Analysis of micromechanisms of metastasis using NOD/SCID-hu and GFP-transfectant human colorectal carcinoma cells

【目的】本项目旨在研究肿瘤转移的微观机制。【材料与方法】采用四种人结直肠癌细胞系CCL188、CX-1、MIP101和克隆A。将CRC细胞分别悬浮于PBS、Matrigel或Cellmatrix （I型胶原）中，在裸鼠盲肠中原位植入CRC细胞。采用RT-PCR、ELISA和免疫组织化学分析结直肠癌细胞中血管生成因子的表达。用稳定的gfp转染细胞系，用CD31免疫染色和活体荧光显微镜（IVFM）观察肿瘤血管生成。【结果】CCL188和CX-1细胞系接种于裸鼠脾脏后，几乎100%的小鼠产生肝脏肿瘤，而其他两种细胞系很少形成肝脏肿瘤。将人结直肠癌细胞原位植入裸鼠体内时，matrigel悬浮细胞产生局部肿瘤的效果明显高于悬浮在PBS或Cellmatrix中的细胞。Matrigel也仅通过两种高转移性CRC细胞系促进自发转移到肝脏。然而，Matrigel原位植入CRC细胞不会增加腹膜转移。4种结直肠癌细胞系均不同程度地释放VEGF，其中CCL188和CX-1释放的VEGF量大于MIP101和克隆a。抗小鼠CD31免疫染色显示，结直肠癌细胞植入3天后，肿瘤血管生成受到诱导。这一结果与转染GFP的体外培养下观察到的肿瘤血管生成的结果很好地吻合。两种高度转移的细胞系诱导的微血管密度高于其他两种细胞系。人结直肠癌细胞与正常人骨髓细胞共植入未诱导出来源于人内皮细胞的肿瘤血管。【结论】Matrigel对裸鼠原位移植人CRC细胞的局部肿瘤生长和转移有促进作用。结直肠癌细胞产生VEGF可能与肿瘤中诱导的微血管密度有关。少

[Purpose] The purpose of this project was to study the micromechanisms of cancer metastasis. [Materials and Methods] Four human colorectal carcinoma (CRC) cell lines, CCL188, CX-1, MIP101 and Clone A, were used. Orthotopic implantation of CRC cells in the cecum of nude mice were carried out with the CRC cells suspeneded either n PBS, Matrigel or Cellmatrix (type I collagen). Expression of angiogenic factors in the CRC cells were analyzed by RT-PCR, ELISA and immunohistochemistry. Tumor angiogenesis was observed by CD31 immuno-staining and intravital fluorescence microscopy (IVFM) with stable GFP-transfectants of each cell line. [Results] When inoculated into the spleens of nude mice CCL188 and CX-1 produced tumors in the liver in almost 100% of mice whereas the other two cell lines rarely formed hepatic tumors. When human CRC cells were implanted orthotopically in nude mice Matrigel-suspended cells produced local tumors with significantly higher efficacy than the cells suspended in PBS … More or Cellmatrix. Matrigel also enhanced spontaneous metastasis to the liver only by the two highly metastatic CRC cell lines. However, orthotopic implantation of CRC cells with Matrigel did not increase peritoneal metastasis. AH the four CRC cell lines released VEGF with different extents while CCL188 and CX-1 did greater amount than did by MIP101 and Clone A. Anti-mouse CD31 immno-staining showed induction of tumor angiogenesis 3 days after implantation of CRC cells. The results were well compatible with the findings of tumor angiogenesis observed under the IVFM with GFP transfectants. The two highly metastatic cell lines induced higher density of microvessels than the other two cell lines did. Co-implantation of human CRC cells with normal human bone marrow cells did not induce tumor vessels of human endothelial cell origin. [Conclusions] Matrigel enhances local tumor growth and metastasis of orthotopically implanted human CRC cells in nude mice. There may be a correlation between the production of VEGF by CRC cells and the density of microvessels induced in the tumors. Less