PMC

Heterogeneous expression of VEGFR2 in melanoma and colorectal cancer. Dual immunofluorescent staining for VEGFR2 (red) and CD31 (green) in human cancer specimens shows VEGFR2 negative blood vessels (arrows) and VEGFR2 positive blood vessels (asterisks). (A-C) Metastatic malignant melanoma and (D-F) colorectal carcinoma have been divided into their respective channels (overlaid; green; red images). Scale bar = 50 μm.

Microvessel density (MVD) as CD31 positive profiles in SW480 and WM239 subcutaneous xenografts; Mean (SEM) are plotted. There were significant reductions in MVD in both SW480 (A) and WM239 (B) xenografts between CTX and control groups (*p < 0.05). Western blotting of tumor lysates for TSP-1 revealed significant increases in TSP-1 levels in CTX treated SW480 tumors compared to control (C; *p < 0.05), but significant decreases in CTX treated WM239 tumors compared to control (D; *p < 0.05).

Effect of low dose metronomic cyclophosphamide therapy on vascular VEGFR2 status. Examples of VEGFR2 positive blood vessels (chevrons) and VEGFR2 negative blood vessels (arrows) are shown in WM239 (A) treated and (B) control and SW480 (C) treated and (D) control subcutaneous xenografts. Scale bar = 50 μm. Quantification of immunofluorescence showed significant decreases in the density of VEGFR2 positive vessels in CTX treated SW480 (E) and WM239 (F) tumors (*p < 0.05). Total VEGFR2 protein levels in tissue lysates were significantly reduced in SW480 tumors treated with CTX (*p < 0.05), but not in treated WM239 tumors (G, H).

Mice were treated orally with 20 mg/kg/day of cyclophosphamide in their drinking water. Tumor growth curves of low dose metronomic cyclophosphamide (CTX) treated and control xenografts of (A) SW480, and (B) WM239 cells; Mean (SEM) is plotted. No significant differences in growth rates were observed between control and treated mice for either SW480 or WM239 xenografts. Cancer cell toxicity to CTX was evaluated in vitro using the MTT assay (C). Apoptosis was quantified in tumor sections by TUNEL reaction, and while rare TUNEL positive nuclei were seen in cancer cells (white arrow in D), we did not find any TUNEL positive endothelial cells (black arrows), nor any significant differences in apoptotic cells between control and CTX tumors (E). Scale bar = 25 μm.

Effect of low dose metronomic cyclophosphamide therapy on vascular mural cell recruitment. A) Dual immunofluorescent staining for vascular alpha smooth muscle actin (α-SMA; red) and CD31 (green) showing blood vessels positive (chevrons) and negative (arrows) for this mural cell marker. Scale bar = 50 μm. B) CTX treated SW480 xenograft tumors had significantly increased proportion of α-SMA positive blood vessels compared to control (*p < 0.05). No significant difference was observed between CTX and control WM239 tumors. The ratio between the mural cell marker protein desmin and pan endothelial marker CD31 was assessed by western blotting of whole tumor lysates (C) LDM CTX treatment resulted in increased desmin content realtive to CD31 for SW480 but not WM239 xenografts.

Effect of low dose metronomic cyclophosphamide therapy on tumor hypoxia. HIF1-α levels in tumor lysates were not significantly different between CTX and control tumors for either SW480 (A) or WM239 (B). Representative images of tumor cross sections immunostained for CA-IX (dark reaction product) and counterstained with hematoxylin: WM239 CTX (C) and control (D); SW480 CTX (E) and control (F). Insert of panel (C) is negative control without primary antibody. Scale bars = 300 μm. G) Quantification of hypoxic regions (Mean; SEM) in SW480 and WM239 xenografts from CTX and control mice. Treated WM239 xenograft tumors had significantly lower average percent hypoxic regions than control (*p < 0.05). No significant difference was observed between CA-IX staining in SW480 CTX and control tumors. H, I) Measurement of conditioned medium by ELISA demonstrates that 24 hour treatment in vitro with CoCl₂ induced up-regulation in VEGF expression in both cell lines (H; SW480; I, WM239), while treatment with low dose 4-HC does not. Combining these treatments had no additive effect over the induction produced by CoCl₂ alone.