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Abstract Background The cRGD peptide is a promising probe for early non-invasive detection of tumors. This study aimed to demonstrate how RAFT-c(-RGDfK-)4, a molecule allowing a tetrameric presentation of cRGD, improved cRGD-targeting potential using in vivo models of αVβ3-positive or negative tumors. Results We chose the human embryonic kidney cells HEK293(β3) (high levels of αVβ3) or HEK293(β1) (αVβ3-negative but expressing αV and β1) engrafted subcutaneously (s.c.) in mice. Non-invasive in vivo optical imaging demonstrated that as compared to its monomeric cRGD analogue, Cy5-RAFT-c(-RGDfK-)4 injected intravenously had higher uptake, prolonged retention and markedly enhanced contrast in HEK293(β3) than in the HEK293(β1) tumors. Blocking studies further demonstrated the targeting specificity and competitive binding ability of the tetramer. Conclusion In conclusion, we demonstrated that Cy5-RAFT-c(-RGDfK-)4 was indeed binding to the αVβ3 receptor and with an improved activity as compared to its monomeric analog, confirming the interest of using multivalent ligands. Intravenous injection of Cy5-RAFT-c(-RGDfK-)4 in this novel pair of HEK293(β3) and HEK293(β1) tumors, provided tumor/skin ratio above 15. Such an important contrast plus the opportunity to use the HEK293(β1) negative control cell line are major assets for the community of researchers working on the design and amelioration of RGD-targeted vectors or on RGD-antagonists.

The modular synthesis and biological characterization of template assembled cyclopeptides as a multimeric system for targeting and endocytosis of cells expressing alphavbetta(sub 3) integrin are reported. The alphavbetta(sub 3) integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis.

Background: The cRGD peptide is a promising probe for early non-invasive detection of tumors. This study aimed to demonstrate how RAFT-c(-RGDfK-)4, a molecule allowing a tetrameric presentation of cRGD, improved cRGD-targeting potential using in vivo models of αVβ3-positive or negative tumors. Results: We chose the human embryonic kidney cells HEK293(β3) (high levels of αVβ3) or HEK293(β1) (αVβ3-negative but expressing αV and β1) engrafted subcutaneously (s.c.) in mice. Non-invasive in vivo optical imaging demonstrated that as compared to its monomeric cRGD analogue, Cy5-RAFT-c(-RGDfK-)4 injected intravenously had higher uptake, prolonged retention and markedly enhanced contrast in HEK293(β3) than in the HEK293(β1) tumors. Blocking studies further demonstrated the targeting specificity and competitive binding ability of the tetramer. Conclusion: In conclusion, we demonstrated that Cy5-RAFT-c(-RGDfK-)4 was indeed binding to the αVβ3 receptor and with an improved activity as compared to its monomeric analog, confirming the interest of using multivalent ligands. Intravenous injection of Cy5-RAFT-c(-RGDfK-)4 in this novel pair of HEK293(β3) and HEK293(β1) tumors, provided tumor/skin ratio above 15. Such an important contrast plus the opportunity to use the HEK293(β1) negative control cell line are major assets for the community of researchers working on the design and amelioration of RGD-targeted vectors or on RGD-antagonists. [ABSTRACT FROM AUTHOR]

We reported that regioselectively addressable functionalized template (RAFT)-c(-RGDfK-)4 presenting four cyclic (Arg-Gly-Asp) (cRGD) peptides targets integrin αVβ3 with an improved specificity compared with monomeric cRGD. In this study, we improved this vector by creating a "stealth" molecule in which a fluorescence quencher (Q) is linked to Cy5 via a disulfide bond (-SS-). RAFT-c(-RGDfK-)4-Cy5-SS-Q fluorescence is quenched unless activated by reduction during internalization. RAFT-c(-RGDfK-)4-Cy5-SS-Q fluorescence was negligible when compared with the control but totally recovered after cleavage of the disulfide bridge. Confocal microscopy showed that only the intracellular Cy5 signal could be detected using RAFT-c(-RGDfK-)4-Cy5-SS-Q, confirming that uncleaved extracellular molecules are not visible. Whole-body imaging of mice bearing subcutaneous tumors injected intravenously with RAFT-c(-RGDfK-)4-Cy5-SS-Q showed a very significant enhancement of the fluorescent contrast in tumors compared with the unquenched molecule. Histology of the tumor confirmed the intracellular accumulation of Cy5. These results demonstrate that the presence of a labile disulfide bridge between the targeting vector and a drug mimetic is an efficient way to deliver a dye, or a drug, intracellularly. In addition, this quenched RAFT-c(-RGDfK-)4-Cy5-SS-Q probe is a very powerful vector for imaging tumor masses and investigating in vivo RGD-mediated internalization. [ABSTRACT FROM AUTHOR]

Our group has developed a new molecular tool based on the use of a regioselectively addressable, functionalized template (RAFT) scaffold, where four cyclic (Arg-Gly-Asp) (cRGD) peptide motifs were grafted. The aim of this study was to determine whether RAFT-c(-RGDfK-)4 combined with optical imaging could allow noninvasive detection of deep ovarian metastases. Human ovarian adenocarcinoma IGROV1 cells expressing low levels of integrin αVβ3 (the main receptor for the cRGD peptide) were used for in vitro and in vivo assays in combination with Cy5-labeled RAFT-c(-RGDfK-)4, cRGD, or RAFT-c(-RβADfK-)4. In vivo fluorescence imaging was performed on subcutaneous (SC) tumors and intraperitoneal IGROV1 metastases in nude mice. The accumulation of RGD-Cy5 conjugates in cultured cells or in tumor tissues was examined using confocal laser scanning microscopy. RAFT-c(-RGDfK-)4 exhibited stronger staining in vitro, enhanced tumor-to-background ratio for SC tumors, and allowed early detection of 1- to 5-mm large intraabdominal nodules using noninvasive optical imaging. Histological study revealed that RAFT-c(-RGDfK-)4 accumulated into tumor neovasculature but also into tumor cells. Our data demonstrate that a Cy5-labeled RAFT-c(-RGDfK-)4 is an efficient optical probe for early and noninvasive tumor detection. [ABSTRACT FROM AUTHOR]

We reported that regioselectively addressable functionalized template (RAFT)-c(-RGDfK-)4 presenting four cyclic (Arg-Gly-Asp) (cRGD) peptides targets integrin αVβ3 with an improved specificity compared with monomeric cRGD. In this study, we improved this vector by creating a "stealth" molecule in which a fluorescence quencher (Q) is linked to Cy5 via a disulfide bond (-SS-). RAFT-c(-RGDfK-)4-Cy5-SS-Q fluorescence is quenched unless activated by reduction during internalization. RAFT-c(-RGDfK-)4-Cy5-SS-Q fluorescence was negligible when compared with the control but totally recovered after cleavage of the disulfide bridge. Confocal microscopy showed that only the intracellular Cy5 signal could be detected using RAFT-c(-RGDfK-)4-Cy5-SS-Q, confirming that uncleaved extracellular molecules are not visible. Whole-body imaging of mice bearing subcutaneous tumors injected intravenously with RAFT-c(-RGDfK-)4-Cy5-SS-Q showed a very significant enhancement of the fluorescent contrast in tumors compared with the unquenched molecule. Histology of the tumor confirmed the intracellular accumulation of Cy5. These results demonstrate that the presence of a labile disulfide bridge between the targeting vector and a drug mimetic is an efficient way to deliver a dye, or a drug, intracellularly. In addition, this quenched RAFT-c(-RGDfK-)4-Cy5-SS-Q probe is a very powerful vector for imaging tumor masses and investigating in vivo RGD-mediated internalization. [ABSTRACT FROM AUTHOR]

Our group has developed a new molecular tool based on the use of a regioselectively addressable, functionalized template (RAFT) scaffold, where four cyclic (Arg-Gly-Asp) (cRGD) peptide motifs were grafted. The aim of this study was to determine whether RAFT-c(-RGDfK-)4 combined with optical imaging could allow noninvasive detection of deep ovarian metastases. Human ovarian adenocarcinoma IGROV1 cells expressing low levels of integrin αVβ3 (the main receptor for the cRGD peptide) were used for in vitro and in vivo assays in combination with Cy5-labeled RAFT-c(-RGDfK-)4, cRGD, or RAFT-c(-RβADfK-)4. In vivo fluorescence imaging was performed on subcutaneous (SC) tumors and intraperitoneal IGROV1 metastases in nude mice. The accumulation of RGD-Cy5 conjugates in cultured cells or in tumor tissues was examined using confocal laser scanning microscopy. RAFT-c(-RGDfK-)4 exhibited stronger staining in vitro, enhanced tumor-to-background ratio for SC tumors, and allowed early detection of 1- to 5-mm large intraabdominal nodules using noninvasive optical imaging. Histological study revealed that RAFT-c(-RGDfK-)4 accumulated into tumor neovasculature but also into tumor cells. Our data demonstrate that a Cy5-labeled RAFT-c(-RGDfK-)4 is an efficient optical probe for early and noninvasive tumor detection. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate prognostic and predictive molecular biomarkers in early-stage non-small cell lung carcinoma (NSCLC) receiving neoadjuvant chemotherapy., Experimental Design: The IFCT-0002 trial compared two neoadjuvant regimens in 528 stages I to II NSCLC patients. DNA extraction of snap-frozen surgical samples taken from 208 patients receiving gemcitabine-cisplatin or paclitaxel-carboplatin regimens allowed for the identification of 3p allelic imbalance, Ras association domain family 1A (RASSF1A) and death-associated protein kinase 1 (DAPK1) promoter methylation, and epidermal growth factor receptor, K-ras, and TP53 mutations. Multivariate analysis identified prognostic and predictive effects of molecular alterations. A Bootstrapping approach was used to assess stability of the prognostic models generating optimism corrected indexes., Results: RASSF1A methylation correlated significantly with shorter disease-free survival (DFS; adjusted HR = 1.88, 95% CI: 1.25-2.82, P = 0.0048) and shorter median overall survival (OS; adjusted HR = 2.01, 95% CI: 1.26-3.20, P = 0.020). A computed bootstrap resampling strategy led to a prognostic model, including RASSF1A, DAPK1, and tumor stage, dividing patients into three prognostic groups, with median OS ranging from 34 months for high-risk patients (HR for death = 3.85, 95% CI: 1.79-6.40) to more than 84 months for moderate (HR = 1.85, 95% CI: 0.97-3.52) and low-risk patients (reference group; P = 0.00044). In addition, RASSF1A methylation predicted longer DFS in patients treated with paclitaxel-carboplatin compared with gemcitabine-cisplatin (adjusted HR = 0.47, 95% CI: 0.23-0.97, P(interaction) = 0.042)., Conclusions: Following neoadjuvant chemotherapy, RASSF1A methylation negatively impacted prognosis of early-stage NSCLC. Along with DAPK1 methylation and tumor stage, RASSF1A methylation allowed definition of three subgroups with strikingly different prognosis. Conversely, significantly longer DFS following paclitaxel-based neoadjuvant chemotherapy for patients whose tumors showed RASSF1A methylation suggested its predictive interest in stages I and II NSCLC., (©2012 AACR.)

Background: The cRGD peptide is a promising probe for early non-invasive detection of tumors. This study aimed to demonstrate how RAFT-c(-RGDfK-)4, a molecule allowing a tetrameric presentation of cRGD, improved cRGD-targeting potential using in vivo models of alphaVbeta3-positive or negative tumors., Results: We chose the human embryonic kidney cells HEK293(beta3) (high levels of alphaVbeta3) or HEK293(beta1) (alphaVbeta3-negative but expressing alphaV and beta1) engrafted subcutaneously (s.c.) in mice. Non-invasive in vivo optical imaging demonstrated that as compared to its monomeric cRGD analogue, Cy5-RAFT-c(-RGDfK-)4 injected intravenously had higher uptake, prolonged retention and markedly enhanced contrast in HEK293(beta3) than in the HEK293(beta1) tumors. Blocking studies further demonstrated the targeting specificity and competitive binding ability of the tetramer., Conclusion: In conclusion, we demonstrated that Cy5-RAFT-c(-RGDfK-)4 was indeed binding to the alphaVbeta3 receptor and with an improved activity as compared to its monomeric analog, confirming the interest of using multivalent ligands. Intravenous injection of Cy5-RAFT-c(-RGDfK-)4 in this novel pair of HEK293(beta3) and HEK293(beta1) tumors, provided tumor/skin ratio above 15. Such an important contrast plus the opportunity to use the HEK293(beta1) negative control cell line are major assets for the community of researchers working on the design and amelioration of RGD-targeted vectors or on RGD-antagonists.

During malignant transformation, the malignant cell accumulates epigenetic abnormalities that do not alter the DNA sequence but are transmissible during divisions and modify genes expression. The methylation of CpG islands in the tumor suppressor genes (TS genes) promoters inhibits their transcription ; it is a mecanism of gene inactivation as frequent as allelic deletions. The methylation profile (or panel of methylated genes in a tumor), similarly to allelic deletions, varies with the tumor histology. Within head and neck squamous cell carcinoma (oral cavity, larynx and oropharynx), 19 genes have been analysed, among them 5 are frequently methylated, i.e. : p16, ECAD, DAPK, MGMT et TIMP3. The method of methylation analysis, based on a bisulfite treatment followed by a PCR amplification, is sensitive and specific enough to allow the detection of abnormalities in biological fluid that drain the tumor or in circulating tumoral DNA. In the head and neck squamous cell carcinoma, correlation between the methylation profile in tumor and paired saliva is excellent ; thus methylation analysis in saliva is a very promising approach for early cancer detection in high risk patients or for the post treatment follow up and rapid diagnosis of relapse. The methylation signature might also reflect the tumor prognosis and complete the histology to define the diagnosis. Finally, DNA methylation is reversible with demethylating agents, a new avenue for cancer therapy in association with conventional chemotherapy.

We reported that regioselectively addressable functionalized template (RAFT)-c(-RGDfK-)(4 )presenting four cyclic (Arg-Gly-Asp) (cRGD) peptides targets integrin alpha(V)beta(3) with an improved specificity compared with monomeric cRGD. In this study, we improved this vector by creating a "stealth" molecule in which a fluorescence quencher (Q) is linked to Cy5 via a disulfide bond (-SS-). RAFT-c(-RGDfK-)(4)-Cy5-SS-Q fluorescence is quenched unless activated by reduction during internalization. RAFT-c(-RGDfK-)(4)-Cy5-SS-Q fluorescence was negligible when compared with the control but totally recovered after cleavage of the disulfide bridge. Confocal microscopy showed that only the intracellular Cy5 signal could be detected using RAFT-c(-RGDfK-)(4)-Cy5-SS-Q, confirming that uncleaved extracellular molecules are not visible. Whole-body imaging of mice bearing subcutaneous tumors injected intravenously with RAFT-c(-RGDfK-)(4)-Cy5-SS-Q showed a very significant enhancement of the fluorescent contrast in tumors compared with the unquenched molecule. Histology of the tumor confirmed the intracellular accumulation of Cy5. These results demonstrate that the presence of a labile disulfide bridge between the targeting vector and a drug mimetic is an efficient way to deliver a dye, or a drug, intracellularly. In addition, this quenched RAFT-c(-RGDfK-)(4)-Cy5-SS-Q probe is a very powerful vector for imaging tumor masses and investigating in vivo RGD-mediated internalization.

We study herein the multivalency effect of a cluster of alphaVbeta3-ligands held on a cyclodecapeptide template. An array of RAFT(c[-RGDfK-])n derivatives containing from one to sixteen clustered RGD motifs were synthesized and comparatively assayed in vitro on alphaVbeta3-expressing cells. Efficient inhibition of the alphaVbeta3-specific 23C6 monoclonal antibody fixation was observed with ligands displaying three and four copies of the cyclo[-RGDfK-] peptide.

Extracorporeal photochimiotherapy (ECP) is based on the exposure of peripheral blood mononuclear cells to the photosensitizing agent (psoralen or 8MOP) and UVA radiation. Mononuclear cells are harvested by cytapheresis and reinfused to the patient after irradiation. This cell therapy has been shown to be effective in the treatment of selected diseases mediated by clonal T cells proliferation such as Sezary T cells lymphoma, rejection after solid organ transplantation and graft-versus-host disease but results obtained in autoimmune diseases are less convincing. ECP is well tolerated with very few side effects and can be combined with immunosuppressive drugs. Two methods of ECP are currently used: in the first one, the whole procedure is performed with the same equipment whereas in the second one, the cytapheresis is performed on a conventional cell separator and treated with an independent UVA irradiation: Experimental data and clinical results suggest that PCE might induced an immune response against pathological T cells clones. However, technical differences in the methods of PCE and weak knowledge on its mechanism of action impair the standardization and evaluation of this cell therapy process as well as its clinical development., (John Libbey Eurotext 2003)

MYCN oncogene amplification is an established indicator of the aggressiveness of neuroblastomas; it is used internationally for stratifying patients for therapy. The present study shows that high levels of MYCN DNA sequences are present in the peripheral blood of patients with MYCN-amplified neuroblastomas. Circulating MYCN DNA may be a powerful and noninvasive prognostic marker at the time of diagnosis. Furthermore, preliminary data strongly suggest that the release of MYCN sequences in the peripheral blood is an early process in disease progression, permitting us to propose this novel marker for the follow-up of patients after chemotherapy.