primary breast cancer and its lymph node metastases[J]. Breast Cancer Res Treat, 2004,84(2):117-130
[23] Warso M, Maniotis AJ,Chen X, et al. Prognostic significance of periodic acid-Schiff-positive patterns in primary cutaneous melanoma. [J]. Clin Cancer Res ,2001,7(3):473-477.
[24] Herbst RS, Yano S, Kuniyasu H, et al .Differential expression of E-cadherin and type IV collagenase genes predicts outcome in patients with stage I non-small cell lung carcinoma [J]. Clin Cancer Res,2000,6(3):790-797.
[25] Chang YS, di Tomaso E, McDonald DM, et al .Mosaic blood vessels in tumors: frequency of cancer cells in contact with flowing blood[J]. Proc Natl Acad Sci U S A.,2000,97(26):14608-14613
[26] Folkman J. Can mosaic tumor vessels facilitate molecular diagnosis of cancer[J]? Proc Natl Acad Sci U S A, 2001,98(2):398-400.
[27] Maniotis AJ, Chen X, Garcia C. et al, Control of melanoma morphogenesis, endothelial survival, and perfusion by extracellular matrix[J]. Lab Invest. 2002 ,82(8): 1031-1043.
[28] Seftor EA, Meltzer PS, Kirschmann DA, et al, Molecular determinants of human uveal melanoma invasion and metastasis[J]. Clin Exp Metastasis, 2002,19(3):233-246.
[29] Folberg R, Chen X, Boldt HC, et al,Microcirculation patterns other than loops and networks in choroidal and ciliary body melanomas[J].Ophthalmology,2001,108(5):996-1001.
[30] Shirakawa K, Kobayashi H, Heike Y, et al, Hemodynamics in vasculogenic mimicry and angiogenesis of inflammatory breast cancer xenograft[J]. Cancer Res,2002,62(2):560-566.
[31] Ruf W, Seftor EA, Petrovan RJ, et al. Differential role of tissue factor pathway inhibitors 1 and 2 in melanoma vasculogenic mimicry[J]. Cancer Res, 2003,63(17):5381-5389.
[32] Hao XS,Sun BC,Zhang SW,et al. Correlation between the expression of collgen IV,VEGF and vasculogenic mimicry[J]. Zhonghua Zhong liu za zhi,2003,25(6):524-526.
[33] Eftor RE, Seftor EA, Koshikawa N, et al. Cooperative interactions of laminin 5 gamma2 chain, matrix metalloproteinase-2, and membrane type-1-matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma[J].Cancer Res,2001,61(17):6322-6327.
[34] Kobayashi H, Shirakawa K, Kawamoto S, et al. Rapid accumulation and internalization of radiolabeled herceptin in an inflammatory breast cancer xenograft with vasculogenic mimicry predicted by the contrast-enhanced dynamic MRI with the macromolecular contrast agent G6-(1B4M-Gd)(256) [J]. Cancer Res,2002,62(3):860-866.
[35] Seftor EA, Meltzer PS, Schatteman GC, et al. Expression of multiple molecular phenotypes by aggressive melanoma tumor cells: role in vasculogenic mimicry[J]. Crit Rev Oncol Hematol,2002 ,44(1):17-27.
[36] Hendrix MJ, Seftor EA, Kirschmann DA, et al. Molecular biology of breast cancer metastasis. Molecular expression of vascular markers by aggressive breast cancer cells[J]. Breast Cancer Res,2000,2(6):417-422.
[37] Bittner M, Meltzer P, Chen Y, et al .Jiang Y Molecular classification of cutaneous malignant melanoma by gene expression profiling[J]. Nature, 2000,406(6795):536-540.
[38] Hendrix MJ, Seftor EA, Hess AR, et al. Vasculogenic mimicry and tumour-cell plasticity: lessons from melanoma[J]. Nat Rev Cancer, 2003,3(6):411-421.
[39] Timar J, Toth J, Dome B, et al. Tumoral sinuses or vascular channels in tumors[J]. Magy Onkol, 2000, 44(2):105-107.
[40] Hendrix MJ, Seftor EA, Meltzer PS, et al. Expression and functional significance of VE-cadherin in aggressive human melanoma cells: role in vasculogenic mimicry[J]. Proc Natl Acad Sci, 2001,98(14):8018-8023.
[41] Hess AR, Seftor EA, Seftor RE, et al. Phosphoinositide 3-kinase regulates membrane Type 1-matrix metalloproteinase (MMP) and MMP-2 activity during melanoma cell vasculogenic mimicry[J]. Cancer Res, 2003, 63(16):4757-4762.
[42] Hendrix MJ, Seftor EA, Kirschmann DA, et al. Remodeling of the microenvironment by aggressive melanoma tumor cells [J].Ann N Y Acad Sci,2003,995:151-161.
[43] Hendrix MJ, Seftor EA, Hess AR, et al. Molecular plasticity of human melanoma cells [J]. Oncogene, 2003,22(20):3070-3075.
[44] Straume O, Akslen LA. Importance of vascular phenotype by basic fibroblast growth factor, and influence of the angiogenic factors basic fibroblast growth factor/fibroblast growth factor receptor-1 and ephrin-A1/EphA2 on melanoma progression[J]. Am J Pathol,2002,160(3):1009-1019.
[45] Colognato H, Yurchenco PD .Form and function: the laminin family of heterotrimers[J]. Dev Dyn,2000,218(2):213-234.
[46] Eftor RE, Seftor EA, Kirschmann DA, et al. Targeting the tumor microenvironment with chemically modified tetracyclines: inhibition of laminin 5 gamma2 chain promigratory fragments and vasculogenic mimicry[J]. Mol Cancer Ther ,2002, 1(13):1173-1179.
[47] Sood AK, Fletcher MS, Coffin JE, et al. Functional role of matrix metalloproteinases in ovarian tumor cell plasticity[J]. Am J Obstet Gynecol, 2004, 190(4):899-909.
[48] ischel EG, Riewald M, Huang HY, et al. Tumor cell adhesion and migration supported by interaction of a receptor protease complex with its inhibitor[J]. J Clin Invest, 1999,104(9):1213-1221.
[49] uf W, Seftor EA, Petrovan R J, et al. Differential role of tissue factor pathway inhibitors 1 and 2 in melanoma vasculogenic mimicry[J]. Cancer Res, 2003, 63(17):5381-5389.
[50] Shinoda E, Yui Y, Hattori R, Tanaka M, et al. Tissue factor pathway inhibitor-2 is a novel mitogen for vascular smooth muscle cells[J]. J Biol Chem. 1999,274(9):5379-5384.
[51] Mori Y, Wada H. TF (tissue factor), TFPI (tissue factor pathway inhibitorO [J] .Nippon Rinsho,2004,62 Suppl 12:629-631.
[52] Hess AR, Seftor EA, Gardner LM, et al. Molecular regulation of tumor cell vasculogenic mimicry by tyrosine phosphorylation: role of epithelial cell kinase (Eck/EphA2) [J].Cancer Res ,2001,61(8):3250-3255.
[52] Sood AK, Seflor EA, Fletcher MS, et al. Molecular determinants of ovarian cancer plasticity [J].Am J Pathol,2001,158(4):1279-1288.
[53] Massi D, Franchi A, Paglierani M, et al Vasculogenic mimicry has no prognostic significance in pT3 and pT4 cutaneous melanoma [J]. Hum Pathol,2004,35(4):496-502.
[54] Sun B, Zhang S, Zhao X, Zhang W, et al .Vasculogenic mimicry is associated with poor survival in patients with mesothelial sarcomas and alveolar rhabdomyosarcomas [J]. Int J Oncol,2004 ,25(6):1609-1614.
[55] McDonald DM, Foss AJ. et al. Endothelial cells of tumor vessels: abnormal but not absent[J]. Cancer Metastasis Rev, 2000,19(1-2):109-120.
[56] Hlatky L, Hahnfeldt P, Folkman J, et al Clinical application of antiangiogenic therapy: microvessel density, what it does and doesn't tell us[J]. J Natl Cancer Inst. 2002,94(12):883-893.
[57] Ferrara N, Winer J, Burton T, et al Expression of vascular endothelial growth factor does not promote transformation but confers a growth advantage in vivo to Chinese hamster ovary cells[J]. Clin Invest,1993,91(1):160-170.
[58] Kalekou H, Miliaras D. Immunohistochemical study of microvessel density, CD44 (standard form), p53 protein and c-erbB2 in gallbladder carcinoma[J]. J Gastroenterol Hepatol,2004,19(7):812-818.
[59] Neuchrist C, Erovic BM, Handisurya A, et al Vascular endothelial growth factor receptor 2 (VEGFR2) expression in squamous cell carcinomas of the head and neck[J]. Laryngoscope,2001,lll(10):1834-1841.
[60] Wagholikar GD, Behari A, Krishnani N, et al .Early gallbladder cancer[J]. J Am Coll Surg,2002,194(2):137-141.
[61] Sasaki R, Takahashi M, Funato O, et al .Hepatopancreatoduodenectomy with wide lymph node dissection for locally advanced carcinoma of the gallbladder-long-term results[J]. Hepatogastroenterology,2002,49(46):912-915.
[62] Lucas M, Rose PE, Morris AG et al. Contrasting effects of HSP72 expression on apoptosis in human umbilical vein endothelial cells and an angiogenic cell line, ECV304[J]. Br J Haematol, 2000,110(4):957-964.
[63] Sood AK, Fletcher MS, Hendrix MJ. et al. The embryonic-like properties of aggressive human tumor cells[J]. J Soc Gynecol Investig,2002,9(1):2-9.
[64] Wu LT, Chung JG, Chen JC, et al. Effect of norcantharidin on N-acetyltransferase activity in HepG2 cells[J]. Am J Chin Med.2001,29(1):161-172.
[65] Bennin DA, Don AS, Brake T,et al. Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G1/S phase cell cycle arrest[J]. J Biol Chem,2002 ,277(30):27449-27467.
[66] Liu XH, Blazsek I, Comisso M, et al. Effects of norcantharidin, a protein phosphatase type-2A inhibitor, on the growth of normal and malignant haemopoietic cells[J]. Eur J Cancer, 1995 ,31A(6):953-963.
[67] McCluskey A, Walkom C, Bowyer MC,et al. Cantharimides: a new class of modified cantharidin analogues inhibiting protein phosphatases 1 and 2A[J]. Bioorg Med Chem Lett,2001 ,ll(22):2941-2946.
[68] McCluskey A, Ackland SP, Gardiner E,et al. The inhibition of protein phosphatases 1 and 2A: a new target for rational anti-cancer drug design? [J] Anticancer Drug Des,2001,16(6):291-303.
[69] Baba Y, Hirukawa N, Tanohira N,et al. Structure-based design of a highly selective catalytic site-directed inhibitor of Ser/Thr protein phosphatase 2B (calcineurin) [J]. J Am Chem Soc, 2003,125(32):9740-9749.
[70] Doerfler P, Forbush KA,Peflmutter RM,et al. Caspase enzyme activity is not essential for apoptosis during thymocyte development [J]. J Immunol,2000,164(11):4071-4079.
[71] An WW, Wang MW, Tashiro SI,et aL Norcantharidin Induces Hurrtan Melanoma A375-S2 Cell Apoptosis through Mitochondrial and Caspase Pathways [J]. J Korean Med Sci, 2004,19(4):560-566.
[1] Maniotis AJ, Folberg R, Hess A, et al. Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry[J]. Am J Pathol ,1999,155(3):739-752.
[2] Lee YJ, Nagai N, Siar CH, et al. Angioarchitecture of primary oral malignant melanomas[J]. J Histochem Cytochem ,2002, 50(11):1555-1562.
[3] Sood AK, Seftor EA, Fletcher MS, et al. Molecular determinants of ovarian cancer plasticity. [J]. Am J Pathol, 2001,158(4):1279-1288.
[4] Hendrix MJ, Seftor EA, Kirschmann DA, et al. Molecular biology of breast cancer metastasis. Molecular expression of vascular markers by aggressive breast cancer cells[J]. Breast Cancer Res ,2002,2(6):417-422.
[5] Shirakawa K, Wakasugi H, Heike Y, et al. Vasculogenic mimicry and pseudo-comedo formation in breast cancer[J]. Int J Cancer, 2002, 99(6):821-828.
[6] Shirakawa K, Kobayashi H, Sobajima J, et al. Inflammatory breast cancer: vasculogenic mimicry and its hemodynamics of an inflammatory breast cancer xenografl model[J]. Breast Cancer Res, 2003, 5(3):136-139.
[7]丁印鲁，李兆亭．ets-1基因的反义寡核甘酸对胃癌体外血管生成拟态的影响[J]．外科理论与实践，2003，8(5)：394-396．
[8] Cai Xuansong, Jia Yongwei, Mei Jiong, et al. Tumor blood vessels formation in osteosarcoma: vasculogenesis mimicry[J]. Chin J Meal,2004,117(1):94-98.
[9] Sharma N, Seftor RE, Seftor EA, et al. Prostatic tumor cell plasticity involves cooperative interactions of distinct phenotypic subpopulations: role in vasculogenic mimicry[J]. Prostate ,2002,50(3):189-201.
[10] Liu C, Huang H, Donate F, et al. Prostate-specific membrane antigen directed selective thrombotic infarction of tumors[J]. Cancer Res, 2002, 62(19):5470-5475.
[11] Buijs JT, Cleton AM, Smit VT, et al. Prognostic significance of periodic acid-Schiff-positive patterns in primary breast cancer and its lymph node metastases[J].. Breast Cancer Res Treat, 2004,84(2):117-130.
[12] Warso M, Maniotis AJ,Chen X, et al. Prognostic significance of periodic acid-Schiff-positive patterns in primary cutaneous melanoma. [J]. Clin Cancer Res ,2001,7(3):473-477.
[13] Folberg R, Hendrix MJ, Maniotis AJ, et al .Vasculogenic mimicry and tumor angiogenesis[J]. Am J Pathol, 2000,156(2):361-381.
[14] Timar J, Toth J, Dome B, et al. Tumoral sinuses or vascular channels in tumors[J]. Magy Onkol, 2000, 44(2):105-107.
[15] Hao XS,Sun BC,Zhang SW, et al. Correlation between the expression of collgen IV,VEGF and vasculogenic mimicry[J]. Zhonghua Zhong liu za zhi,2003,25(6):524-526.
[16] Ruf W, Seftor EA, Petrovan RJ, et al. Differential role of tissue factor pathway inhibitors 1 and 2 in melanoma vasculogenic mimicry[J]. Cancer Res, 2003, 63(17):5381-5389.
[17] Hendrix MJ, Seftor EA, Meltzer PS, et al. Expression and functional significance of VE-cadherin in aggressive human melanoma cells: role in vasculogenic mimicry[J]. Proc Nail Acad Sci, 2001,98(14):8018-8023.
[18] Hess AR, Seftor EA, Seftor RE, et al. Phosphoinositide 3-kinase regulates membrane Type 1-matrix metalloproteinase (MMP) and MMP-2 activity during melanoma cell vasculogenic mimicry[J]. Cancer Res, 2003, 63(16):4757-4762.
[19] Eftor RE, Seftor EA, Kirschmann DA, et al. Targeting the tumor microenvironment with chemically modified tetracyclines: inhibition of laminin 5 gamma2 chain promigratory fragments and vasculogenic mimicry[J]. Mol Cancer Ther ,2002, 1(13):1173-1179.
[20] Error RE, Seftor EA, Koshikawa N, et al. Cooperative interactions of larninin 5 gamma2 chain, matrix metalloproteinase-2, and membrane type-1-matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma[J].Cancer Res,2001,61(17):6322-6327.
[21] Sood AK, Fletcher MS, Coffin JE, et al. Functional role of matrix metalloproteinases in ovarian tumor cell plasticity[J]. Am J Obstet Gynecol, 2004, 190(4):899-909.
[22] Fischel EG, Riewald M, Huang HY, et al. Tumor cell adhesion and migration supported by interaction of a receptor protease complex with its inhibitor[J]. J Clin Invest, 1999,104(9):1213-1221.
[23] Hess AR, Seftor EA, Gardner LM, et al. Molecular regulation of tumor cell vasculogenic mimicry by tyrosine phosphorylation: role of epithelial cell kinase (Eck/EphA2) [J].. Cancer Res ,2001,61(8):3250-3255.
[24] Clarijs R, Schalkwijk L, Ruiter D, et al. Lack of lymphangiogenesis despite coexpression of VEGF-C and its receptor fit-4 in uveal melanoma[J]. Invest Ophthalmol Vis Sci, 2001, 42(7):1422-1428.
[25] Laakkonen P, Porkka K, Hoffman JA, et al. A tumor-homing peptide with a targeting specificity related to lymphatic vessels[J]. Nature Med ,2002,8(7):751-755.
[1] Maniotis AJ, Folberg R, Hess A, et al. Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry[J]. Am J Patho1,1999, 155(3):739-752.
[2] Sood AK, Seftor EA, Fletcher MS, et al. Molecular determinants of ovarian cancer plasticity[J]. Am J Patho1,2001,158(4):1279-1288.
[3] Shirakawa K, Wakasugi H, Heike Y, et al. Vasculogenic mimicry and pseudo-comedo formation in breast cancer[J]. Int J Cancer ,2002,99(6):821-828.
[4] Shirakawa K, Kobayashi H, Sobajima J, et al. Inflammatory breast cancer: vasculogenic mimicry and its hemodynamics of an inflammatory breast cancer xenograft model. Breast[J]. Cancer Res ,2003, 5(3):136-139.
[5] 丁印鲁，李兆亭．ets—1基因的反义寡核甘酸对胃癌体外血管生成拟态的影响[J]．外科理论与实践，2003，8(5)：394-396．
[6] Cai Xuansong, Jia Yongwei, Mei Jiong, et al. Tumor blood vessels formation in osteosarcoma: vasculogenesis mimicry[J]. Chin J Med ,2004,117(1):94-98.
[7] Sharma N, Seftor RE, Seftor EA, et al. Prostatic tumor cell plasticity involves cooperative interactions of distinct phenotypic subpopulations: role in vasculogenic mimicry[J]. Prostate, 2002,50(3):189-201.
[8] Liu C, Huang H, Donate F, et al. Prostate-specific membrane antigen directed selective thrombotic infarction of tumors[J]. Cancer Res ,2002, 62(19):5470-5475.
[9] Warso M, Maniotis AJ,Chen X,et al. Prognostic significance of periodic acid-Schiff-positive patterns in primary cutaneous melanoma[J]. Clin Cancer Res, 2001,7(3):473-477.
[10] Hendrix MJ, Seftor EA, Meltzer PS, et al. Expression and functional significance of VE-cadherin in aggressive human melanoma cells: role in vasculogenic mimicry[J]. Proc Natl Acad Sci ,2001,98(14):8018-8023.
[11] Hess AR, Seflor EA, Gardner LM, et al. Molecular regulation of tumor cell vasculogenic mimicry by tyrosine phosphorylation: role of epithelial cell kinase (Eck/EphA2)[J]. Cancer Res ,2001, 61(8):3250-3255.
[12] Hess AR, Seftor EA, Seftor RE, et al. Phosphoinositide 3-kinase regulates membrane Type 1-matrix metalloproteinase (MMP) and MMP-2 activity during melanoma cell vasculogenic mimicry[J]. Cancer Res, 2003, 63(16):4757-4762.
[13] Sood AK, Fletcher MS, Coffin JE, et al. Functional role of matrix metalloproteinases in ovarian tumor cell plasticity[J]. Am J Obstet Gyneco1,2004,190(4):899-909.
[14] Seflor RE, Seftor EA, Kirschmann DA, et al. Targeting the tumor microenvironment with chemically modified tetracyclines: inhibition of laminin 5 gamma2 chain promigratory fragments and vasculogenic mimicry[J]. Mol Cancer Ther, 2002, 1(13):1173-1179.
[15] Seftor RE, Seftor EA, Koshikawa N, et al. Cooperative interactions of laminin 5 gamma2 chain, matrix metalloproteinase-2, and membrane type-1-matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma[J].Cancer Res,2001,61(17):6322-6327.
[16] Ruf W, Seftor EA, Petrovan RJ, et al. Differential role of tissue factor pathway inhibitors 1 and 2 in melanoma vasculogenic mimicry[J]. Cancer Res , 2003,63(17):5381-5389.
[17] Clarijs R, Schalkwijk L, Ruiter D, et al. Lack of lymphangiogenesis despite coexpression of VEGF-C and its receptor fit-4 in uveal melanoma[J]. Invest Ophthalmol Vis Sci, 2001, 42(7):1422-1428.
[18] Laakkonen P, Porkka K, Hoffman JA, et al. A tumor-homing peptide with a targeting specificity related to lymphatic vessels[J]. Nature Med ,2002,8(7):751-755.
