Induction of Angiogenesis by Hyperplastic Colonic Mucosa Adjacent to Colon Cancer
2000; Elsevier BV; Volume: 157; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)64790-6
ISSN1525-2191
AutoresHiroki Kuniyasu, Wataru Yasui, Hisashi Shinohara, Seiji Yano, Lee M. Ellis, Michael R. Wilson, Corazon D. Bucana, Tadayoshi Rikita, Eiichi Tahara, Isaiah J. Fidler,
Tópico(s)Gastric Cancer Management and Outcomes
ResumoWe determined whether hyperplastic mucosa adjacent to colon cancer contributes to neoplastic angiogenesis. Surgical specimens of human colon cancer (40 Dukes' stage B and 34 Dukes' stage C) were analyzed by immunohistochemistry for expression of proliferative and angiogenic molecules. The mucosa adjacent to Dukes' stage C tumors (but not Dukes' stage B tumors) had a higher Ki-67 labeling index and a higher expression of epidermal growth factor receptor and transforming growth factor-α than distant mucosa. The expression levels of vascular endothelial growth factor, basic fibroblast growth factor, interleukin-8, and the vascular density in the adjacent mucosa were similar to those in the tumor lesions and significantly higher than those in the distant mucosa. The expression of interferon-β inversely correlated with the level of pro-angiogenic molecules and the vascular density. The injection of metastatic human colon cancer cells and murine colon cancer cells into the cecal wall of mice induced hyperplastic changes in the adjacent mucosa which expressed higher levels of epidermal growth factor receptor, basic fibroblast growth factor, and vascular endothelial growth factor, and lower levels of interferon-β than did the control mucosa, which directly correlated with the degree of hyperplasia. These data suggest that metastatic human colon cancer cells can induce hyperplasia in the adjacent mucosa, which in turn produces angiogenic molecules that contribute to neoplastic angiogenesis. We determined whether hyperplastic mucosa adjacent to colon cancer contributes to neoplastic angiogenesis. Surgical specimens of human colon cancer (40 Dukes' stage B and 34 Dukes' stage C) were analyzed by immunohistochemistry for expression of proliferative and angiogenic molecules. The mucosa adjacent to Dukes' stage C tumors (but not Dukes' stage B tumors) had a higher Ki-67 labeling index and a higher expression of epidermal growth factor receptor and transforming growth factor-α than distant mucosa. The expression levels of vascular endothelial growth factor, basic fibroblast growth factor, interleukin-8, and the vascular density in the adjacent mucosa were similar to those in the tumor lesions and significantly higher than those in the distant mucosa. The expression of interferon-β inversely correlated with the level of pro-angiogenic molecules and the vascular density. The injection of metastatic human colon cancer cells and murine colon cancer cells into the cecal wall of mice induced hyperplastic changes in the adjacent mucosa which expressed higher levels of epidermal growth factor receptor, basic fibroblast growth factor, and vascular endothelial growth factor, and lower levels of interferon-β than did the control mucosa, which directly correlated with the degree of hyperplasia. These data suggest that metastatic human colon cancer cells can induce hyperplasia in the adjacent mucosa, which in turn produces angiogenic molecules that contribute to neoplastic angiogenesis. 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We examined the expression of the pro-angiogenic molecules vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and interleukin-8 (IL-8) and the anti-angiogenic regulator interferon-β (IFN-β) in 74 surgical specimens of human colon cancer and found that the hyperplastic mucosa produces high levels of pro-angiogenic molecules. We also implanted murine and human colon cancer cells into the cecal wall of nude mice and found that the growing tumor lesions induce hyperplasia in the adjacent mucosa that in turn expresses high levels of pro-angiogenic molecules directly correlating with a high degree of vascularity. Seventy-four formalin-fixed, paraffin-embedded archival surgical specimens of human primary colon adenocarcinomas that invaded the subserosal layer from four patients treated at The University of Texas M. D. Anderson Cancer Center and 70 patients treated at the J. R. Hiroshima General Hospital of the West Japan Railway Company were chosen at random. In 34 of 74 cases, lymph node metastasis was detected (Dukes' stage C), and 40 cases had no lymph node metastases (Dukes' stage B). For each case, the tumor lesion, the adjacent mucosa (within 2 mm of the tumor), and nonpathological control mucosa (at least 10 cm from the edge of the tumor) were studied. 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The adherent monolayer cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2 in air. All cultures were free of mycoplasma, reovirus type 3, pneumonia virus of mice, K virus, encephalitis virus, lymphocyte choriomeningitis virus, ectromelia virus, and lactate dehydrogenase virus (assayed by M. A. Bioproducts, Walkersville, MD). Specific pathogen-free male BALB/c mice and male athymic NCr-nu/nu mice were purchased from the Animal Production Area of the National Cancer Institute–Frederick Cancer Research and Development Center (Frederick, MD). Animals were maintained according to institutional guidelines in facilities approved by the American Association for Accreditation of Laboratory Animal Care in accordance with current regulation and standards of the United States Department of Agriculture, Department of Health and Human Services, and National Institutes of Health. The mice were used according to the institutional guidelines when they were 8 to 10 weeks old. Modified Eagle's medium, Ca2+- and Mg2+-free Hanks' balanced salt solution, and fetal bovine serum were purchased from M. A. Bioproducts. 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Tumors were harvested 7 to 28 days after injection. Mice were injected intravenously with 0.2 ml saline containing 250 μg anti-5-bromo-2-deoxyuridine (BrdU) (Sigma Chemical Co., St. Louis, MO) 1 hour before being killed. Specimens were fixed in buffered formalin and embedded in paraffin. For both human and mouse studies, consecutive 4-μm sections were cut from each study block. The sections were immunostained by anti-proliferating cell nuclear antigen (PCNA) monoclonal antibody (DAKO Corp., Carpinteria, CA), anti-BrdU monoclonal antibody (Becton-Dickinson, Mountain View, CA), anti-epidermal growth factor receptor (EGF-R) polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-mouse IFN-β polyclonal antibody and anti-human IFN-β polyclonal antibody (Lee Biomolecular Research Laboratories, Inc., San Diego, CA), anti-VEGF/VPF polyclonal antibody (Santa Cruz Biotechnology, Inc.), anti-bFGF monoclonal antibody (Upstate Biotechnology, Inc., Lake Placid, NY), anti-IL-8 polyclonal antibody (Biosource, Camarillo, CA), anti-CD31 monoclonal antibody (DAKO Corp.), and anti-Factor VIII polyclonal antibody (DAKO Corp.). Immunohistochemical staining was performed by the immunoperoxidase technique after antigen retrieval: microwave treatment (1000 W) in citrate buffer for 5 minutes for PCNA; 2 N HCl at 37°C for 30 minutes for BrdU; and pepsin (Biomeda Corp., Foster City, CA) at room temperature for 20 minutes for EGF-R, mouse IFN-β, human IFN-β, VEGF/VPF, IL-8, CD31, and Factor VIII. After peroxidase block by 3. H2O2-methanol for 10 minutes, specimens were blocked with phosphate-buffered saline (PBS. containing 5% normal horse serum and 1% normal goat serum (Vector Laboratories, Inc., Burlingame, CA). The antibodies were used at the following dilutions: 1:50 for PCNA, BrdU, and IL-8; 1:400 for EGF-R, 1:1,000 for mouse IFN-β and human IFN-β; and 1:200 for VEGF/VPF, bFGF, CD31, and Factor VIII. After an overnight incubation at 4°C with primary antibody, specimens were briefly washed with PBS and incubated at room temperature with secondary antibody conjugated with peroxidase: anti-mouse immunoglobulin G (IgG)2a goat antibody (Serotec, Inc., Raleigh, NC) for PCNA; anti-mouse IgG1 antibody (PharMingen, San Diego, CA) for BrdU; anti-mouse IgG antibody (Jackson Immuno Research, West Grove, PA) for bFGF and CD31; and anti-rabbit IgG antibody (Jackson Immuno Research) for EGF-R, mouse IFN-β, human IFN-β, VEGF/VPF, IL-8, and Factor VIII. The specimens were then washed with PBS and color-developed by stable 3,3′-diaminobenzidine solution (Research Genetics, Huntsville, AL). After quantitation by colorimetric scanning using a computer, specimens were counterstained with Meyer-hematoxylin (Sigma Chemical Co.). 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The stock solution was diluted with Probe Diluent (Research Genetics) immediately before use. 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The samples were incubated in alkaline phosphatase-labeled avidin for 30 minutes at 45°C, briefly rinsed in 50 mmol/L Tris buffer (pH 7.6), rinsed with alkaline phosphatase enhancer (Biomeda Corp.) for 1 minute, and incubated with chromogen substrate FastRed (Research Genetics) for 30 minutes at 45°C. A positive reaction in this assay stained red. To provide a control for endogenous alkaline phosphatase, the samples were treated as described above but in the absence of the biotinylated probe, and chromogen was used in the absence of any oligonucleotide probes. The specificity of the hybridization signal was checked using the following controls: 1) RNase to pretreat tissue section; 2) a biotin-labeled sense probe; and 3) a competition assay with unlabeled antisense probe. A markedly decreased or absent signal was obtained after all these treatments. Apoptotic cells in intestinal tissues were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) method as described previously.60Xie K Wang Y Huang S Xu L Bielenberg D Salas T McConkey DJ Jiang W Fidler IJ Nitric oxide-mediated apoptosis of K-1735 melanoma cells is associated with downregulation of Bcl-2.Oncogene. 1997; 15: 771-779Crossref PubMed Scopus (81) Google Scholar Stained sections were examined in a Zeiss photomicroscope (Carl Zeiss Inc., Thornwood, NY) equipped with a three-chip charge-coupled device color camera (model DXC-960 MD; Sony Corp., Tokyo, Japan). The images were analyzed using the Optimas image analysis software (version 5.2; Bothell, WA). The slides to be analyzed were prescreened by one of the investigators to determine the range in staining intensity. Images covering the range of staining intensities were captured electronically. For immunostaining, captured images were converted to gray scale, and the threshold value was set to gray scale. All subsequent images were quantified based on this threshold. The integrated optical density (OD) of each selected field was determined based on its equivalence to the mean log inverse gray value multiplied by the area of the field. Because the samples were not counterstained before image analysis, the OD was due solely to immunoreaction. A total of eight hot spots, each consisting of 20 strong-staining cells,61Kumar R Kuniyasu H Bucana CD Wilson MR Fidler IJ Spatial and temporal distribution of angiogenic factors in tumor progression.Oncol Res. 1998; 10: 301-311PubMed Google Scholar were subjected to measurement of intensities. three in the tumor, three in the control mucosa, and three in the mucosa within 2 mm adjacent to the tumor. Staining intensities in each area were measured only on cytoplasm for in situ hybridization and on cytoplasm and/or membrane for immunohistochemistry. Staining of the cells was then quantified to derive an average value of the area. The representative OD value was the mean of three hot spots for the tumor, and the mean of two mucosa at the oral and anal edges for the adjacent mucosa. The measured OD of each in situ hybridization or immunostained specimen was standardized by comparison with the integrated OD of nonpathological control mucosa of the ascending colon in mouse specimens or of nonpathological control mucosa at lea
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