doi:10.1016/j.yexcr.2004.06.014
Copyright © 2004 Elsevier Inc. All rights reserved.
Angiogenesis gene expression profiling in xenograft models to study cellular interactions
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Victor L.J.L. Thijssena, Ricardo J.M.G.E. Brandwijka, Ruud P.M. Dingsa, b and Arjan W. Griffioena, 
, 
aAngiogenesis Laboratory, Research Institute for Growth and Development (GROW), Departments of Internal Medicine and Pathology, University Maastricht and University Hospital Maastricht, 6202 AZ Maastricht, The Netherlands
bDepartment of Biochemistry, University of Minnesota, Minneapolis, MN 55455, USA
Received 2 April 2004;
revised 11 June 2004.
Available online 20 July 2004.
Abstract
The present study describes a method to simultaneously obtain the angiogenic expression profile in tumor cells and vascular cells of a single tumor. Human- and mouse-specific primers were used for quantitative real-time RT-PCR to determine the expression of vascular endothelial growth factors A, B, C, and D, vascular endothelial growth factor receptors 1, 2, and 3, neuropilin 1 and 2, angiopoietin 1, 2, 3/4, tyrosine kinase receptors 1 and 2, basic fibroblast growth factor (bFGF) in xenograft tumors obtained by injection of human ovarian carcinoma cells in nude mice. In addition, the effect of treatment with anginex and taxol on the expression profile was analyzed. Most factors were expressed higher in vascular cells as compared to tumor cells. In response to treatment, tumor cells significantly upregulated bFGF expression and downregulated VEGF receptor expression. This was accompanied by downregulation of VEGF-B and -D, and upregulation of angiopoietin-3 as well as angiopoetin receptors in nontumor cells. In conclusion, real-time qRT-PCR combined with xenograft tumor models presents a sensitive method to monitor angiogenesis and to analyze interactions between tumor cells and nontumor cells in vivo. The approach can be applied to different research fields in which xenograft models are used.
Keywords: Quantitative real-time RT-PCR; Xenograft; Ovarian carcinoma; Angiogenesis
Fig. 1. VEGFR-1 primer design. (A) Detail of alignment between human and mouse VEGFR-1 cDNA (overall homology: 83%). The box indicates a region of relatively low homology (71%). Selected primers are shown in bold. (B) Characteristics of the selected forward and reverse primers with respect to primer requirements.
Fig. 2. Analysis of primer specificity and sensitivity. (A) Agarose gel electrophoresis of amplicons generated during PCR with primers specific for human (h) or mouse (m) isoforms on cloned cDNA fragments of mouse and human receptor TIE-2 and the ligands ANG-1, ANG-3, or ANG-4. cDNA fragments were cloned from either mouse B16F10 tumor tissue or human breast tumor tissue. M = 100 bp marker. (B) Amplification plots generated during quantitative real-time PCR on the dilution series of human and mouse isoforms of TIE-2 and ANG-1. Cloned fragments were diluted in 10-fold steps down to 1 fg. (C) Standard curves for human and mouse isoforms of TIE-2 and ANG-1. The slope is indicated in brackets. All primers displayed a linear detection range down to 1 fg with an expected slope of approximately −3.3.
Fig. 3. Molecular profiling in xenograft tumors. (A) Quantification of mRNA copy number in tumor cells (upper panel) or in nontumor cells (lower panel) of mice injected subcutaneously with human ovarian carcinoma cells. Copy numbers were calculated from standard curves and normalized to 10,000 copies of cyclophilin. (B) Fold expression in nontumor cells as compared to tumor cells.
Fig. 4. Effect of antitumor treatment. (A) The effect of 20 days of antitumor treatment on tumor volume. Mice were injected subcutaneously with human ovarian carcinoma cells and after 2 weeks treatment with saline, paclitaxel, or anginex was started. *P < 0.05 treatment vs. control. (B) Immunohistochemical staining on 10-μm-thick cryosections of treated and untreated tumors with PE-conjugated rat anti-mouse CD31 antibody. Original magnification is 200×. Scale bar is 50 μm. (C) The microvessel density and vessel characteristics of the different treatment groups. *P < 0.05 treatment vs. control.
Fig. 5. The effect of antitumor treatment on gene expression. Effect of antitumor treatment on the expression of angiogenesis factors in tumor cells (A) and nontumor cells (B). qRT-PCR with human-specific primers was performed on xenograft tumors after 20 days of treatment with saline, paclitaxel, or anginex. *P < 0.01 vs. control, #P < 0.02 vs. control, †P < 0.05 vs. control.
Table 1.
Selected markers and species-specific primers for xenograft expression profiling
Primer sequences are shown as large caps (5′–3′); mismatches of the human sequence with the mouse sequence are shown in bold; h = human; m = mouse; see Table 1 for explanation of abbreviations.
a ANG-3 and ANG-4 represent interspecies orthologues.
Corresponding author. Department of Pathology, Maastricht University and University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands. Fax: +31 43 3876613
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