Comparison of the Spatial Organization in Colorectal Tumors using Second-Order Statistics and Functional ANOVA

International audienceWe propose an automatic method to quantitatively describe the spatial organization governing populations of biological objects, such as cells, which exist in stationary histology images. This quantification is of prime importance when striving to compare different tumoral models in order to evaluate potential therapies. We compare two animal models of colorectal cancer. Our approach is based on the topographic map to automatically extract the location of the relevant biological objects. We describe their spatial organization along a continuous range of scales using second-order statistics. Using a functional analysis of variance test, we show that there are significant differences in these statistics depending on cancer model, and on the day after tumor implant

Caractérisation de modèles de tumeurs murines et leurs applications en thérapie anti-angiogénique vectorisée

The first part of this study was to assess and compare fifteen widely used tumour models to select the ones most suitable for angiogenesis research. Two tumours were selected: a lung tumour (Lewis lung carcinoma, LLC) for its high vascularization and a colon carcinoma (colon tumour 26, C26) because it expresses both integrin αvβ3 and E-selectin. CT26 tumours were implanted subcutaneously (s.c.) in Balb/c mice for the ectopic model, or into the caecum for the orthotopic model. Tumours were evaluated by histology, spectrofluorescence, bioluminescence (BLI), magnetic resonance imaging (MRI) and ultrasound imaging (US). BLI was found to be more appropriate for early tumour detection, whereas MRI and US afforded excellent non invasive imaging techniques to accurately follow tumour growth of both ectopic and orthotopic tumour models. These models were very useful to investigate antiangiogenic therapies, in particular during the preclinical development of fisetin as an antiangiogenic agent. The natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) has been shown to possess antiangiogenic and anticancer properties. Because of the limited water solubility of fisetin, our aim was to design and optimize a liposomal formulation that could facilitate its in vivo administration. Low dose of liposomal fisetin (21 mg/kg) improved fisetin antitumoral activity in LLC tumor bearing mice compared to the administration of free fisetin. This liposomal formulation could therefore advantageously be employed to improve the antiangiogenic and anticancer activities of this flavonoid, as well as other flavonoids sharing similar problems of in vivo administrationLa première partie de ce travail de thèse a permis de sélectionner parmi un panel de 15 tumeurs murines, les modèles les plus appropriés pour l'étude de molécules à potentiel anti-angiogénique. Deux tumeurs ont été choisies, la tumeur de poumon (Lewis Lung carcinoma, LLC) pour laquelle une forte vascularisation a été observée et la tumeur de côlon (colon tumor 26, C26) pour laquelle l'expression des intégrines αvβ3 et de la sélectine-E a été observée. Les modèles de côlon C26 implantés en sous-cutané (ectopique) et sur le caecum (orthotopique) ont été développés et caractérisés par différentes modalités d'imagerie. L'imagerie optique s'est montrée adaptée uniquement pour la détection précoce des tumeurs, tandis que l'imagerie par résonance magnétique et l'imagerie par ultrason se sont avérées des techniques très adaptées pour le suivi de la croissance des tumeurs implantées en ectopique et en orthotopique. Le développement et la caractérisation de ces modèles se sont révélés très utiles pour la deuxième partie de nos travaux, notamment pour l'évaluation antitumorale d'un flavonoïde la fisétine. La fisétine est un flavonoïde naturel (3,3',4',7-tetrahydroxyflavone) ayant montré une activité anti-angiogénique et des propriétés anticancéreuses.Etant donné sa faible solubilité dans l'eau, une forme liposomale a été développée afin de faciliter son administration systémique.Une efficacité antitumorale plus importante de la fisétine liposomale comparée à la fisétine libre a été obtenue après injection d'une faible dose (21 mg/kg) sur la lignée tumorale LLC. Cette formulation pourrait donc avantageusement être employée pour la formulation de composés de la famille des flavonoïde

Caractérisation de modèles de tumeurs murines et leurs applications en thérapie anti-angiogénique vectorisée

The first part of this study was to assess and compare fifteen widely used tumour models to select the ones most suitable for angiogenesis research. Two tumours were selected: a lung tumour (Lewis lung carcinoma, LLC) for its high vascularization and a colon carcinoma (colon tumour 26, C26) because it expresses both integrin αvβ3 and E-selectin. CT26 tumours were implanted subcutaneously (s.c.) in Balb/c mice for the ectopic model, or into the caecum for the orthotopic model. Tumours were evaluated by histology, spectrofluorescence, bioluminescence (BLI), magnetic resonance imaging (MRI) and ultrasound imaging (US). BLI was found to be more appropriate for early tumour detection, whereas MRI and US afforded excellent non invasive imaging techniques to accurately follow tumour growth of both ectopic and orthotopic tumour models. These models were very useful to investigate antiangiogenic therapies, in particular during the preclinical development of fisetin as an antiangiogenic agent. The natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) has been shown to possess antiangiogenic and anticancer properties. Because of the limited water solubility of fisetin, our aim was to design and optimize a liposomal formulation that could facilitate its in vivo administration. Low dose of liposomal fisetin (21 mg/kg) improved fisetin antitumoral activity in LLC tumor bearing mice compared to the administration of free fisetin. This liposomal formulation could therefore advantageously be employed to improve the antiangiogenic and anticancer activities of this flavonoid, as well as other flavonoids sharing similar problems of in vivo administrationLa première partie de ce travail de thèse a permis de sélectionner parmi un panel de 15 tumeurs murines, les modèles les plus appropriés pour l'étude de molécules à potentiel anti-angiogénique. Deux tumeurs ont été choisies, la tumeur de poumon (Lewis Lung carcinoma, LLC) pour laquelle une forte vascularisation a été observée et la tumeur de côlon (colon tumor 26, C26) pour laquelle l'expression des intégrines αvβ3 et de la sélectine-E a été observée. Les modèles de côlon C26 implantés en sous-cutané (ectopique) et sur le caecum (orthotopique) ont été développés et caractérisés par différentes modalités d'imagerie. L'imagerie optique s'est montrée adaptée uniquement pour la détection précoce des tumeurs, tandis que l'imagerie par résonance magnétique et l'imagerie par ultrason se sont avérées des techniques très adaptées pour le suivi de la croissance des tumeurs implantées en ectopique et en orthotopique. Le développement et la caractérisation de ces modèles se sont révélés très utiles pour la deuxième partie de nos travaux, notamment pour l'évaluation antitumorale d'un flavonoïde la fisétine. La fisétine est un flavonoïde naturel (3,3',4',7-tetrahydroxyflavone) ayant montré une activité anti-angiogénique et des propriétés anticancéreuses.Etant donné sa faible solubilité dans l'eau, une forme liposomale a été développée afin de faciliter son administration systémique.Une efficacité antitumorale plus importante de la fisétine liposomale comparée à la fisétine libre a été obtenue après injection d'une faible dose (21 mg/kg) sur la lignée tumorale LLC. Cette formulation pourrait donc avantageusement être employée pour la formulation de composés de la famille des flavonoïde

Bioavailability of Polyphenol Liposomes: A Challenge Ahead

Dietary polyphenols, including flavonoids, have long been recognized as a source of important molecules involved in the prevention of several diseases, including cancer. However, because of their poor bioavailability, polyphenols remain difficult to be employed clinically. Over the past few years, a renewed interest has been devoted to the use of liposomes as carriers aimed at increasing the bioavailability and, hence, the therapeutic benefits of polyphenols. In this paper, we review the causes of the poor bioavailability of polyphenols and concentrate on their liposomal formulations, which offer a means of improving their pharmacokinetics and pharmacodynamics. The problems linked to their development and their potential therapeutic advantages are reviewed. Future directions for liposomal polyphenol development are suggested

Improved antiangiogenic and antitumour activity of the combination of the natural flavonoid fisetin and cyclophosphamide in Lewis lung carcinoma-bearing mice.

International audiencePURPOSE: The natural flavonoid fisetin was recently identified as a lead compound that stabilizes endothelial cell microtubules. In this study, we investigated the antiproliferative and antiangiogenic properties of fisetin in vitro and in vivo. METHODS: Fisetin cytotoxicity was evaluated using Lewis lung carcinoma cells (LLC), endothelial cells and NIH 3T3 cells. Endothelial cell (EC) migration and capillary-like structure formation were evaluated using EAhy 926 cells. In vivo tumour growth inhibition studies were performed using LLC-bearing mice treated with fisetin and/or cyclophosphamide (CPA). RESULTS: The fisetin IC(50) was 59 μM for LLC and 77 μM for EC cells, compared to 210 μM for normal NIH 3T3 cells (24 h). Fisetin inhibited EC migration and capillary-like structure formation at non-cytotoxic concentrations (22-44 μM). In mice, fisetin inhibited angiogenesis assessed using the Matrigel plug assay. In LLC-bearing mice, fisetin produced a 67% tumour growth inhibition (223 mg/kg, intraperitoneal), similar to the 66% produced by low-dose CPA (30 mg/kg, subcutaneous). When fisetin and CPA were combined, however, a marked improvement in antitumour activity was observed (92% tumour growth inhibition), with low systemic toxicity. Tumour histology showed decreased microvessel density with either fisetin or CPA alone, and a dramatic decrease after the fisetin/CPA combination. CONCLUSIONS: We have shown that fisetin not only displays in vitro and in vivo antiangiogenic properties, but also can markedly improve the in vivo antitumour effect of CPA. We propose that this drug combination associating a non-toxic dietary flavonoid with a cytotoxic agent could advantageously be used in the treatment of solid tumours

Co–encapsulation of flavonoids with anti–cancer drugs: A challenge ahead

International audienc

Vascular density and endothelial cell expression of integrin alpha v beta 3 and E-selectin in murine tumours.

International audienceThe endothelial cell adhesion molecules, including the integrin alpha v beta 3 (αvβ3) and E-selectin, are involved in the process of angiogenesis required for tumour growth, cell migration and metastasis. The purpose of this study was to assess and compare widely used tumour models to select the ones most suitable for angiogenesis research. Fifteen murine tumours were selected including melanoma (B16), colon (C26, C38, C51), mammary (MA13, MA16, MA16/Adr, MA17, MA17/Adr, MA25, MA44), pancreatic (PO2, PO3), Glasgow osteogenic sarcoma (GOS) and Lewis lung carcinoma (LLC). The tumour vascular density, assessed using the platelet endothelial cell adhesion molecule 1 (PECAM-1; CD31) immunostaining, revealed that B16 melanoma was poorly vascularized (15 %) were the pancreatic tumours (PO2 and PO3), the sarcoma (GOS) and the lung tumour (LLC). The integrin αvβ3 and E-selectin, evaluated by immunohistology, showed that 7/15 tumours expressed the αvβ3 integrin which was homogeneously distributed on all tumour sections (B16, C26, MA17/Adr, MA25, MA44, PO2, LLC). E-selectin was expressed in 4/15 tumours and its expression was restricted to the tumour periphery. Only 2/15 tumours (B16 and C26) were shown to express both integrin αvβ3 and E-selectin. In conclusion, these data not only contribute to a better understanding of the tumour biology of murine tumours but can also guide the choice of appropriate models for antiangiogenic therapy, for selective drug delivery to tumours and the validation of tumour imaging modalities targeting these endothelial cell adhesion molecules

EVALUATION OF ANTIVASCULAR COMBRETASTATIN A4P EFFICACY BY USING SUPERSONIC SHEAR IMAGING TECHNIQUE OF ECTOPIC COLON CARCINOMA CT26

International audienceA recent ultrasound imaging technique, called shear wave elastography, showed its ability to image and quantify mechanical properties of biological tissues, such as prostate or liver. In the present study this technique was used to evaluate the relation between tumor growth, stiffness and tumor reduction upon treatment with combretastatin (CA4P) in allografted colon tumor CT26, in mice. During twelve days, CT26 tumor growth (n=52) was imaged by ultrasound, and shear modulus was quantified, showing a good correlation between tumor volume and stiffness (r = 0.59). At day 12, the treatment was initiated and monitored every day during 4 days. Following the treatment, the tumor volume has decreased, still the elasticity parameter of the tumor volume was steady throughout the treatment. After segmentation using the shear modulus map, a detailed analysis showed a decrease in the stiffness after treatment as compared to the control group. This reduction in the mechanical properties was shown to correlate with tissue reorganization, in particular fibrosis and necrosis, assessed by histology

Lipopolythiourea Transfecting Agents: Lysine Thiourea Derivatives

International audienceSynthetic vectors represent an alternative to recombinant viruses for gene transfer. We have recently explored the transfection potential of a class of noncationic lipids bearing thiourea moieties as DNA associating headgroups. The encouraging results obtained with lipopolythioureas derived from serinol prompted us to investigate further this family of vectors. In the present study, we considered the transfection properties of a series of derivatives based on a different thiourea polar headgroup bearing a lysine scaffold. The synthesis of these compounds could be readily achieved in 3 steps with good yields. We found that these lipopolythioureas (LPT) might be considered as alternative systems for gene transfection, since their activity reached the same magnitude range as cationic vectors in the presence of serum. LPT with 14-carbon length chains appeared to be more efficient as transfecting agent than the ones with shorter chains. Toxicity studies proved that the hydration film 2 method led to particles well tolerated both by the cells in vitro and by the mice in vivo. The ability to induce gene expression in vivo was demonstrated by intratumoral injection. Finally, biodistribution studies showed that the quantity recovered in the blood circulation, 2h after systemic injection, was improved as compared to cationic lipids