Impact of VEGF-dependent tumour micro-environment on EDB fibronectin expression by subcutaneous human tumour xenografts in nude mice.
Fibronectin (FN) is an extracellular matrix cell-adhesive glycoprotein. The alternative spliced isoform EDB-FN (extra domain B containing FN) is highly expressed in tumour blood vessels and stroma and represents a candidate for tumour targeting. To investigate the impact of different angiogenic micro-environments on EDB-FN expression, we used a tumour model in which human endometrial adenocarcinoma Tet-FGF2 cells overexpressing fibroblast growth factor-2 (FGF2) driven by the tetracycline-responsive promoter were further transfected with a VEGF antisense cDNA, generating AS-VEGF/Tet-FGF2 cells. In this model, the expression of FGF2 plus VEGF results in fast-growing, highly vascularized Tet-FGF2 tumours. Down-regulation of FGF2 production by tetracycline administration and/or of VEGF production by AS-VEGF transduction inhibited tumour growth and vascularization, with profound changes in tumour micro-environment. Quantitative RT-PCR analysis using human EDB-FN primers shows that subcutaneous grafting in immunodeficient mice is per se sufficient to cause a dramatic up-regulation of EDB-FN expression by these cells, as well as by human oesophageal cancer KYSE 30 cells and renal carcinoma Caki-1 cells. However, in vivo down-regulation of VEGF expression, as occurs in AS-VEGF/Tet-FGF2 tumours, and to a lesser extent of FGF2 expression, as occurs in tetracycline-treated Tet-FGF2 tumour-bearing animals, causes significant inhibition of EDB-FN production in tumour grafts, as shown by immunohistochemistry and quantitative RT-PCR analysis. Accordingly, treatment of Tet-FGF2 tumour-bearing animals with the neutralizing anti-murine VEGF receptor-2 antibody DC101, or of Caki-1 tumour-bearing animals with the anti-VEGF antibody bevacizumab, inhibited EDB-FN expression in tumour grafts. EDB-FN down-regulation was paralleled by a decrease in vascularity, thus confirming EDB-FN as a marker of tumour angiogenesis. These data demonstrate that the angiogenic micro-environment, and in particular the VEGF/VEGFR-2 system, plays a key role in modulating EDB-FN expression by tumour cells in vivo. This may have implications for the design of therapeutic strategies targeting EDB-FN in combination with anti-angiogenic and/or cytotoxic drugs.
Coltrini
Daniela
D
Department of Biomedical Sciences and Biotechnology, University of Brescia, 25123 Brescia, Italy.
Ronca
Roberto
R
Belleri
Mirella
M
Zardi
Luciano
L
Indraccolo
Stefano
S
Scarlato
Valentina
V
Giavazzi
Raffaella
R
Presta
Marco
M
D000230:Adenocarcinoma / Q000098:blood supply / Q000378:metabolism* / Q000473:pathology; D020533:Angiogenesis Inhibitors / Q000627:therapeutic use; D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use; D061067:Antibodies, Monoclonal, Humanized; D000068258:Bevacizumab; D016889:Endometrial Neoplasms / Q000098:blood supply / Q000378:metabolism* / Q000473:pathology; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007680:Kidney Neoplasms / Q000098:blood supply / Q000473:pathology / Q000517:prevention & control; D051379:Mice; D008819:Mice, Nude; D009363:Neoplasm Proteins / Q000378:metabolism / Q000502:physiology; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014183:Transplantation, Heterologous; D042461:Vascular Endothelial Growth Factor A / Q000502:physiology*
D020533:Angiogenesis Inhibitors; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D005353:Fibronectins; D009363:Neoplasm Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D012334:RNA, Neoplasm; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2; D000068258:Bevacizumab