Search

Purpose: Patient-derived xenograft models are considered to represent the heterogeneity of human cancers and advanced preclinical models. Our consortium joins efforts to extensively develop and characterize a new collection of patient-derived colorectal cancer (CRC) models. Experimental Design: From the 85 unsupervised surgical colorectal samples collection, 54 tumors were successfully xenografted in immunodeficient mice and rats, representing 35 primary tumors, 5 peritoneal carcinoses and 14 metastases. Histologic and molecular characterization of patient tumors, first and late passages on mice includes the sequence of key genes involved in CRC (i.e., APC, KRAS, TP53), aCGH, and transcriptomic analysis. Results: This comprehensive characterization shows that our collection recapitulates the clinical situation about the histopathology and molecular diversity of CRC. Moreover, patient tumors and corresponding models are clustering together allowing comparison studies between clinical and preclinical data. Hence, we conducted pharmacologic monotherapy studies with standard of care for CRC (5-fluorouracil, oxaliplatin, irinotecan, and cetuximab). Through this extensive in vivo analysis, we have shown the loss of human stroma cells after engraftment, observed a metastatic phenotype in some models, and finally compared the molecular profile with the drug sensitivity of each tumor model. Through an experimental cetuximab phase II trial, we confirmed the key role of KRAS mutation in cetuximab resistance. Conclusions: This new collection could bring benefit to evaluate novel targeted therapeutic strategies and to better understand the basis for sensitivity or resistance of tumors from individual patients. Clin Cancer Res; 18(19); 5314–28. ©2012 AACR.

International audience; Electrical transport through molecular monolayers being very sensitive to disorder effects, admittance and current density characteristics of Hg // C12H25 – n Si junctions incorporating covalently bonded n-alkyl molecular layers, were investigated at low temperature (150-300 K), in the as-grafted state and after ageing at the ambient. This comparison reveals local oxidation effects both at the submicron scale in the effective barrier height distribution and at the molecular scale in the dipolar relaxation. In the bias range dominated by thermionic emission and modified by the tunnel barrier attenuation, , where is the thickness of the molecular tunnel barrier and is the inverse attenuation length at zero applied bias, some excess current is attributed to a distribution of low barrier height patches. Complementary methods are used to analyze the current density J(V, T) characteristics of Metal-Insulator-Semiconductor tunnel diodes. Assuming a Gaussian distribution of barrier heights centered at provides an analytical expression of the effective barrier height, ; this allows fitting of the distribution standard deviation and tunnel parameter over a wide temperature range. In a more realistic modeling including the voltage dependence of barrier height and circular patch area, the so-called “pinch-off” effect is described by a distribution of parameter which combines interface potential modulation and patch area variations. An arbitrary distribution of  values, fitted to low-temperature J(V) data, is equally well described by Gaussian or exponential functions. Ageing in air also increases the interface oxidation of Si substrate and affects the density of localized states near mid gap, which typically rises to the high 1011 eV-1.cm-2 range, as compared with < 1011 eV-1.cm-2 in the as-grafted state. The bias-independent relaxation observed near 1 kHz at low temperature may be attributed either to dipoles in the alkyl chain induced by the strong permanent dipoles of interface silicon oxide or to a local relaxation of water molecules trapped at the OML / silicon interface. The respective roles of SiO2 formation and water physisorption on the decrease of patch barrier height are also discussed.

International audience

International audience; Electrical transport in ultrathin Metal-insulator-semiconductor (MIS) tunnel junctions is analyzed using the temperature dependence of current density and admittance characteristics, as illustrated by Hg//C12H25 - n Si junctions incorporating n-alkyl molecular layers (1.45 nm thick) covalently bonded to Si(111). The voltage partition is obtained from J(V, T) characteristics, over eight decades in current. In the low forward bias regime (0.2-0.4 V) governed by thermionic emission, the observed linear T-dependence of the effective barrier height, qΦEFF(T) = qΦB+(kT)β0dT, provides the tunnel barrier attenuation, exp(-β0dT), with β0= 0.93 Å−1 and the thermionic emission barrier height, ΦB = 0.53 eV. In the high-forward-bias regime (0.5-2.0 V), the bias dependence of the tunnel barrier transparency, approximated by a modified Simmons model for a rectangular tunnel barrier, provides the tunnel barrier height, ΦT = 0.5 eV; the fitted prefactor value, G0 = 10−10 Ω−1, is four decades smaller than the theoretical Simmons prefactor for MIM structures. The density distribution of defects localized at the C12H25 - n Si interface is deduced from admittance data (low-high frequency method) and from a simulation of the response time τR(V) using Gomila's model for a non equilibrium tunnel junction. The low density of electrically active defects near mid-gap (DS

International audience; c-Yes, a member of the Src tyrosine kinase family, is found highly activated in colon carcinoma but its importance relative to c-Src has remained unclear. Here we show that, in HT29 colon carcinoma cells, silencing of c-Yes, but not of c-Src, selectively leads to an increase of cell clustering associated with a localisation of β-catenin at cell membranes and a reduction of expression of β-catenin target genes. c-Yes silencing induced an increase in apoptosis, inhibition of growth in soft-agar and in mouse xenografts, inhibition of cell migration and loss of the capacity to generate liver metastases in mice. Re-introduction of c-Yes, but not c -Src, restores transforming properties of c-Yes depleted cells. Moreover, we found that c-Yes kinase activity is required for its role in β-catenin localisation and growth in soft agar, whereas kinase activity is dispensable for its role in cell migration. We conclude that c-Yes regulates specific oncogenic signalling pathways important for colon cancer progression that is not shared with c-Src.

International audience

International audience

International audience

International audience; The electronic properties of redox-active transition metal clusters (Re6Se8) covalently immobilized on modified Si(111) surfaces through linear alkyl spacers have been studied as a function of the cluster coverage (1 × 1013-6 × 1013 cm-2). The latter is controlled by using Si(111)/H surfaces modified by dense mixed alkyl/acid-terminated monolayers with variable fraction of the acid grafting sites from 5 to 100% in solution. Quantitative X-ray photoemission analysis, spectroscopic ellipsometry, and scanning tunnelling microscopy indicate a covalent attachment of a submonolayer to densely packed monolayer of Re6Se8 clusters, while the vibrational Raman signature confirms the cluster integrity within the monolayer. Electrical band gaps as deduced from scanning tunnelling spectroscopy have been obtained for low Re6Se8 cluster coverage. Using ultraviolet photoemission spectroscopy, electronic properties such as ionization potential changes and energy level alignments at organic/inorganic interfaces are studied. We show that the lowest unoccupied molecular orbital of the Re6Se8 cluster is close to the bottom of the Si conduction band. At high cluster coverage, this affects the current-voltage characteristics measured using a weakly interacting top mercury contact onto the organic monolayer/silicon junctions. Indeed, on n-type silicon, the high level current at low bias and the shape of the conductance G(V) curve indicate a Schottky barrier height lowering. On the other hand, the current-voltage characteristics are the same for both acid-terminated and low coverage Re6Se8 cluster junctions at low bias; the high Schottky barrier height limits the current at low bias. When the forward bias increases, the current is tunnelling limited. As expected from the band alignment deduced from photoemission data, the opposite behavior is obtained on p-type silicon.

We present results on the photorefractive performance of two different classes of organic materials. One of them is based on the space-charge field induced reorientation of the optical axis of chiral smectic A phases. In this case the orientational effect is linear in the field and it is due to the so-called electroclinic effect, in contrast with the quadratic effect present in nematics and associated with dielectric anisotropy. Besides presenting data on the photorefractive properties of these new mesophases, we will consider a simple model which describes their performance as a function of several material and geometrical parameters. In the second part of the paper we introduce cyclopalladated complexes as a new class of multifunctional photorefractive materials. Such molecules form amorphous phases which are photoconducting and exhibit a field dependent refractive index. Their efficiency is among the best known to date for organic materials and the simple synthetic route makes us foresee a fast optimization of cyclometallated compounds for photorefractive applications.

Stress-induced activation of an acidic sphingomyelinase leading to generation of ceramide, an important lipid mediator, has been associated with apoptosis; however, the implication of this hydrolase has been questioned. The present study aimed at re-evaluating the role of this lysosomal enzyme in apoptosis initiated by different apoptotic inducers. The sensitivity of a series of acid sphingomyelinase-deficient cell lines derived from Niemann-Pick disease patients to stress-induced apoptosis was investigated. We have now shown that stress stimuli, such as anthracyclines, ionizing radiation, and Fas ligation trigger similar apoptotic hallmarks in normal and acid sphingomyelinase-deficient cell lines. Retrovirus-mediated gene correction of enzyme deficiency in Niemann-Pick cells does not modify response to apoptosis. Ceramide production is comparable in normal and Niemann-Pick cells, and increased activity of neutral sphingomyelinase is observed. Thus, our findings cast serious doubts that lysosomal sphingomyelinase activation is responsible for stress-induced apoptosis of cultured cells.

The possibility that the sphingomyelin (SM)-ceramide pathway is activated by CD40, a transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily and that plays a critical role in the regulation of immune responses has been investigated. We demonstrate that incubation of Epstein-Barr virus-transformed lymphoid cells with an anti-CD40 antibody acting as an agonist results in the stimulation of a neutral sphingomyelinase, hydrolysis of cellular SM, and concomitant ceramide generation. In addition, SM degradation was observed in acid sphingomyelinase-deficient cells, as well as after ligation by soluble CD40 ligand. The anti-CD40 antibody, as well as the soluble CD40 ligand induced a decrease in thymidine incorporation and morphological features of apoptosis, which were mimicked by cell-permeant or bacterial sphingomyelinase-produced ceramides. Stable expression of a dominant-negative form of the FAN protein (factor associated with neutral sphingomyelinase activation), which has been reported to mediate tumor necrosis factor-induced activation of neutral sphingomyelinase, significantly inhibited CD40 ligand-induced sphingomyelinase stimulation and apoptosis of transformed human fibroblasts. Transformed fibroblasts from FAN knockout mice were also protected from CD40-mediated cell death. Finally, anti-CD40 antibodies were able to co-immunoprecipitate FAN in control fibroblasts but not in cells expressing the dominant-negative form of FAN, indicating interaction between CD40 and FAN. Altogether, these results strongly suggest that CD40 ligation can activate via FAN a neutral sphingomyelinase-mediated ceramide pathway that is involved in the cell growth inhibitory effects of CD40.

The mechanism(s) by which ionizing radiation (IR) induces cell death is of fundamental importance in understanding cell sensitivity and resistance. Here we evaluated the response to IR of two subclones of the autonomous human erythromyeloblastic cell line TF-1: TF-1-34 (which expresses CD34) and TF-1-33 (which lacks CD34). In clonogenic assays, TF-1-34 cells were found to be relatively less sensitive to IR compared to TF-1-33 cells based on the D0 determination (3.01 vs 1.56 Gy). Furthermore, after IR at 12 Gy, TF-1-33 cell viability decreased by approximately 50% within 24 h, whereas TF-1-34 cell growth was unaffected during this time. Gradual loss of TF-1-34 cell viability was observed only after 48 h. Morphological and molecular analysis revealed that TF-1-33 cells died of apoptosis, and TF-1-34 cells of delayed reproductive cell death. While IR produced comparable amounts of DNA double strand breaks (DSB) in both cell lines, TF-1-34 retained DSB much longer than TF-1-33 suggesting that radioresistance and the defective apoptotic response of TF-1-34 cells was not related to a higher DNA repair capacity. However, the lack of an apoptotic response in TF-1-34 was correlated to the absence of a sphingomyelin (SM)-ceramide (CER) signaling pathway. Indeed, IR triggered in TF-1-33 cells but not in TF-1-34, SM hydrolysis (25% at 12 Gy) and CER generation (50%) through the activation of neutral but not acid sphingomyelinase. Synthetic cell permeate CER induced apoptosis in both TF-1-33 and TF-1-34 cells. This study indicates that alterations of the SM-CER signaling pathway can significantly influence the cell death process as well as the survival of acute myeloid leukemia cells after IR exposure.

Aberrant activity of receptor tyrosine kinases (RTKs) has been associated with tumor progression in a wide variety of human malignancies, making them promising drug targets for cancer therapy. Although, in some instances, specific inhibition of just one of these RTKs suffices for inhibition of tumor progression, in the majority of cases, targeting more than one RTK could be required for therapeutic efficacy. Moreover, increased expression or activation of RTKs is often associated with resistance to standard chemotherapy agents or signal transduction modulators. S 49076 is a novel, potent, ATP-competitive tyrosine kinase inhibitor of the RTKs MET, AXL and FGFR. S 49076 blocks autophosphorylation of these RTKs and their downstream signaling in cells with IC50 values of less than 50 nM in the case of MET and AXL, and at below 200 nM for FGFR1/2/3. In vitro, S 49076 inhibits the proliferation of MET- and FGFR2- dependent gastric cancer cells, blocks MET-driven migration of lung carcinoma cells and inhibits colony formation of hepatocarcinoma cells overexpressing FGFR2 and AXL. In vivo, oral administration of S 49076 inhibits MET autophosphorylation and tumor growth in subcutaneous GTL-16 human gastric carcinoma and U87-MG human glioblastoma at 6 mg/kg/day. In FGFR2-dependent SNU-16 gastric tumors S 49076 inhibits FGFR2 autophosphorylation, downstream signaling and tumor growth at 25 mg/kg/day. In a panel of 53 patient-derived tumors and 14 cell lines of diverse origin growing in three-dimensional in vitro culture, twenty-five (37%) were found to be sensitive to S 49076 at 1 µM or less. In the majority of cases, analysis of the expression, activation and mutation status of MET, AXL, FGFR1/2/3 and other target kinases of S 49076 as well as that of major signaling proteins enabled hypotheses to be made concerning the sensitivity or resistance to S 49076. Moreover, in many of these resistant tumors, a rationale for association of S 49076 with other targeted therapies emerged. Examples of the efficacy of such combined therapies will be shown. Based on these preclinical studies showing a favorable and novel pharmacological profile of S 49076, a phase I study is currently underway in patients with advanced solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 848. doi:1538-7445.AM2012-848

Proteins of the Bcl-2 family are central regulators of programmed cell death. Pro-survival Bcl-2 proteins, such as Bcl-2, Bcl-xL and Mcl-1 are often over-expressed in human tumours and participate in tumour initiation, progression and chemo-resistance. Therefore drugs targeting these pro-survival Bcl-2 proteins represent a promising therapeutic approach for cancer treatment. The most advanced drug targeting this protein family is ABT-263, a potent Bcl-2 and Bcl-xL inhibitor showing anti-tumour efficacy in preclinical models of leukaemia and small cell lung carcinoma. Survival of circulating platelets has been shown to be highly Bcl-xL dependent; consequently the dose-limiting toxicity of ABT-263 is an on-target peripheral thrombocytopenia. We have used a range of biophysical methods to guide the structure-based generation of a significant number of small molecules,* which bind with high affinity (MW < 780; KD < 1 nM) to the BH3 binding groove of Bcl-2, and with high selectivity versus other members of the Bcl-2 family (selectivity > 100 fold). In cellular assays, our lead compounds efficiently displace Bax from Bcl-2 with near complete inhibition of Bcl-2 / Bax co-immunoprecipitation at 100 nM. These compounds are strong inducers of cell death in Bcl-2 dependent cellular models such as the acute myeloblastic leukaemia (AML) cell line RS4;11, affording sub-10 nM IC50's for the most potent compounds. In vivo, in agreement with their mechanism of action, these Bcl-2 selective inhibitors, given either intravenously or orally, elicit a rapid (30 min iv, and 2 hours po) and strong apoptotic response in mouse xenografts of the RS4:11 cell line. When the most potent compounds are given orally to RS4;11 xenograft-bearing mice, apoptosis in tumor cells is induced more than 15 fold (at 25 mg/kg) and more than 20 fold (at 50 mg/kg) compared to untreated mice. Importantly, in agreement with the selectivity of the compounds for Bcl-2 versus Bcl-xL, no platelet loss was observed in mice treated with our compounds, in sharp contrast to ABT-263. Finally, we observe very robust anti-tumor activity when a lead compound is given orally at 50 mg/kg and 100 mg/kg (with complete regression at 100 mg/kg) in an RS4;11 mouse xenograft model. This anti-tumor activity was similar whether the compound was dosed daily or twice a week over two weeks. Altogether our data demonstrate that highly Bcl-2 selective antagonists show anti-tumor activity and no platelet toxicity, in contrast to Bcl-2 / Bcl-xL dual inhibitors. Such compounds represent promising drug candidates for the treatment of Bcl-2 dependent malignancies such as chronic lymphocytic leukaemia (CLL) and other leukaemias and lymphomas. * Chemical structures of compounds will not be disclosed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2782. doi:1538-7445.AM2012-2782

Receptor tyrosine kinases (RTKs) are key regulators of a multitude of cell processes, including survival, proliferation, migration, invasion and angiogenesis. Aberrant activity of certain of these receptor tyrosine kinases has been associated with tumor progression in a wide variety of human malignancies, making them promising drug targets for cancer therapy. Although, in some instances, specific inhibition of just one of these RTKs suffices for inhibition of tumor progression, in the majority of cases, targeting more than one RTK could be required for therapeutic efficacy. These RTKs include MET, the receptor for hepatocyte growth factor (HGF), the fibroblast growth factor receptor, FGFR, and AXL. Dysregulation of MET activity, due to its overexpression or mutation, or overexpression of its ligand, has been consistently associated with aggressive phenotype, resistance to certain anti-cancer therapies and poor outcome. The FGFRs have a well-documented role in tumor angiogenesis, and, more recently, have been implicated directly in tumor cell survival, proliferation and metastasis in a wide range of tumor types. AXL is one of a family of three tyrosine kinase receptors (TAM family) involved in the pathogenesis of several human cancers and has also recently incited interest as a cancer therapeutic target. S 49076 is a novel, potent, ATP-competitive tyrosine kinase inhibitor of MET, FGFR1/2/3 and AXL. S 49076 blocks autophosphorylation of these RTKs and their downstream signaling in cells with IC50 values of between 1 and 200 nM depending on the target and cell line. Furthermore, in kinase binding assays, S 49076 also binds to clinically-relevant MET mutated isoforms and a number of other kinases implicated in cancer pathology at concentrations of less than 100 nM. S 49076 is not, however, a potent inhibitor of VEGFR2. Although VEGFR2 is implicated in tumor angiogenesis, it also plays a major physiological role in the maintenance of vascular tone, and its inhibition by other RTK inhibitors has limited dosing of these molecules in the clinic. The unique inhibition profile of S 49076 may allow inhibition of oncogenic RTKs potentially without toxic effects encountered by VEGFR2 inhibitors. In vitro, S 49076 inhibits the proliferation of MET- and FGFR2-dependent gastric cancer cells, blocks MET-driven migration of lung carcinoma cells and inhibits colony formation of AXL-overexpressing hepatocarcinoma cells. In vivo, oral administration of S 49076 inhibits > 80% MET autophosphorylation in subcutaneous GTL-16 human gastric carcinoma tumors at 3 mg/kg. In both this GTL-16 model and in the MET-dependent U87-MG human glioblastoma model, S 49076 inhibits > 80% tumor growth at 6 mg/kg/day. S 49076 also inhibits FGFR2 autophosphorylation, downstream signaling and tumor growth in FGFR2-dependent SNU-16 gastric tumors, and tumor growth in LS-174T colon carcinoma. Based on these preclinical studies showing a favorable and novel pharmacological profile and the potential of S 49076 as innovative anticancer agent, a phase I study is planned soon to evaluate an oral formulation of S 49076 in patients with advanced solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A238.

International audience; The shape of tunnel barrier junctions is derived from experimental current density versus bias, J(V), using the normalized differential conductance, NDC=d log J/d log V, to discriminate barrier height, ΦT, and barrier width, dT, effects. Parameterization of the Simmons model for a rectangular tunnel barrier, with NDC≈dTV/(ΦT-qV)1/2, provides physical (dT,ΦT) values for Hg||monomolecular layer--n Si(111) junctions incorporating functionalized n-alkyl layers covalently bonded to silicon.

Well characterized models representing the heterogeneity of human colorectal cancers (CRC) are needed to develop effective therapeutic agents. Establishment of such tools will allow a better prediction of the clinical outcome, taking into account the diversity of each patient tumor phenotype and genotype. For this purpose and with the financial support of the French Ministry of Industry, we have associated efforts from hospitals, academic groups, biotech and pharmaceutical companies. From May 2007 to January 2009, 86 surgical specimens [59 primary (P) tumors, 19 metastasis (M), and 8 peritoneal carcinomatosis (C)] were collected from CRC patients (with informed consents and negative HBV, HCV, and HIVs serologies). Tumor samples were subcutaneously xenografted in nude mice. The take rate was 57% (P), 55% (C), and 82% (M) with a difference in the take rate between tumor stages (p= 0.0184). We further engrafted in nude mice, SCID mice and nude rats. No significant difference in the take rate and growth was observed between the 2 mouse strains. Most of the mouse-growing tumors (95%) were successfully engrafted in nude rats, however their growth was significantly slower showing a more pronounced stromal component when compared with mice. Characteristics of our models are in accordance with the CRC clinical heterogeneity. Sequence analysis of 54 models has been performed and mutations were observed for KRAS (46%), APC (44%), TP53 (59%), BRAF (11%), PI3KCA (11%), FBXW7 (6%), CTNNB1 (2%), EGFR (2%), and 5/21 present a MSI status. Besides molecular markers, we compared the gene copy number using CGH technology before and after engraftment. The results exhibit high similarity between early passages of xenografts and the original clinical tumor samples. The histological structure and molecular profile were preserved, indicating the relevance of these models. Nevertheless the quality control is required to follow potential molecular deviation after multiple in vivo passages. The established models are being evaluated for ex vivo and in vivo sensitivities to colon anticancer drugs (5-FU, oxaliplatin, irinotecan and cetuximab). In vivo studies performed in our panel show 14/19 models sensitive to 5-FU, 1/19 to oxaliplatin, 7/8 to irinotecan, and 6/19 to cetuximab. We will present the correlation between pharmacological studies, molecular profile and patient clinical history. Preclinical studies on patient-derived tumor models will bring benefits to evaluate novel targeted therapeutic strategies and potentially help the stratification strategy for cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4169.

This paper presents a method for natural texture analysis in a multiscale context. The texture signature is taken to be an anisotropic fractal feature related to the fractal dimension. By computing this signature in different orientations and through several pyramid levels, an attribute vector is obtained. The experimental results of texture segmentation by using Bayesian classifier show the method is efficient.