Inhibition of the alpha-nu integrins with a cyclic RGD peptide impairs angiogenesis, growth and metastasis of solid tumours in vivo

Abstract

Anti-angiogenetic cancer therapy is a potential new form for treatment of solid tumours. The alpha(v)-integrins (alpha(v)beta3, alpha(v)beta5) mediate the contact of activated endothelial cells to proteins of the extracellular matrix during tumour angiogenesis as a prerequisite for survival of endothelial cells. The aim of this study was to investigate the effects of application of a methylated cyclic RGD-peptide as an alpha(v)-integrin antagonist on angiogenesis, microcirculation, growth and metastasis formation of a solid tumour in vivo. Experiments were performed in the dorsal skinfold preparation of Syrian Golden hamsters bearing the amelanotic hamster melanoma A-Mel-3. Animals were injected intraperitoneally with a methylated cyclic RGD-peptide every 12 h, the control group received an inactive peptide. Microcirculatory parameters of tumour angiogenesis including functional vessel density, red blood cell velocity, vessel diameter and leucocyte-endothelium interaction were analysed using intravital microscopy. In an additional study the effects on growth and metastasis of subcutaneous A-Mel-3 were quantified. Functional vessel density was markedly reduced on day 3 in treated animals compared to controls (37.2 +/- 12.1 vs 105.2 +/- 11.2 cm(-1); mean +/- s.e.m.; P<0.05) and increased subsequently in both groups. Red blood cell velocity at day 3 was below values of controls (0.026 +/- 0.01 vs 0.12 +/- 0.03 mm x s(-1); P<0.05). No differences were observed in vessel diameters and leucocyte-endothelium interaction was almost absent in both groups. Furthermore, growth and metastasis of subcutaneous tumours after administration of the cyclic RGD-peptide was significantly delayed in comparison to controls (P<0.05). Inhibition of alpha(v)-integrins by a cyclic RGD-peptide resulted in significant reduction of functional vessel density, retardation of tumour growth and metastasis in vivo. Taken together, these results implicate RGD-peptides as agents which have anti-tumour and anti-metastatic activity in vivo.

Copyright 2002 Cancer Research UK

Figures

Figure 1

Images of tumour microcirculation 3 days after tumour cell implantation acquired by intravital microscopy after injection of the plasma marker FITC-Dextran. (A) Tumour vasculature of animals treated with the control peptide EMD135981 already appears as dense, chaotic structured network. (B) In the RGD peptide treated group (EMD121974) only individual sprouts as signs of early angiogenesis were visible. Bar, 50 μm.

Figure 2

Functional vessel density (mean±s.e.m.) of control group and RGD treated group. Measurements were performed 3, 5, 9 and 13 days after tumour cell implantation by intravital microscopy. Animals were treated every 12 h (30 mg kg−1) beginning at day 1 after tumour cell implantation until day 13. *P<0.05 vs corresponding controls; #P<0.05 vs day 9 and 13; +P<0.05 vs day 3 and 13.

Figure 3

Red blood cell velocity (mean±s.e.m.) of control group and RGD treated group. *P<0.05 vs corresponding controls; #P<0.05 vs treatment day 9 and day 13.

Figure 4

Vessel diameters (mean±s.e.m.) as measured by intravital microscopy of control group and RGD treated group.

Figure 5

Images of the A-Mel-3 tumour in the dorsal skinfold chamber acquired by video macroscopy. (A) Tumour of control group 5 days after tumour cell implantation. Arrows define the tumour margin. (C) The same tumour 4 days later. (B) Corresponding tumour of RGD treated group on day 5 and (D) same tumour on day 9 after tumour cell implantation. Bar 1 mm.

Figure 6

Area of the A-Mel-3 tumour of the control group and the RGD treated group in the dorsal skinfold chamber measured on day 3, 5, 9 and 13 after tumour cell implantation (mean±s.e.m.). *P<0.05 vs corresponding controls.

Figure 7

Tumour growth curves of animals with subcutaneously implanted solid A-Mel-3 tumours. Changes in tumour volume are presented for control animals and following application of cyclic RGD peptide. Values are indicated as mean±s.e.m. *P<0.05 vs controls.

Figure 8

Metastasis formation presented as a Kaplan–Meier curve. Animals of the control group and animals of the RGD peptide treated group were examined for axillar and inguinal lymph node metastases during evaluation of tumour growth. Metastasis formation was significantly delayed following therapy with the RGD peptide. *P<0.05 vs controls.