Oncolytic attenuated measles virus encoding NY-ESO-1 induces HLA I and II presentation of this tumor antigen by melanoma and dendritic cells.

Antitumor virotherapy stimulates the antitumor immune response during tumor cell lysis induced by oncolytic viruses (OVs). OV can be modified to express additional transgenes that enhance their therapeutic potential. In this study, we armed the spontaneously oncolytic Schwarz strain of measles viruses (MVs) with the gene encoding the cancer/testis antigen NY-ESO-1 to obtain MVny. We compared MV and MVny oncolytic activity and ability to induce NY-ESO-1 expression in six human melanoma cell lines. After MVny infection, we measured the capacity of melanoma cells to present NY-ESO-1 peptides to CD4 + and CD8 + T cell clones specific for this antigen. We assessed the ability of MVny to induce NY-ESO-1 expression and presentation in monocyte-derived dendritic cells (DCs). Our results show that MVny and MV oncolytic activity are similar with a faster cell lysis induced by MVny. We also observed that melanoma cell lines and DC expressed the NY-ESO-1 protein after MVny infection. In addition, MVny-infected melanoma cells and DCs were able to stimulate NY-ESO-1-specific CD4 + and CD8 + T cells. Finally, MVny was able to induce DC maturation. Altogether, these results show that MVny could be an interesting candidate to stimulate NY-ESO-1-specific T cells in melanoma patients with NY-ESO-1-expressing tumor cells.

Impact of automated methods for quantitative evaluation of immunostaining: Towards digital pathology.

Introduction: We sought to develop a novel method for a fully automated, robust quantification of protein biomarker expression within the epithelial component of high-grade serous ovarian tumors (HGSOC). Rather than defining thresholds for a given biomarker, the objective of this study in a small cohort of patients was to develop a method applicable to the many clinical situations in which immunomarkers need to be quantified. We aimed to quantify biomarker expression by correlating it with the heterogeneity of staining, using a non-subjective choice of scoring thresholds based on classical mathematical approaches. This could lead to a universal method for quantifying other immunohistochemical markers to guide pathologists in therapeutic decision-making. Methods: We studied a cohort of 25 cases of HGSOC for which three biomarkers predictive of the response observed ex vivo to the BH3 mimetic molecule ABT-737 had been previously validated by a pathologist. We calibrated our algorithms using Stereology analyses performed by two experts to detect immunohistochemical staining and epithelial/stromal compartments. Immunostaining quantification within Stereology grids of hexagons was then performed for each histological slice. To define thresholds from the staining distribution histograms and to classify staining within each hexagon as low, medium, or high, we used the Gaussian Mixture Model (GMM). Results: Stereology analysis of this calibration process produced a good correlation between the experts for both epithelium and immunostaining detection. There was also a good correlation between the experts and image processing. Image processing clearly revealed the respective proportions of low, medium, and high areas in a single tumor and showed that this parameter of heterogeneity could be included in a composite score, thus decreasing the level of discrepancy. Therefore, agreement with the pathologist was increased by taking heterogeneity into account. Conclusion and discussion: This simple, robust, calibrated method using basic tools and known parameters can be used to quantify and characterize the expression of protein biomarkers within the different tumor compartments. It is based on known mathematical thresholds and takes the intratumoral heterogeneity of staining into account. Although some discrepancies need to be diminished, correlation with the pathologist's classification was satisfactory. The method is replicable and can be used to analyze other biological and medical issues. This non-subjective technique for assessing protein biomarker expression uses a fully automated choice of thresholds (GMM) and defined composite scores that take the intra-tumor heterogeneity of immunostaining into account. It could help to avoid the misclassification of patients and its subsequent negative impact on therapeutic care.

Frequent Homozygous Deletions of Type I Interferon Genes in Pleural Mesothelioma Confer Sensitivity to Oncolytic Measles Virus.

INTRODUCTION: Oncolytic immunotherapy is based on the use of nonpathogenic replicative oncolytic viruses that infect and kill tumor cells exclusively. Recently, we found that the spontaneous oncolytic activity of the Schwarz strain of measles virus (MV) against human malignant pleural mesothelioma (MPM) depends on defects in the antiviral type I interferon (IFN-I) response in tumor cells. METHODS: In this study, we studied three independent human MPM bio-collections to identify the defects in the IFN-I responses in tumor cells. RESULTS: We show that the most frequent defect is the homozygous deletions (HDs) of all the 14 IFN-I genes (IFN-α and IFN-ß) that we found in more than half of MV-sensitive MPM cell lines. These HDs occur together with the HDs of the tumor suppressor gene CDKN2A also located in the 9p21.3 chromosome region. Therefore, the IFN-I-/- MPM cell lines develop a partial and weak IFN-I response when they are exposed to the virus compared with that of normal cells and MV-resistant MPM cell lines. This response consists of the expression of a restricted number of IFN-stimulated genes that do not depend on the presence of IFN-I. In addition, the IFN-I-/- MPM cell lines infected by MV also develop a pro-inflammatory response associated with stress of the endoplasmic reticulum. CONCLUSION: Our study emphasizes the link between HDs of IFN-I encoding genes and the CDKN2A gene in MPM and sensitivity to MV oncolytic immunotherapy.

Oncolytic viruses sensitize human tumor cells for NY-ESO-1 tumor antigen recognition by CD4+ effector T cells.

Oncolytic immunotherapy using oncolytic viruses (OV) has been shown to stimulate the antitumor immune response by inducing the release of tumor-associated antigens (TAA) and danger signals from the dying infected tumor cells. In this study, we sought to determine if the lysis of tumor cells induced by different OV: measles virus, vaccinia virus, vesicular stomatitis virus, herpes simplex type I virus, adenovirus or enterovirus, has consequences on the capacity of tumor cells to present TAA, such as NY-ESO-1. We show that the co-culture of NY-ESO-1neg/HLA-DP4pos melanoma cells with NY-ESO-1pos/HLA-DP4neg melanoma cells infected and killed by different OV induces an intercellular transfer of NY-ESO-1 that allows the recognition of NY-ESO-1neg/HLA-DP4pos tumor cells by an HLA-DP4/NY-ESO-1(157-170)-specific CD4+ cytotoxic T cell clone, NY67. We then confirmed this result in a second model with an HLA-DP4+ melanoma cell line that expresses a low amount of NY-ESO-1. Recognition of this cell line by the NY67 clone is largely increased in the presence of OV productive infection. Altogether, our results show for the first time another mechanism of stimulation of the anti-tumor immune response by OV, via the loading of tumor cells with TAA that sensitizes them for direct recognition by specific effector CD4+ T cells, supporting the use of OV for cancer immunotherapy.

Atelocollagen-mediated in vivo siRNA transfection in ovarian carcinoma is influenced by tumor site, siRNA target and administration route.

Ovarian cancer is the leading cause of death from gynecological malignancies worldwide, and innate or acquired chemoresistance of ovarian cancer cells is the major cause of therapeutic failure. It has been demonstrated that the concomitant inhibition of Bcl-xL and Mcl-1 anti-apoptotic activities is able to trigger apoptosis in chemoresistant ovarian cancer cells. In this context, siRNA-mediated Bcl­xL and Mcl-1 inhibition constitutes an appealing strategy by which to eliminate chemoresistant cancer cells. However, the safest and most efficient way to vectorize siRNAs in vivo is still under debate. In the present study, using in vivo bioluminescence imaging, we evaluated the interest of atelocollagen to vectorize siRNAs by intraperitoneal (i.p.) or intravenous (i.v.) administration in 2 xenografted ovarian cancer models (peritoneal carcinomatosis and subcutaneous tumors in nude mice). Whereas i.p. administration of atelocollagen-vectorized siRNA in the peritoneal carcinomatosis model did not induce any gene downregulation, a 70% transient downregulation of luciferase expression was achieved after i.v. injection of atelocollagen-vectorized siRNA in the subcutaneous (s.c.) model. However, the use of siRNA targeting Bcl-xL or Mcl-1 did not induce target-specific downregulation in vivo in nude mice. Our results therefore show that atelocollagen complex formulation, the administration route, tumor site and the identity of the siRNA target influence the efficiency of atelocollagen­mediated siRNA delivery.

Plasma membrane reorganization links acid sphingomyelinase/ceramide to p38 MAPK pathways in endothelial cells apoptosis.

The p38 MAPK signaling pathway is essential in the cellular response to stress stimuli, in particular in the endothelial cells that are major target of external stress. The importance of the bioactive sphingolipid ceramide generated by acid sphingomyelinase is also firmly established in stress-induced endothelial apoptotic cell death. Despite a suggested link between the p38 MAPK and ceramide pathways, the exact molecular events of this connection remain elusive. In the present study, by using two different activators of p38 MAPK, namely anisomycin and ionizing radiation, we depicted how ceramide generated by acid sphingomyelinase was involved in p38 MAPK-dependent apoptosis of endothelial cells. We first proved that both anisomycin and ionizing radiation conducted to apoptosis through activation of p38 MAPK in human microvascular endothelial cells HMEC-1. We then found that both treatments induced activation of acid sphingomyelinase and the generation of ceramide. This step was required for p38 MAPK activation and apoptosis. We finally showed that irradiation, as well as treatment with exogenous C16-ceramide or bacterial sphingomyelinase, induced in endothelial cells a deep reorganization of the plasma membrane with formation of large lipid platforms at the cell surface, leading to p38 MAPK activation and apoptosis in endothelial cells. Altogether, our results proved that the plasma membrane reorganization leading to ceramide production is essential for stress-induced activation of p38 MAPK and apoptosis in endothelial cells and established the link between the acid sphingomyelinase/ceramide and p38 MAPK pathways.

Oxidative stress disassembles the p38/NPM/PP2A complex, which leads to modulation of nucleophosmin-mediated signaling to DNA damage response.

Oxidative stress is a leading cause of endothelial dysfunction. The p38 MAPK pathway plays a determinant role in allowing cells to cope with oxidative stress and is tightly regulated by a balanced interaction between p38 protein and its interacting partners. By using a proteomic approach, we identified nucleophosmin (NPM) as a new partner of p38 in HUVECs. Coimmunoprecipitation and microscopic analyses confirmed the existence of a cytosolic nucleophosmin (NPM)/p38 interaction in basal condition. Oxidative stress, which was generated by exposure to 500 µM H2O2, induces a rapid dephosphorylation of NPM at T199 that depends on phosphatase PP2A, another partner of the NPM/p38 complex. Blocking PP2A activity leads to accumulation of NPM-pT199 and to an increased association of NPM with p38. Concomitantly to its dephosphorylation, oxidative stress promotes translocation of NPM to the nucleus to affect the DNA damage response. Dephosphorylated NPM impairs the signaling of oxidative stress-induced DNA damage via inhibition of the phosphorylation of ataxia-telangiectasia mutated and DNA-dependent protein kinase catalytic subunit. Overall, these results suggest that the p38/NPM/PP2A complex acts as a dynamic sensor, allowing endothelial cells to react rapidly to acute oxidative stress.-Guillonneau, M., Paris, F., Dutoit, S., Estephan, H., Bénéteau, E., Huot, J., Corre, I. Oxidative stress disassembles the p38/NPM/PP2A complex, which leads to modulation of nucleophosmin-mediated signaling to DNA damage response.

Quantifying and correcting for tail vein extravasation in small animal PET scans in cancer research: is there an impact on therapy assessment?

BACKGROUND: Tail vein injection under short anesthesia is the most commonly used route for administering radiopharmaceuticals. However, the small caliber of the vein in rodents may lead to tracer extravasation and thereby compromise quantitative accuracy of PET. We aimed to evaluate a method for correction of interstitial radiotracer leakage in the context of pre-clinical therapeutic response assessment. METHODS: In two separate studies involving 16 nude rats, a model of human ovarian cancer was xenografted and each was treated with a Phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor or used as a control. Tracer injections were performed via the tail vein by a single operator. Two observers qualitatively evaluated the resulting images and if appropriate drew a volume of interest (VOI) over the injection site to record extravasated activities. Uncorrected and corrected tumors' mean standardized uptake value (SUV)mean was computed (corrected injected activity = calibrated activity - decay corrected residual syringe activity - decay corrected tail extravasated activity). Molecular analyses were taken as a gold standard. The frequency and magnitude of extravasation were analyzed, as well as the inter-observer agreement and the impact of the correction method on tumor uptake quantification. RESULTS: Extravasation never exceeded 20 % of the injected dose but occurred in more than 50 % of injections. It was independent of groups of animals and protocol time points with p values of 1.00 and 0.61, respectively, in the first experiment and 0.47 and 0.13, respectively, in the second experiment. There was a good inter-observer agreement for qualitative analysis (kappa = 0.72) and a moderate agreement when using quantitative analysis (ρ c = 0.94). In both experiments, there was significant difference between uncorrected and corrected SUVmean. Despite this significant difference, mean percent differences between uncorrected and corrected SUVmean in the first and the second experiments were -3.61 and -1.78, respectively. Concerning therapy assessment, in both experiments, significant differences in median %SUVmean between control and treated groups were observed over all time points with either uncorrected and corrected data (p < 0.05). CONCLUSIONS: Although extravasation is common and can be reproducibly corrected, this is probably not required for validation of response to drugs that induce large SUV changes. However, further studies are required to evaluate the impact of extravasation in situations where less marked metabolic responses are observed or important extravasations occur.

NEMA NU 4-Optimized Reconstructions for Therapy Assessment in Cancer Research with the Inveon Small Animal PET/CT System.

PURPOSE: We compared conventional filtered back-projection (FBP), two-dimensional-ordered subsets expectation maximization (OSEM) and maximum a posteriori (MAP) NEMA NU 4-optimized reconstructions for therapy assessment. PROCEDURES: Varying reconstruction settings were used to determine the parameters for optimal image quality with two NEMA NU 4 phantom acquisitions. Subsequently, data from two experiments in which nude rats bearing subcutaneous tumors had received a dual PI3K/mTOR inhibitor were reconstructed with the NEMA NU 4-optimized parameters. Mann-Whitney tests were used to compare mean standardized uptake value (SUV(mean)) variations among groups. RESULTS: All NEMA NU 4-optimized reconstructions showed the same 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) kinetic patterns and detected a significant difference in SUV(mean) relative to day 0 between controls and treated groups for all time points with comparable p values. CONCLUSION: In the framework of therapy assessment in rats bearing subcutaneous tumors, all algorithms available on the Inveon system performed equally.

Identification of predictive factors of response to the BH3-mimetic molecule ABT-737: an ex vivo experiment in human serous ovarian carcinoma.

Ovarian cancers are addicted to Bcl-xL and Mcl-1, antiapoptotic members of the Bcl-2 family. Bcl-xL can be inhibited by the BH3-mimetic ABT-737. In vitro, ABT-737 can induce apoptosis of cancer cells, and its activity is potentiated by Mcl-1 inactivation. Herein, we assessed the sensitivity of human ovarian tumor nodes to ABT-737 when combined with carboplatin, which can indirectly inhibit Mcl-1. Fresh samples from 25 patients with high-grade serous ovarian cancer (HGSOC) who were chemo-naïve and had undergone surgery were prospectively exposed ex vivo to ABT-737 ± carboplatin. The treatment effect was studied on sliced tumor nodes by assessment of cleaved-caspase 3 immunostaining. We also studied the association between baseline Bcl-2 family protein expression (via immunohistochemistry) and the response of nodes to treatment. ABT-737 induced apoptosis as a single agent but its efficacy was not improved by the addition of carboplatin. Bim was frequently expressed (20/25) and its absence or low expression was associated with the absence of response to ABT-737, p value = 0.019 by Fisher's test and sensitivity = 93%, (95% confidence interval, 66-100). Moreover, we observed that in tumors in which Bim was expressed, a low expression of phospho-Erk1/2 or Mcl-1 improved the proportion of responses. This pilot study showed that ABT-737 has promise as monotherapy for HGSOC in a specific subgroup of tumors. Bim, Mcl-1, and phospho-Erk1/2 appeared to be relevant biomarkers that could be used for the selection of patients in the design of clinical trials using Navitoclax (an orally available compound related to ABT-737).

(18)F-FDG Is a Surrogate Marker of Therapy Response and Tumor Recovery after Drug Withdrawal during Treatment with a Dual PI3K/mTOR Inhibitor in a Preclinical Model of Cisplatin-Resistant Ovarian Cancer.

AIM: Targeting the phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway is a potential means of overcoming chemoresistance in ovarian cancer. We investigated the capability of (18)F-fluororodeoxyglucose ((18)F-FDG) small-animal positron emission tomography (SA-PET) to predict the effects of a dual PI3K/mTOR inhibitor (BEZ-235) in a cisplatin-resistant ovarian cancer model. METHODS: In a first experiment, nude rats bearing subcutaneous SKOV3 tumors received BEZ-235 for 3 days given alone or after paclitaxel and were compared to controls (either untreated or that were given the excipients of paclitaxel and BEZ-235). SA-PET was performed at baseline, on day 3, and day 7. In a second experiment aiming at further exploring the kinetics of (18)F-FDG tumor uptake during the first 48 hours following drug cessation, untreated controls were compared to rats receiving BEZ-235, which were imaged at baseline, on day 3, on day 4, and on day 5. SA-PET results were compared to cell proliferation assessment (Ki-67), PI3K/mTOR downstream target expression studies (pAKT and phospho-eukaryotic translation initiation factor 4E-binding protein 1), and apoptosis evaluation (cleaved caspase-3). RESULTS: In the first experiment, BEZ-235, compared to untreated controls, induced a marked decrease in (18)F-FDG uptake on day 3, which was correlated to a significant decrease in cell proliferation and to a significant PI3K/mTOR pathway inhibition. No tumor necrosis or apoptosis occurred. Four days following treatment cessation, tumor recovery (in terms of PI3K/mTOR inhibition and cell proliferation) occurred and was identified by (18)F-FDG SA-PET. Paclitaxel plus BEZ-235 showed results similar to BEZ-235 alone. In the second experiment, PI3K/mTOR pathways exhibited partial recovery as early as 24 hours following treatment cessation, but both (18)F-FDG SA-PET and cell proliferation remained unchanged. CONCLUSIONS: (18)F-FDG SA-PET is a surrogate marker of target inhibition during treatment with BEZ-235 and predicts tumor recovery 4 days after drug withdrawal, but not during the first 48 hours following drug cessation, when a lag between PI3K/mTOR pathway recovery and metabolic recovery is observed. (18)F-FDG SA-PET could be used for therapy monitoring of PI3K/mTOR inhibitors, but our results also raise questions regarding the potential impact of the delay between PET imaging and the last drug intake on the accuracy of FDG imaging.

Biodistribution and imaging of [99mTc]-HYNIC-RGD in MDA-MB-231 and NTERA-2 cancer cell xenografts.

PURPOSE: Increased expression of αvß3 integrins is involved in tumour angiogenesis. Targeting these receptors with a dedicated peptide containing the RGD sequence may provide information about the receptor status in and around the tumour and about the angiogenesis process involved. The aim of this study was to compare the uptake of [Tc]-HYNIC-RGD in two types of tumours that either express or do not express the αvß3 receptor (NTERA-2 and MDA-MB-231, respectively) and discuss the use of this tracer in experimental models of angiogenesis. PROCEDURES: Uptake of intravenously injected [Tc]-HYNIC-RGD was studied ex vivo and in vivo. Histological analysis of excised tumours was carried out. Percentages of the injected dose uptake per gram of tissue were compared between the two types of tumours and in the periphery and centre of each tumour. RESULTS: [Tc]-HYNIC-RGD was rapidly cleared from blood circulation and excreted through the kidneys. Residual activity was retained in the intestine. Tumour uptake of [Tc]-HYNIC-RGD was high and homogeneous for αvß3-positive cell lines (1.94±0.26%ID/g). Tumour uptake was weak and distributed only in the tumour periphery for αvß3-negative cell lines (0.10±0.02%ID/g, tumour periphery; 0.06±0.01%ID/g, tumour core; P=0.01). These results correlated with vessel distribution. CONCLUSION: Uptake of [Tc]-HYNIC-RGD was more intense in αvß3-positive cell lines than in αvß3-negative cell lines, but tracer distribution was more representative of angiogenic locations in αvß3-negative cell lines. Further clinical and preclinical studies are needed on the use of RGD-related tracers.

Platinum compounds sensitize ovarian carcinoma cells to ABT-737 by modulation of the Mcl-1/Noxa axis.

Ovarian cancer is the leading cause of death from gynecological cancer. The anti-apoptotic protein Bcl-x(L) is frequently overexpressed in ovarian carcinoma which correlates with chemotherapy resistance. It has been demonstrated that Bcl-x(L) cooperates with another anti-apoptotic protein, Mcl-1, to protect ovarian cancer cells against apoptosis, and that their concomitant inhibition induces massive cell death. Here, we examined the interest of ABT-737, a potent BH3-mimetic molecule targeting Bcl-x(L), both alone and in combination with Mcl-1 modulators, in ovarian cancer cell lines. As a single agent, ABT-737 was ineffective at promoting cell death in the four cell lines we tested in vitro. However, the specific inhibition of Mcl-1 by siRNA dramatically increased the sensitivity of chemoresistant cells to ABT-737. Platinum compounds also sensitize to ABT-737 by dose-dependently decreasing Mcl-1 expression or by increasing the expression of pro-apoptotic BH3-only proteins Noxa and, to a lower extent, Bim. Furthermore, we demonstrated that Noxa accumulation was involved in apoptosis occurring in response to the combination of ABT-737 and platinum compounds, since cells were protected from apoptosis by its silencing. Moreover, the combination was also highly cytotoxic ex vivo in sliced SKOV3 tumor nodes. However we observed in these slices a strong basal expression of Noxa and apoptotic cell death in response to ABT-737 alone. Therefore, we have revealed that the modulation of the Mcl-1/Noxa axis by platinum compounds results in a strong sensitization of chemoresistant ovarian carcinoma cells to ABT-737, which could constitute a promising therapeutic in these cancers.

Molecular mechanism of hypoxia-induced chondrogenesis and its application in in vivo cartilage tissue engineering.

Cartilage engineering is one of the most challenging issue in regenerative medicine, due to its limited self-ability to repair. Here, we assessed engineering of cartilage tissue starting from human bone marrow (hBM) stem cells under hypoxic environment and delineated the mechanism whereby chondrogenesis could be conducted without addition of exogenous growth factors. hBM stem cells were cultured in alginate beads and chondrogenesis was monitored by chondrocyte phenotypic markers. Activities and roles of Sox and HIF-1α transcription factors were investigated with complementary approaches of gain and loss of function and provided evidences that HIF-1α is essential for hypoxic induction of chondrogenesis. Thereafter, hBM cells and human articular chondrocytes (HAC) underwent chondrogenesis by 3D and hypoxic culture for 7 days or by ectopic expression of HIF-1α. After subcutaneous implantation of 3 weeks into athymic mice, tissue analysis showed that hypoxia or HIF-1α overexpression is effective and sufficient to induce chondrocyte phenotype in hBM cells, without use of exogenous growth factors. Therefore, this study brings interesting data for a simple and affordable system in biotechnology of cartilage engineering.

αvß3 imaging can accurately distinguish between mature teratoma and necrosis in 18F-FDG-negative residual masses after treatment of non-seminomatous testicular cancer: a preclinical study.

PURPOSE: We assessed whether imaging α(v)ß(3) integrin could distinguish mature teratoma from necrosis in human non-seminomatous germ cell tumour (NSGCT) post-chemotherapy residual masses. METHODS: Human embryonal carcinoma xenografts (six/rat) were untreated (controls) or treated to form mature teratomas with low-dose cisplatin and all-trans retinoic acid (ATRA) over a period of 8 weeks. In another group, necrosis was induced in xenografts with high-dose cisplatin plus etoposide (two cycles). (18)F-Fluorodeoxyglucose ((18)F-FDG) small animal positron emission tomography (SA PET) imaging was performed in three rats (one control and two treated for 4 and 8 weeks with cisplatin+ATRA). Imaging of α(v)ß(3) expression was performed in six rats bearing mature teratomas and two rats with necrotic lesions on a microSPECT/CT device after injection of the tracer [(99m)Tc]HYNIC-RGD [6-hydrazinonicotinic acid conjugated to cyclo(Arg-Gly-Asp-D-Phe-Lys)]. Correlative immunohistochemistry studies of human and mouse α(v)ß(3) expression were performed. RESULTS: Cisplatin+ATRA induced differentiation of the xenografts. After 8 weeks, some glandular structures and mesenchymal cells were visible; in contrast, control tumours showed undifferentiated tissues. SA PET imaging showed that mature teratoma had very low avidity for (18)F-FDG [mean standardised uptake value (SUV(mean)) = 0.48 ± 0.05] compared to untreated embryonal carcinoma (SUV(mean) = 0.92 ± 0.13) (p = 0.005). α(v)ß(3) imaging accurately distinguished mature teratoma (tumour to muscle ratio = 4.29 ± 1.57) from necrosis (tumour to muscle ratio = 1.3 ± 0.26) (p = 0.0002). Immunohistochemistry studies showed that α(v)ß(3) integrin expression was strong in the glandular structures of mature teratoma lesions and negative in host stroma. CONCLUSION: Imaging α(v)ß(3) integrin accurately distinguished mature teratoma from necrosis following cisplatin-based treatment in human NSGCT xenografts.

Initial formation of IGROV1 ovarian cancer multicellular aggregates involves vitronectin.

Ovarian cancer progression is frequently associated with the development of malignant ascites. Multicellular aggregates of carcinoma cells (spheroids) found within ascites are thought to be able to promote peritoneal carcinomatosis. We have previously demonstrated the involvement of the vitronectin/alphav integrin adhesive system in the dissemination of ovarian cancer cells and continue to investigate the influence of these molecules by studying their role(s) in spheroid behavior. The aim of this study was to generate ovarian cancer multicellular aggregates and to focus on the role of vitronectin and alphav integrins in their initiation. IGROV1 cancer cells cultured in the absence of adhesive substratum formed multicellular aggregates comparable to spheroids. After 21 days, a fraction of the cells within clusters remained viable and proliferated recurrently. Within the multicellular aggregates, vitronectin and alphav integrins were co-localized at intercellular sites, suggesting their involvement in cell-cell interactions. Initial formation of IGROV1 aggregates was inhibited using anti-vitronectin and anti-alphav integrin blocking antibodies or the cyclic peptide cRGDfV. Vitronectin expression persisted during cluster disaggregation on fibronectin. These results demonstrate the ability of IGROV1 cells to generate multicellular aggregates and point to a contributory role for the vitronectin/alphav integrin system in the initial step of this process. These events could represent a prerequisite for further dissemination.

Mesothelial vitronectin stimulates migration of ovarian cancer cells.

Ovarian carcinomas, the most fatal gynaecological malignancies, are associated with poor prognosis predominantly because of a high recurrence rate. Ovarian cancer cells spread widely throughout the abdominal cavity leading to peritoneal metastasis. The influence of the mesothelial microenvironment on the biological mechanisms leading to cancer cell colonization of the mesothelium is poorly understood. This study aims to investigate whether mesothelial secretions affect the migration of ovarian cancer cells and focuses on the role of the adhesive molecule Vn (vitronectin) and its integrin receptors. An in vitro co-culture model indicated that clusters of IGROV1 and SKOV3 cells adhere to MeT-5A mesothelial cells preferentially at intercellular sites, invade the mesothelial monolayer and alter the integrity of the mesothelium. In addition, mesothelial CM (cell-conditioned medium) induces migration of IGROV1 and SKOV3 cells in Boyden chambers and wound healing assays. Furthermore, blocking molecules directed against vitronectin or its alphav integrin receptor decrease mesothelial-CM-induced migration by approximately 40% and 60-70% for IGROV1 and SKOV3 ovarian cancer cells, respectively, in Boyden chamber assays. Wound healing assays that allow cell migration to be measured over 24 h periods demonstrated that blocking molecules prevent the migration of IGROV1 and SKOV3 cells. Vitronectin is present in CM MeT-5A (mesothelial conditioned medium) and in metastatic peritoneal tissue sections. The expression of vitronectin at the periphery of mesothelial cells and within ovarian cancer cell clusters suggests a potential role for this molecule during intraperitoneal implantation of ovarian cancer cells. Vitronectin could represent a target for the development of anti-adhesive strategies to impede ovarian cancer dissemination.

Mcl-1 is an important determinant of the apoptotic response to the BH3-mimetic molecule HA14-1 in cisplatin-resistant ovarian carcinoma cells.

Chemoresistance of ovarian carcinoma has been associated previously to the absence of Bcl-x(L) expression downregulation in response to cisplatin. Among BH3-mimetic molecules constituting promising anticancer agents able to inhibit the activity of antiapoptotic Bcl-2 family proteins, we evaluated the effect of one of them, HA14-1, on various ovarian carcinoma cell lines. In response to HA14-1, the cisplatin-resistant IGROV1-R10 cell line underwent massive cell death, whereas other cell lines presented a partial response (IGROV1, SKOV3, and A2780) or did not respond to this molecule (OAW42 and OAW42-R). However, the expression of HA14-1 targets (Bcl-2 and Bcl-x(L)) did not correlate to these different responses. In contrast, cell death was associated with the disappearance of Mcl-1 after exposure to HA14-1. We showed that, in the HA14-1 nonresponsive cell lines (SKOV3 and OAW42), small interfering RNA-mediated Mcl-1 downregulation allowed HA14-1-induced massive apoptosis in the absence of chemotherapy. Furthermore, cisplatin-induced Mcl-1 downregulation was also able to sensitize highly chemoresistant SKOV3 cells to HA14-1. Taken together, these results show that Bcl-x(L) and Mcl-1 are able to cooperate to protect ovarian carcinoma cells against oncogenic stress or chemotherapy-induced apoptosis and suggest that the development of multitargeted strategies directed against these two antiapoptotic proteins may constitute a major challenge for the therapeutic care of chemoresistant ovarian carcinomas. BH3-mimetic compounds represent promising tools for this purpose either on their own (direct or indirect pan-inhibitors) or in combination with new drugs aiming to inactivate Mcl-1.

Early evaluation of the effects of chemotherapy with longitudinal FDG small-animal PET in human testicular cancer xenografts: early flare response does not reflect refractory disease.

AIM: We aimed to evaluate the usefulness of FDG PET in the early prediction of the effects of chemotherapy on human testicular cancer xenografts. MATERIAL AND METHODS: Nude rats bearing subcutaneous human embryonal carcinoma xenografts received either cisplatin (5 mg/kg) or saline serum. Small-animal PET studies were performed on days 0, 2, 4 and 7 and compared to immunochemistry studies, flow cytometry studies and hexokinase assays. RESULTS: Cisplatin treatment resulted in biphasic FDG uptake evolution: a peak was observed on day 2, followed by a marked decrease on day 7 despite an insignificant change in tumour volume. Similarly, a peak in cyclin A immunostaining was observed on days 2 and 4), followed by a significant decrease on day 7. Flow cytometry showed that the cyclin A peak was not related to increased cell proliferation but was due to a transient S and G(2)/M cell cycle arrest. A marked increase in cell apoptosis was observed from day 2 to day 7. GLUT-1 showed a significant decrease on day 7. Macrophagic infiltrate remained stable except for an increase observed on day 7. In control tumours, continuous growth was observed, all immunostaining markers remaining stable over time. Hexokinase activity was significantly lower on day 7 in treated tumours than in controls. CONCLUSION: FDG PET may be useful in the early evaluation of treatment in patients with testicular cancer. In our model, a very early increased [(18)F]-FDG uptake was related to a transient cell cycle arrest and early stage apoptosis but did not reveal refractory disease.

Vitronectin and its receptors partly mediate adhesion of ovarian cancer cells to peritoneal mesothelium in vitro.

Epithelial ovarian cancer cells metastasize by implanting onto the peritoneal mesothelial surface of the abdominal cavity. Adhesive molecules that lead to this implantation remain unclear. The aim of our study was to focus on the role of vitronectin (Vn) and its receptors, alpha(v) integrins and urokinase plasminogen activator receptor (uPAR), in the interactions of ovarian adenocarcinoma cells (IGROV1 and SKOV3 cell lines) with mesothelial cells (MeT-5A cell line and primary cultures). For all cell lines, immunofluorescence staining disclosed the presence of Vn over the whole cell surface and in thin continuous deposits underlining the cell periphery. Recruitment of Vn receptors to cell-cell contact sites was also revealed. We developed two distinct methods for the evaluation of in vitro cell-cell adhesion using cocultures of the tumor and mesothelial cells. Both adhesion assays revealed a strong ability of ovarian cancer cells to adhere preferentially to mesothelial intercellular junctions. Adhesion of ovarian carcinoma cells to mesothelial cells was significantly inhibited using anti-Vn-, -alpha(v)-integrin- and -uPAR-blocking antibodies or cyclic peptide cRGDfV. These results evidence the ability of ovarian carcinoma cells to bind to peritoneal mesothelium in vitro and strongly suggest that Vn and its receptors contribute to this crucial event.