Fabien Gueugnon

Identification of Novel Markers for the Diagnosis of Malignant Pleural Mesothelioma

The diagnosis of malignant pleural mesothelioma is difficult, with the most common differential diagnoses being benign pleural diseases and metastatic adenocarcinomas (ADCA). To identify novel markers that would be able to improve diagnostic accuracy, we performed a genome-wide gene expression analysis on tumor cell lines established from pleural effusions (malignant pleural mesothelioma and lung ADCA). This analysis led to the identification of genes encoding novel and pertinent cellular and soluble markers, for which the expression was validated by real-time RT-PCR. Immunohistochemical staining of tumor biopsy specimens with anti-type III collagen antibodies showed positive labeling for mesothelioma cells but not for ADCA cells. Using enzyme-linked immunosorbent assay, we showed that the C-C motif chemokine 2 (CCL2) concentration was significantly higher in pleural effusions from patients with mesothelioma (n = 61) than in subjects with ADCA (n = 25) or with benign pleural effusions (n = 15): median (interquartile range) = 2.99 ng/ml (1.76 to 6.01) vs 0.99 ng/ml (0.51 to 1.83) and 1.47 ng/ml (0.80 to 1.56), respectively, P < 0.0001. Conversely, the galectin-3 concentration was lower in mesothelioma: 11.50 ng/ml (6.73 to 23.53) vs 24.74 ng/ml (20.42 to 70.35) and 17.64 ng/ml (14.81 to 24.68), respectively, P < 0.0001. The areas under the curve for CCL2 were 0.8030 and 0.7716 for the differentiation of mesothelioma from ADCA or benign pleural effusions, respectively. Similarly, the areas under the curve obtained for galectin-3 were 0.7980 and 0.6923, respectively. In conclusion, type III collagen, CCL2, and galectin-3 are promising new diagnostic markers for mesothelioma.

Nanoparticles Produced by Ring-Opening Metathesis Polymerization Using Norbornenyl-poly(ethylene oxide) as a Ligand-Free Generic Platform for Highly Selective In Vivo Tumor Targeting

We described a norbornenyl-poly(ethylene oxide) nanoparticles ligand-free generic platform, made fluorescent with straightforward preparation by ring-opening metathesis polymerization (ROMP). Our method allowed to easily obtain a drug delivery system (DDS) with facilitated functionalization by means of azide-alkyne click chemistry and with a high selectivity for the tumor in vivo, while cellular internalization is obtained without cell targeting strategy. We demonstrated that our nanoparticles are internalized by endocytosis and colocalized with acidic intracellular compartments in two models of aggressive tumoral cell lines with low prognostic and limited therapeutic treatments. Our nanoparticles could be of real interest to limit the toxicity and to increase the clinical benefit of drugs suffering rapid clearance and side effects and an alternative for cancers with poorly efficient therapeutic solutions by associating the drug delivery in the tumor tissue with an acid-sensitive release system.

A 5-aza-2′-deoxycytidine/valproate combination induces cytotoxic T-cell response against mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive tumour with a limited response to conventional therapy. The aim of this study was to evaluate the anticancer effect of a DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-azaCdR), and two histone deacetylase inhibitors, valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA). Human mesothelioma cells were treated with each epigenetic drug, either alone or in combinations. The cytotoxic effects on treated cells and the expression of specific tumour antigens were evaluated. The recognition of treated cells by a specific CD8+ T-cell clone was also measured. Additionally, the effect of combined treatments was tested in a murine model of mesothelioma. We showed that VPA and SAHA synergised with 5-azaCdR to kill MPM cells and induce tumour antigen expression in the remaining living tumour cells. As a consequence, tumour cells expressing these antigens were recognised and lysed by specific CD8+ cytotoxic T-cells. In vivo, treatment with 5-azaCdR/VPA inhibited tumour growth, and promoted lymphocyte infiltration and an immune response against tumour cells. Appropriate epigenetic drug combinations, in addition to inducing mesothelioma cell death, also affect the immunogenic status of these cells. This property could be exploited in clinical investigations to develop MPM treatments combining chemotherapeutic and immunotherapeutic approaches.

CCL2, Galectin-3, and SMRP Combination Improves the Diagnosis of Mesothelioma in Pleural Effusions

Introduction: Malignant pleural mesothelioma (MPM) is a highly aggressive tumor with poor prognosis. One major challenge for this disease is the development of new, early, and highly reliable diagnostic markers. The aim of this study was to compare the diagnostic value of the chemokine chemokine (C-C motif) ligand 2 (CCL2), galectin-3, and the secretory leukocyte peptidase inhibitor (SLPI) with soluble mesothelin-related peptides (SMRP), and to evaluate the diagnostic performance of marker combinations. Methods: The levels of the different markers were measured by enzyme-linked immunosorbent assay in pleural fluids from patients with MPM (n = 61), adenocarcinomas (ADCA, n = 25), or with benign pleural effusions (BPE, n = 15). Results: SMRP, SLPI, and CCL2 concentrations were significantly higher in pleural effusions from mesothelioma patients. Conversely, galectin-3 levels seemed to be elevated in patients with pulmonary ADCA. Receiver operating characteristic curve analysis revealed that SMRP (area under the curve [AUC] = 0.9059), CCL2 (AUC = 0.7912), galectin-3 (AUC = 0.7584), and SLPI (AUC = 0.7219) were potentially interesting biomarkers for the differentiation of MPM patients from those with BPE or ADCA. Of interest, we showed that the combination of SMRP/CCL2/galectin-3 greatly improved MPM diagnosis (AUC = 0.9680), when compared with SMRP alone. Conclusion: The combination of SMRP/CCL2/galectin-3 seems to represent a promising panel of biomarkers for the reliable diagnosis of MPM in pleural fluids.

Vorinostat–Polymer Conjugate Nanoparticles for Acid-Responsive Delivery and Passive Tumor Targeting

In vivo histone deacetylase (HDAC) inhibition by vorinostat under clinically acceptable dosing is limited by its poor pharmacokinetics properties. A new type of nontoxic pH-responsive delivery system has been synthesized by ring-opening metathesis polymerization, allowing for the selective distribution of vorinostat in mesothelioma tumors in vivo and subsequent histone reacetylation. The delivery system is synthesized by generic click chemistry, possesses native stealth properties for passive tumor targeting, and does not need additional chemistry for cellular internalization. Although vorinostat alone at 50 mg/kg in mice showed no effect, our new delivery system with 2 mg/kg vorinostat promoted histone reacetylation in tumors without side effects, demonstrating that our strategy improves the activity of this HDAC inihibitor in vivo.

Downregulation of MUC1 expression and its recognition by CD8+ T cells on the surface of malignant pleural mesothelioma cells treated with HDACi

Research into new treatments against malignant pleural mesothelioma (MPM) is of great interest, as this aggressive cancer is often resistant to conventional therapies. One potential strategy is the use of epigenetic drugs, such as 5‐aza‐2′‐deoxycytidine (5‐azaCdR), a DNA‐hypomethylating drug, and valproate (VPA), a histone deacetylase inhibitor (HDACi). Indeed, these drugs not only trigger MPM cell death, but also induce the expression of cancer testis antigens recognized by CD8+ T cells, such as New York‐esophageal cancer‐1 (NY‐ESO‐1). The objective of this study was to assess effects of these drugs on the expression and recognition by CD8+ T cells of Mucin1 (MUC1), a tumor‐associated antigen that is overexpressed by MPM. MPM tumor cell lines were treated with epigenetic drugs, alone or in combination. MUC1 expression by MPM cells, and its recognition by a MUC1‐specific CD8+ T‐cell clone, was downregulated by HDACi when used alone or in combination with 5‐azaCdR. This effect was not due to a blocking of the HLA class I presentation pathway in treated MPM cells, as NY‐ESO‐1 induced by 5‐azaCdR alone, or with VPA, was recognized by a NY‐ESO‐1‐specific T‐cell clone. This study suggests that the choice of tumor antigens could be critical for strategies combining epigenetic drugs with immunotherapy.

Downregulation of the neonatal Fc receptor expression in non-small cell lung cancer tissue is associated with a poor prognosis.

Lung cancer is the leading cause of cancer-related death worldwide. Although the recommended tumor, node and metastasis (TNM) classification and stage determination are important to select therapeutic options for patients with non-small cell lung carcinoma (NSCLC), additional molecular markers are required to indicate the prognosis, in particular within a specific stage, and help with the management of patients. Because neonatal Fc receptor (FcRn) has recently been involved in colon cancer immunosurveillance, we measured its expression in non-cancerous and NSCLC lung tissues and evaluated its prognostic value in overall survival for patient with NSCLC. FcRn expression was determined at both mRNA and protein levels on cancerous and adjacent non-cancerous tissues from 80 NSCLC patients. In NSCLC, FcRn was mainly found in resident and tumor infiltrating immune cells. The corresponding mRNA and protein were significantly less abundant in lung tumor than non-cancerous tissue. Moreover, analysis of our cohort and datasets from the public data bases show that FCGRT mRNA down-regulation is a robust and independent, unfavorable predictive factor of NSCLC patient survival. We conclude that FCGRT mRNA expression may be a useful additional marker for immunoscoring, reflecting tumor immune system, and help in the decision-making process for NSCLC patients.

Interleukin-22 receptor is overexpressed in nonsmall cell lung cancer and portends a poor prognosis

The recent decade has witnessed a change of view about cancer mechanisms. Considerable evidence now suggests that the tumour microenvironment status is well correlated with disease outcome. The presence of particular immune cell types or molecules determines whether a pro- or antitumour immune response predominates within the microenvironment [1]. Accordingly, improved understanding of the factors that modulate the tumour microenvironment will be critical for the development of effective future strategies for cancer management. High expression of IL-22R1 in nonsmall cell lung cancer is an independent indicator of poor overall survival http://ow.ly/WZxKh

Altered expression of the CCN genes in the lungs of mice in response to cigarette smoke exposure and viral and bacterial infections.

The CCN proteins are key signaling and regulatory molecules involved in many biological functions and contribute to malignant and non-malignant lung diseases. Despite the high morbidity and mortality of the lung respiratory infectious diseases, there is very little data related to the expression of the CCNs during infection. We investigated in mice the pulmonary mRNA expression levels of five CCNs (1 to 5) in response to influenza A virus (IAV) and bacterial agents (Nontypeable Haemophilus influenzae (NTHi), lipopolysaccharide (LPS) and lipoteichoic acid (LTA)). IAV, NTHi, LPS or LTA were instilled intranasally into mice. Mice were also exposed for 4days or 8weeks to cigarette smoke alone or prior infection to IAV in order to determine if CS modifies the CCN response to a viral infection. All challenges induced a robust inflammation. The mRNA expression of CCN1, CCN2 and CCN3 was decreased after short exposure to CS whereas prolonged exposure altered the expression of CCN1, CCN3 and CCN4. Influenza A virus infection increased CCN1, 2, 4 and 5 mRNA levels but expression of CCN3 was significantly decreased. Acute CS exposure prior infection had little effect on the expression of CCN genes but prolonged exposure abolished the IAV-dependent induction. Treatment with LPS or LTA and infection with NTHi revealed that both Gram-positive and Gram-negative bacteria rapidly modulate the expression of the CCN genes. Our findings reveal that several triggers of lung inflammation influence differently the CCN genes. CCN3 deserves special attention since its mRNA expression is decreased by all the triggers studied.

Kallikrein-related peptidase 5 and seasonal influenza viruses, limitations of the experimental models for activating proteases

Abstract Every year, influenza A virus (IAV) affects and kills many people worldwide. The viral hemagglutinin (HA) is a critical actor in influenza virus infectivity which needs to be cleaved by host serine proteases to exert its activity. KLK5 has been identified as an activating protease in humans with a preference for the H3N2 IAV subtype. We investigated the origin of this preference using influenza A/Puerto Rico/8/34 (PR8, H1N1) and A/Scotland/20/74 (Scotland, H3N2) viruses. Pretreatment of noninfectious virions with human KLK5 increased infectivity of Scotland IAV in MDCK cells and triggered influenza pneumonia in mice. These effects were not observed with the PR8 IAV. Molecular modeling and in vitro enzymatic studies of peptide substrates and recombinant HAs revealed that the sequences around the cleavage site do not represent the sole determinant of the KLK5 preference for the H3N2 subtype. Using mouse Klk5 and Klk5-deficient mice, we demonstrated in vitro and in vivo that the mouse ortholog protease is not an IAV activating enzyme. This may be explained by unfavorable interactions between H3 HA and mKlk5. Our data highlight the limitations of some approaches used to identify IAV-activating proteases.