Assessment of the direct effects of DDAH I on tumour angiogenesis in vivo

Figures

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  Fig. 1. In vitro characterization of C6 DDAH cells in comparison with parental cells. Cells were treated for 4 days with 2 µg/ml DOX. For the last 48 h, cells were also stimulated with cytokines (10 ng/ml TNF-α, 1000 U/ml IFN-γ) and 5 µg/ml LPS. a iNOS (131 kDa) and c DDAH I (34 kDa) expression determined by western blot and integrated density of individual bands (corrected for loading). b NOx production. d l-citrulline production evaluated after 4-h incubation with ADMA substrate. Results are mean + 1 SEM of three separate experiments (*P < 0.05, **P < 0.01, ***P < 0.001)

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  Fig. 2. In vivo characterization of C6 DDAH tumour xenografts compared with D27 tumour xenografts. C6 DDAH cells were pre-treated with 2 µg/ml DOX in the medium for 5 days (group A). Mice with C6 DDAH tumours were given 0.2 mg/ml DOX in 5% (w/v) sucrose or 5% (w/v) sucrose alone (n = 6 per group) in the drinking water ad libitum. Mice with D27 (C6 constitutively DDAH overexpressing) tumours were given drinking water alone (n = 4). a, b Example western blots of tumour homogenates and mean box plot distributions of the integrated densities of the individual bands for DDAH I (a) and iNOS (b) corrected using α-tubulin and α-actin respectively. c l-citrulline production and (d) NOx production by tumours, determined from homogenates. e TDTs in days. Results are the mean + 1 SEM of measurements made from all tumours in each group (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, One-Way ANOVA with Bonferroni’s multiple comparison post-test)

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  Fig. 3. Investigation of the effect of DDAH I on VEGF expression in vitro and in vivo. a VEGF levels in culture medium from cells treated with 2 µg/ml DOX for 6 days. For the last day, cells were also stimulated with cytokines (10 ng/ml TNF-α, 1000 U/ml IFN-γ) and 5 µg/ml LPS. Culture medium was collected from the last 24 h of treatment. b VEGF expression in tumour homogenates. C6 DDAH tumours (group A) derived from animals provided with drinking water containing 0.2 mg/ml DOX in 5% (w/v) sucrose or 5% (w/v) sucrose alone (n = 6 per group). Mice with D27 tumours were given drinking water alone (n = 4). Results are mean + 1 SEM of duplicate measurements from three separate in vitro experiments or from all tumours in each group (*P < 0.05, ****P < 0.0001, One-Way ANOVA with Bonferroni’s multiple comparison post-test)

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  Fig. 4. The effects of DDAH I up-regulation on tumour vascular development as determined by MRI of C6 DDAH xenografts (group A). a Representative T2-weighted images, parametric maps of baseline R2*air, ΔR2*CB in response to carbogen and fractional blood volume (fBV) acquired from the central tumour slice. ROIs encompassing the whole tumour volume are demarcated on the T2-weighted images. Box plot distributions of the median R2*air (b) and ΔR2*CB (c) values measured over the whole tumour volume of D27 (n = 4) and C6 DDAH ± DOX (n = 6) xenografts. Median fBV values (d) and median ΔR2*CB values (e) in regions where fBV is higher than the median fBV value of all the data of C6 DDAH − DOX (n = 4) and C6 DDAH + DOX (n = 3) tumours

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  Fig. 5. Histological assessment of the vasculature of C6 DDAH ± DOX (group A) and D27 xenografts. a Representative composite images from frozen whole C6 DDAH ± DOX tumour sections stained with the perfusion marker Hoechst 33342 (blue fluorescence) and the hypoxia marker pimonidazole (green fluorescence). b H&E-stained frozen sections from C6 DDAH ± DOX and D27 tumours indicating necrotic regions (arrows). c Composite images from frozen C6 DDAH ± DOX and D27 tumour sections stained with the endothelial cell marker CD31 (green fluorescence) and the perivascular cell marker α-SMA (red fluorescence). Scale bar is 1 mm

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  Fig. 6. Quantification of histological markers of tumour vasculature for C6 DDAH ± DOX (group A) and D27 xenografts. a Hoechst 33342 perfused area, b pimonidazole adduct area, c necrotic area, d endothelial cell content (CD31 stained area), e perivascular cell coverage (α-SMA stained area), and f fraction of mature vessels (α-SMA/CD31 ratio) of tumour xenografts. Results are means + 1 SEM of three sections per tumour for n = 4 in the D27 group and n = 6 in the C6 DDAH ± DOX groups (*P < 0.05, **P < 0.01, ***P < 0.001)