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. 2006 Jun 19;94(12):1845-52.
doi: 10.1038/sj.bjc.6603186. Epub 2006 May 23.

Angiogenesis in a human neuroblastoma xenograft model: mechanisms and inhibition by tumour-derived interferon-gamma

D Ribatti  1 , B Nico, A Pezzolo, A Vacca, R Meazza, R Cinti, B Carlini, F Parodi, V Pistoia, M V Corrias
Affiliations

Angiogenesis in a human neuroblastoma xenograft model: mechanisms and inhibition by tumour-derived interferon-gamma

D Ribatti et al. Br J Cancer. .

Abstract

Tumour progression in neuroblastoma (NB) patients correlates with high vascular index. We have previously shown that the ACN NB cell line is tumorigenic and angiogenic in immunodeficient mice, and that interferon-gamma (IFN-gamma) gene transfer dampens ACN tumorigenicity. As IFN-gamma represses lymphocyte-induced tumour angiogenesis in various murine models and inhibits proliferation and migration of human endothelial cells, we have investigated the antiangiogenic activity of tumour-derived IFN-gamma and the underlying mechanism(s). In addition, we characterised the tumour vasculature of the ACN xenografts, using the chick embryo chorioallantoic membrane assay. We show that the ACN/IFN-gamma xenografts had a lower microvessel density and less in vivo angiogenic potential than the vector-transfected ACN/neo. The vascular channels of both xenografts were formed by a mixed endothelial cell population of murine and human origin, as assessed by the FICTION (fluorescence immunophenotyping and interphase cytogenetics) technique. With respect to ACN/neo, the ACN/IFN-gamma xenografts showed more terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling-positive human and murine endothelial cells, suggesting that inhibition of angiogenesis by IFN-gamma was dependent on the induction of apoptosis, likely mediated by nitric oxide. Once the dual origin of tumour vasculature is confirmed in NB patients, the xenograft model described here will prove useful in testing the efficacy of different antiangiogenic compounds.

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Figures

Figure 1
Figure 1
Immunohistochemical analysis of ACN/neo (A) and ACN/IFN-γ (B) tumours removed 14 days after injection. The microvessel density, determined by means of an anti-CD31 antibody recognising endothelial cells, was significantly (P<0.001) higher in ACN/neo than in ACN/IFN-γ tumours. Original magnification: × 160.
Figure 2
Figure 2
Immunofluorescence performed on ACN/IFN-γ paraffin-embedded sections. (A) Section simultaneously incubated with a rat anti-mouse CD34 and a mouse anti-human CD31 antibody developed, respectively, with a goat FITC-conjugated anti-rat and an Alexa Fluor® rabbit anti-mouse (human CD31 positive: red; murine CD34 positive: green). Original magnification: × 1000. (B) Section incubated as in (A) where a microvessel composed of both red and green cells is shown.
Figure 3
Figure 3
FICTION analysis of ACN/IFN-γ paraffin-embedded sections. (A) ACN/IFN-γ sections hybridised with the murine Cot-1 probe. The murine cell line TAP-1 hybridised with the Cot-1 probe is shown in the inset. (B) Identification of microvessel of mouse origin with anti-mouse CD34 (green) and a Cot-1 probe (red). (C) Human metaphase hybridised with the centromere-specific human chromosome 1 probe (red). Murine metaphase hybridised with the same probe is shown in the upper inset. ACN cells hybridised with the same probe are shown in the lower inset. (D) Identification of microvessels of human origin with anti-human CD31 (green) and the centromere-specific human chromosome 1 probe (red). Original magnification: (A) × 200, (B–D) × 1000.
Figure 4
Figure 4
Immunofluorescence and TUNEL assay. ACN/IFN-γ (A, B) and ACN/neo (C, D) deparaffinized sections were subject to TUNEL assay (green) and then stained by immunofluorescence in red using either anti-murine CD34 (A, C) or anti-human CD31 (B, D) mAbs. Colocalisation of red and green (yellow) indicated apoptosis of endothelial cells. Original magnification: (A–D) × 1000.
Figure 5
Figure 5
(A) A 12-day-old chick embryo CAM incubated on day 8 for 4 days with a gelatin sponge loaded with 500 ng hrFGF-2. Note numerous allantoic blood vessels with a radially arranged ‘spoked wheel’ pattern around the sponge. (B) Histological section of the sponge shown in (A). Note numerous small blood vessels among the sponge trabeculae intermingled with a collagenous matrix. (C) A 12-day-old CAM incubated on day 8 for 4 days with bioptic specimen of ACN/IFN-γ tumour xenograft, showing few vessels around the graft. (D) Immunohistochemical analysis of the xenograft, showing few CD31-positive blood vessels. (E) A 12-day-old CAM incubated on day 8 for 4 days with bioptic specimen of ACN/neo tumour xenograft, showing numerous blood vessels around the graft. (F) Immunohistochemical analysis of the xenograft, showing numerous CD31-positive blood vessels. The microvessel density, determined by means of an anti-CD31 antibody recognising endothelial cells, is significantly (P<0.001) lower in ACN/IFN-γ than in ACN/neo xenografts. Original magnification: (A, C, E) × 50; (B, D, F) × 400.
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