To test the impact on the angiogenic switch, 5-week-old transgenic mice, whose islets had not yet activated the angiogenic switch, were treated with SU5416, a small synthetic inhibitor of VEGF-R2 signalling, until the first tumours appeared in control mice at 10.5 weeks of age a, To reduce tumour growth, 10-week-old transgenic mice with small solid tumours were treated until the appearance of end-stage disease (13.5 weeks of age, b). Numbers of angiogenic islets (a) and tumour burden (b) in transgenic and control Rip1-Tag2 mice are shown. Values are means ± s.d. (n =7–10).

A, VEGF localizes with its receptor concomitant with the angiogenic switch. Immunolocalization of VEGF, VEGFR-2 and the VEGF–VEGF-R2 complex is shown for hyperplastic, pre-angiogenic islets (left panels) and tumours (right panels). Immunohistochemical analysis was carried out using specific antibodies against VEGF, VEGF-R2 or VEGF–VEGF-R2. VEGF was detected at high levels in β-cells of pre-angiogenic hyperplastic islets and in angiogenic tumours (a). VEGF-R2 is localized on endothelial cells and it antibody therefore visualizes the capillary network in both hyperplastic islets and tumours (b). VEGF–VEGFR2 on endothelial cells was detected only in the angiogenic and tumour stages but not in hyperplastic, non-angiogenic islets (c). B, Inhibition of secreted VEGF impairs the angiogenic response. Non-angiogenic islets (a) and angiogenic islets in the absence (b) or presence (c) of VEGF-neutralizing antibodies were embedded into a three-dimensional collagen gel containing endothelial cells. Angiogenic islets, but not non-angiogenic islets, provoked an angiogenic response. Angiogenesis was delayed and diminished when anti-VEGF antibodies were added.

The activity of plasminogen activators and gelatinases in tissue lysates from non-transgenic islets, angiogenic islets, and tumours was revealed by zymography. a, MMP-2 and MMP-9 were upregulated concomitant with the angiogenic switch and persisted in tumours. Both enzymes were found as inactive higher-M_r and active lower-M_r. As controls, gelatinase activity in MMP-2ko/ko and MMP-9ko/ko tumours is shown. b, The serine proteinases urokinase-plasminogen activator (uPA) and tissue-plasminogen activator (tPA) were constitutively present during islet carcinogenesis, before and after the angiogenic switch. Both the inactive high-M_r and the active lower-M_r forms of urokinase are evident.

A, Effect of treatment with gelatinase on angiogenic activity of non-transgenic normal islets. a, untreated islets; b, MMP-2-treated islets; c, MMP-9-treated islets; d, MMP-9-treated islets in a collagen matrix without endothelial cells; e, MMP-9-treated islets in the presence of the MMP inhibitor GM6001; f, MMP-9-treated islets in the presence of a VEGF-neutralizing antibody. Gelatinases were used at 1 mg ml⁻¹. B, Determination by ELISA of VEGF levels in conditioned media of protease-treated and non-treated islets. Untreated and MMP-2-treated islets release similar, small amounts of VEGF. Treatment with MMP-9 caused a roughly twofold increase in VEGF release; this effect was abolished in the presence of GM6001. Conditioned media of angiogenic islets and tumours served as a positive, but non-quantitative, control.

Non-angiogenic islets did not exhibit MMP-9-positive cells (a). MMP-9 was expressed by infiltrating cells (black arrows) in angiogenic islets (b) and in tumours (c) and was associated with the basement membrane (blue arrow) and extracellular matrix (green arrow), as visualized by immunohistochemistry. Insets, higher magnification of single MMP-9-expressing cells, proximal to capillaries.

Genetic and pharmacogenetic analyses were carried out to test the impact on the angiogenic switch and tumour growth in proteinase-deficient mice. Rip1-Tag2 mice were either crossed successively with MMP-9, MMP-2 or urokinase gene-knockout mice to produce homozygous knockouts, or treated with BB-94/Batimastat (a broad-spectrum MMP-I) or R94138 (a selective inhibitor of MMP-9). Data for SU5416 from are reproduced here to allow comparison. For the pharmacological analysis, 5-week-old transgenic mice were treated from 5.0–10.5 weeks of age in a prevention trial (a), and from 10.0–13.5 weeks of age in an intervention trial (b). Homozygous deficient or MMPI-treated Rip1-Tag2 mice were analyzed at two distinct stages of tumour progression and compared with control Rip1-Tag2 mice with regard to the frequency of angiogenic switching (at 10.5 weeks of age, a) and cumulative tumour volume (tumour burden), a measure of relative tumour-growth rates (at 13.5 weeks of age, b). Values are means ± s.d. (n=7–14) and are compared with those of control RIP1-Tag2 mice.

A, Histopathology of control (a), uPA-ko/ko (b), MMP-9-ko/ko (c) and MMP-2-ko/ko (d) tumours. Asterisks indicate exocrine pancreas. Shadowed arrows point to invasiveness and black arrows to haemorrhage formation. B, Proliferation and apoptotic rates in tumours of MMP-2-ko/ko or MMP-9 ko/ko RIP1-Tag2 mice, or RIP1-Tag2 mice that were treated in an experimental trial with SU5416, as compared to standard RIP1-Tag2 controls. Apoptotic cells were detected by TUNEL staining; proliferating cells were identified by PCNA staining. The significance of results was determined using a non-parametric bootstrap analysis.C, Formation of VEGF–VEGF-R2 complexes in control (a), MMP-2-ko/ko (b) and MMP-9-ko/ko (c) angiogenic islets and in control (d), MMP-2-ko/ko (e) and MMP-9-ko/ko (f) tumours. Black arrowheads in c indicate rare labelled cells.