Valérie Rouffiac

Canstatin Acts on Endothelial and Tumor Cells via Mitochondrial Damage Initiated through Interaction with αvβ3 and αvβ5 Integrins

Canstatin, the noncollagenous domain of collagen type IV α-chains, belongs to a series of collagen-derived angiogenic inhibitors. We have elucidated the functional receptors and intracellular signaling induced by canstatin that explain its strong antitumor efficacy in vivo . For this purpose, we generated a canstatin-human serum albumin (CanHSA) fusion protein, employing the HSA moiety as an expression tag. We show that CanHSA triggers a crucial mitochondrial apoptotic mechanism through procaspase-9 cleavage in both endothelial and tumor cells, which is mediated through cross-talk between α v β 3 - and α v β 5 -integrin receptors. As a point of reference, we employed the first three kringle domains of angiostatin (K1-3), fused with HSA, which, in contrast to CanHSA, act only on endothelial cells through α v β 3 -integrin receptor–mediated activation of caspase-8 alone, without ensuing mitochondrial damage. Taken together, these results provide insights into how canstatin might exert its strong anticancer effect.

Early quantitative evaluation of a tumor vasculature disruptive agent AVE8062 using dynamic contrast-enhanced ultrasonography.

Objectives:To evaluate the early tumor vasculature disrupting effects of the AVE8062 molecule and the feasibility of dynamic contrast-enhanced ultrasonography (DCE-US) in the quantitative assessment of these effects. Material and Methods:AVE8062 was administered at a single dose (41 mg/kg) to 40 melanoma-bearing nude mice, which were all imaged before and after drug administration (5 + 15 minutes, 1, 6, and 24 hours). Using an ultrasound scanner (Aplio, Toshiba), intratumor vessels were counted in power Doppler mode and tumor microvasculature was assessed in a specific harmonic mode associated with a perfusion and quantification software for contrast-uptake quantification (Sonovue, Bracco). The peak intensity (PI), time-to-PI (TPI), and full-width at half maximum (FWHM) were extracted from the time-intensity curves expressed as linear raw data. Histologic analysis evaluated microvessel density (MVD) and necrosis at each time point studied. Statistical significance was estimated (paired sum rank and Mann–Whitney tests) to evaluate drug activity and to compare its efficacy at the different time points. Results:In power Doppler mode, intratumoral vessels depletion started 15 minutes postinjection (32%, P = 0.004) and the decrease was maximal at 6 hours (51%, P = 0.002). PI decreased by 3.5- and 45.7-fold at 1 and 6 hours, respectively, compared with preinjection values (P = 0.016 and P = 0.008). The decrease at 6 hours was significantly different from the variation at 1 hour (P = 0.0012) and at 24 hours (P = 0.0008). TPI and FWHM showed a significant increase exclusively at 6 hours (P = 0.0034, P = 0.0039). Histology revealed significantly decreased MVD and increased necrosis at 24 hours (P < 0.01). Conclusion:DCE-US allowed quantitative in vivo evaluation of the functional effects of AVE8062, which was found most effective on tumoral microvasculature 6 hours after its administration. A clinical phase-1 study of AVE8062 is ongoing using the same ultrasonography methodology before and 6 and 24 hours postadministration.

Angiogenesis and tumor growth inhibition by a matrix metalloproteinase inhibitor targeting radiation-induced invasion

In this study, we have evaluated the interactions between ionizing radiation and a matrix metalloproteinase (MMP) inhibitor. Using Matrigel invasion assays, we show that ionizing radiation induced a dose-dependent increase in the invasive phenotype of cultured B16 melanoma cells and that conditioned medium from these irradiated B16 cells promoted endothelial cell [human microvascular endothelial cells (HMEC)] invasiveness. To determine whether the radiation-induced changes in invasive phenotype could be due to changes in MMP activation, we have tested the ability of the MMP inhibitor Metastat to modulate the ionizing radiation–induced invasive phenotype using both an in vitro melanoma model and a mouse s.c. tumor model. In these studies, Metastat inhibited the ionizing radiation–induced invasive phenotype in cultured B16 cells and similarly inhibited the increase in HMEC invasion induced by conditioned medium from irradiated B16 cells. Conversely, ionizing radiation increased B16 MMP-2 activity and the conditioned medium from irradiated B16 induced HMEC MMP-2 activity. To further investigate the interaction between ionizing radiation and MMP activation, we then studied the effects of ionizing radiation on downstream effectors of the MMP system. We found that ionizing radiation induced vascular endothelial growth factor (VEGF) secretion by B16 melanoma cells and that this secretion was inhibited by Metastat. Similarly, conditioned medium from irradiated B16 was also able to increase VEGF secretion in HMECs. Moreover, ionizing radiation–induced melanoma cell invasiveness was partially inhibited by an anti-VEGF monoclonal antibody. In vivo, ionizing radiation plus concomitant Metastat yielded the greatest growth inhibition of melanoma s.c. tumors and this effect correlated with inhibition of angiogenesis as measured by both Doppler ultrasonography and platelet/endothelial cell adhesion molecule-1 staining. Finally, ionizing radiation modulated MMP-2, VEGF, and VEGF receptor expression in these tumor samples using immunohistochemistry. Taken together, these results suggest that there is an ionizing radiation–induced tumor survival pathway and a possible paracrine ionizing radiation–induced stimulatory pathway emanating from tumor cells toward the endothelial bed that is impeded when Metastat is given simultaneously. This model could provide in vivo evidence of the antitumor efficacy of combining a MMP inhibitor with ionizing radiation to target radiation-induced invasion and angiogenesis.

Évaluation fonctionnelle et précoce des traitements en cancérologie : intérêt des produits de contraste ultrasonores

Resume L’evaluation precoce et fonctionnelle des nouveaux traitements en cancerologie represente un enjeu majeur. Actuellement, les progres technologiques en echographie-Doppler permettent d’etudier la microvascularisation des tumeurs superficielles et profondes afin d’evaluer l’efficacite des nouvelles therapeutiques: 1) visant a bloquer l’angiogenese tumorale (Neovastat, Sugen, TNFα…) ou 2)detruisant physiquement la vascularisation des lesions hepatiques par radiofrequence. Il est possible de mettre en evidence des flux lents dans des microvaisseaux tumoraux et de predire l’efficacite therapeutique en fonction de la modification de cette microvascularisation avant meme qu’il n’y ait une modification du volume tumoral.L’injection de produits de contraste renforce l’efficacite de la technique et le developpement de logiciels de perfusion optimise cette detection.

HIGH-FREQUENCY SONOGRAPHY AND COLOR DOPPLER IN THE MANAGEMENT OF PIGMENTED SKIN LESIONS

We aimed to evaluate high-frequency sonography (HFS) coupled with color Doppler in the management of pigmented skin lesions (PSL). HFS examination was performed in 111 patients with 130 PSL. A color Doppler study was conducted in 107 lesions, to assess intralesional vascularization. Imaging findings were compared with histologic diagnosis. In the case of melanoma, sonographic and histologic maximum thickness measurements were compared. HFS showed 114 of the 130 lesions. Among the detected lesions, HFS alone provided 100% sensitivity and 100% specificity in the distinction of melanoma/nevi from other lesions, and 100% sensitivity and 32% specificity in the distinction of melanomas from nonmelanoma lesions. Sonographic and histologic measurement of melanoma thickness strongly correlated (r > 0.96, p < 0.001). Color Doppler detection of intralesional vessels had a 100% specificity and 34% sensitivity in the distinction of melanomas from other PSL. HFS coupled with color Doppler is a simple, reliable tool for PSL management.

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.

In vivo echographic evidence of tumoral vascularization and microenvironment interactions in metastatic orthotopic human neuroblastoma xenografts.

Human neuroblastoma (NB) is the second most frequent solid tumor of childhood and represents a highly heterogeneous disease at clinical and biologic levels. Little progress has been made to improve the poor prognosis of patients with high‐stage NB. Tumor progression and metastatic dissemination still represent major obstacles to the successful treatment of advanced stage disease. In order to develop and evaluate new, targeted, therapeutic strategies, fully defined and biologically relevant in vivo models of NB are strongly needed. We have developed an orthotopic model of metastatic human NB in the nude mouse, using 2 well‐characterized NB cell lines. Tumor growth, vascular properties and metastatic patterns were investigated using a sensitive and newly developed in vivo echographic technology in addition to immunohistochemistry and PCR analyses. Results show that implantation of low numbers of NB cells directly into the adrenal gland of nude mice resulted in rapid and homogeneous tumor growth without tumor morbidity. Nude mice were shown to rapidly develop highly vascularized adrenal tumors that selectively metastasized to the liver and bone marrow. In addition, the newly formed mouse vessels in orthotopic but not in heterotopic tumors, were found to express the highly angiogenic αvβ3 integrin marker, indicating the development of a truly malignant neovasculature in orthotopic conditions only. This observation confirms the impact of the regional microenvironment on tumor biology and suggests the existence of cross‐talk with the tumor cells. In conclusion, such model faithfully reproduces the growth, vascular and metastatic patterns as observed in patients. It therefore represents a powerful and biologically relevant tool to improve our understanding of the biology of NB and to develop and assess new antiangiogenic and metastasis‐targeted therapies.

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.

In vitro echogenicity characterization of poly[lactide-coglycolide] (PLGA) microparticles and preliminary in vivo ultrasound enhancement study for ultrasound contrast agent application

Objectives:This work includes (1) the characterization of a reproducible poly[lactide-coglycolide] (PLGA) microparticle preparation with an optimial mean diameter and size distribution and (2) the preliminary in vivo ultrasonographic investigation of PLGA microparticles. Methods:A first series of PLGA microparticle preparations (1 to 15 &mgr;m) was acoustically characterized on a hydrodynamic device to select the most appropriate for ultrasound contrast agent application. Preparations of 3-&mgr;m microparticles were selected, characterized at different doses, and then injected into 20 melanoma grafted mice for contrast-enhanced power Doppler ultrasonography evaluation. Results:The 3-&mgr;m microparticles (3.26-&mgr;m mean diameter with 0.41-&mgr;m standard deviation) led to in vitro enhancement of 18.3 dB at 0.62 mg/mL. In vivo experiments showed 47% enhancement of intratumoral vascularization detection after PLGA injection, significantly correlated (P < 0.0001) with preinjection intravascularization and tumoral volume. No toxicity was histologically observed. Conclusion:The 3-&mgr;m PLGA microparticles provided significant enhancement in vitro and in vivo without any toxicity.

The anti-angiogenic activity of IL-12 is increased in iNOS−/− mice and involves NK cells

We have previously reported that the in vivo transfer of murine interleukin-12 (IL-12) gene using a Semliki Forest virus vector induced tumor regression through inhibition of tumor blood vessel formation. To examine whether IL-12 anti-angiogenic activity interferes with the NO pathway, we used inducible nitric oxide synthase-deficient mice (iNOS−/−) and demonstrated that the anti-tumor effect of IL-12 is more pronounced in these mice. In addition, despite the increased level of intratumoral VEGF in iNOS−/− mice, IL-12 induced a stronger inhibition of blood vessel formation. Histological analysis of SFV-IL-12-treated tumors showed an increase in natural killer (NK) perivascular infiltration in iNOS−/− as compared to control mice. In vitro IL-12-stimulated murine splenic NK cells displayed significant killing activity towards established murine endothelial cells used as targets. These studies indicate that the anti-angiogenic activity of IL-12 interferes with iNOS pathway and involves NK cell recruitment.