Canstatin gene electrotransfer combined with radiotherapy: preclinical trials for cancer treatment.

Given as a prophylactic treatment, a single muscle electrogene transfer of plasmid coding canstatin fused to human serum albumin (CanHSA), slowed down the development of two xenografted human carcinomas from mammary (MDA-MB-231) and prostate origin (PC-3) in nude mice and delayed lung metastatic spreading of B16F10 melanoma cells in syngenic mice. No effect was observed with unfused canstatin. The long lasting circulating blood level of CanHSA (20 ng ml(-1)) resulted in a profound disorganization of the tumor blood vessel network. However, when used as a curative treatment, on well-established tumors, CanHSA electrogenetherapy was ineffective in reducing tumor growth. As radiation is known to increase the alpha v beta3 and alpha v beta5 integrins, which are canstatin receptors, to extend the use of CanHSA electrogenetherapy, as a curative treatment, we explored the combination of CanHSA and ionizing radiation. We demonstrated a better efficacy (P=0.01) of the bitherapy over irradiation alone, as a result of strong vessel disorganization and dramatic increase of tumor cells apoptosis. This extremely simple virus free curative protocol could open the door to potential clinical applications, especially for prostate cancer that often develops radioresistance.

A gene transfer comparative study of HSA-conjugated antiangiogenic factors in a transgenic mouse model of metastatic ocular cancer.

Different antiangiogenic and antimetastatic recombinant adenoviruses were tested in a transgenic mouse model of metastatic ocular cancer (TRP1/SV40 Tag transgenic mice), which is a highly aggressive tumor, developed from the pigmented epithelium of the retina. These vectors, encoding amino-terminal fragments of urokinase plasminogen activator (ATF), angiostatin Kringles (K1-3), endostatin (ES) and canstatin (Can) coupled to human serum albumin (HSA) were injected to assess their metastatic and antiangiogenic activities in our model. Compared to AdCO1 control group, AdATF-HSA did not significantly reduce metastatic growth. In contrast, mice treated with AdK1-3-HSA, AdES-HSA and AdCan-HSA displayed significantly smaller metastases (1.19+/-1.19, 0.87+/-1.5, 0.43+/-0.56 vs controls 4.04+/-5.12 mm3). Moreover, a stronger inhibition of metastatic growth was obtained with AdCan-HSA than with AdK1-3-HSA (P=0.04). Median survival was improved by 4 weeks. A close correlation was observed between the effects of these viruses on metastatic growth and their capacity to inhibit tumor angiogenesis. Our study indicates that systemic antiangiogenic factors production by recombinant adenoviruses, particularly Can, might represent an effective way of delaying metastatic growth via inhibition of angiogenesis.

Dynamic assessment of antiangiogenic therapy by monitoring both tumoral vascularization and tissue degeneration.

Tumor growth is dependent both on endothelial and tumor cells. The aim of this study was to investigate dynamically whether changes in tumor vasculature implicate tumor tissue degeneration during antiangiogenic therapies. In order to quantify intra-tumor vascularization and necrosis, we have used ultrasound technology. This study has identified essential parameters needed to quantify specifically and sensitively the number of microvessels and the extent of necrosis in xenografted human carcinomas during natural tumor evolution, using contrast-enhanced high-frequency ultrasonography with (HFCDUS) or without (HFUS) color Doppler. We showed that quantification of intra-tumor microvessels between HFCDUS and immunohistochemistry is correlated using an anti-CD31 antibody. Furthermore, quantification of tumor necrosis with HFUS was confirmed by histological examination of hematoxylin-eosin-saffranin-stained sections over the observation period. Subsequently, for the assessment of novel angiogenic inhibitors, HFCDUS and HFUS were used to elucidate the underlying dynamics linking vessel inhibition and tumor eradication. We describe a novel application for HFCDUS/HFUS that constitutes an effective, convenient, and non-invasive method for clinical assessment of angiogenic inhibitors. In conclusion, we showed that tumor cells abruptly became necrotic following an antivascular therapy, whereas untreated tumors were protected from degeneration by a significant blood supply.

Full kringles of plasminogen (aa 1-566) mediate complete regression of human MDA-MB-231 breast tumor xenografted in nude mice.

Since kringle (K)5, not present in the angiostatin molecule, was shown to be a key functional domain possessing potent antiangiogenic activity, we have evaluated a new plasminogen-derived fragment, consisting of the N-terminal part of human plasminogen, that included the complete secondary structure of K1-5 (aa 1-566). In contrast to other fragments described to date, K1-5 includes cysteine residues at positions 543, 555 and 560 allowing the formation of the three disulfide bonds lying within K5. Vascular endothelial cell proliferation and migration assays revealed that a replication-defective adenovirus (AdK1-5(1-566)), expressing K1-5 (aa 1-566), was dose dependently more potent that AdK1-3(1-354), an adenovirus that expresses only the first three kringles. In contrast to AdK1-3(1-354), a single intratumoral injection of AdK1-5(1-566) into MDA-MB-231 breast human carcinoma tumors was followed by a total regression of 40% of the tumor and by significant arrest of tumor growth (90%), which was correlated with a drastic decrease of functional neovascularization into the tumors. Furthermore, systemic delivery of AdK1-5(1-566) in mice inhibited the lung invasion of melanoma B16-F10 cells by 87%. Our findings provide evidence that the full kringles of plasminogen (aa 1-566) may be much more potent than K1-3 (aa 1-354), for the suppression of angiogenesis, tumor growth and metastatic dissemination.