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Tumor necrosis factor (TNF)-α is present in the microenvironment of human tumors, including malignant pleural effusion (MPE). Although the cytokine is produced in the pleural cavity by both tumor and host cells, its effects on MPE formation are unknown. In these studies, we sought to determine the role of TNF-α in the pathogenesis of MPE and to assess the therapeutic effects of its neutralization in a preclinical model. For this, MPEs were generated in immunocompetent mice using intrapleural injection of mouse lung adenocarcinoma cells. The roles of tumor- and host-derived TNF-α were assessed using combined experimentation with TNF-α gene–deficient mice and in vivo TNF-α neutralization. To expand the scope of preclinical data, TNF-α and vascular endothelial growth factor (VEGF) expression were determined in human cancer cell lines and human MPE. In the MPE model, TNF-α of host and tumor origin was present. TNF-α neutralization significantly limited tumor dissemination, effusion formation, vascular hyperpermeability, TNF-α and VEGF expression, and angiogenesis, thereby improving survival. In contrast, these variables were not different between TNF-α gene–sufficient and TNF-α gene–deficient mice. In mouse cancer cells, TNF-α functioned via nuclear factor-κB– and neutral sphingomyelinase–dependent pathways to induce TNF-α and VEGF, respectively. These results were recapitulated in human cancer cells, and a correlation was detected between TNF-α and VEGF content of human MPE. We conclude that tumor-derived TNF-α is important in the development of MPE in mice, and provide preclinical evidence supporting the efficacy of TNF-α blockade against malignant pleural disease. [Cancer Res 2007;67(20):9825–34]

Supplementary Figure Legend from Tumor Necrosis Factor-α Promotes Malignant Pleural Effusion

Supplementary Figure 1 from Tumor Necrosis Factor-α Promotes Malignant Pleural Effusion

Background and objective Anti-angiogenic treatment has been recently shown to be clinically beneficial for mesothelioma patients. Angiopoietins-1 and -2 are key regulators of tumor angiogenesis. Ang-1 is mainly known to promote angiogenesis and vessel stability, while Ang-2 could serve as an antagonist of Ang-1 causing vessel regression and destabilization or enhance angiogenesis in a context-dependent manner. We hypothesized that Ang-1 would promote and Ang2 would halt experimental mesothelioma by affecting tumor angiogenesis. Methods To examine the effects of angiopoietins in mesothelioma angiogenesis and in vivo growth we constructed Ang-1 or Ang-2 overexpressing AE17 and AB1 mesothelioma cells and implanted them in the respective syngeneic animals. We also explored the clinical relevance of our observations using the human tumoral mRNAseq data available in the TCGA database. Results and conclusions Ang-1 promotes mesothelioma angiogenesis and growth while the effect of Ang-2 is context-dependent. Low Ang-1 levels in human mesotheliomas are associated with the epitheloid subtype. Tumors of high Ang-1, or concurrent high Ang-2 and VEGF expression present high PECAM-1 and CDH5 expression, markers of vascularity and vascular stability, respectively. Our results highlight the importance of angiopoietins in mesothelioma pathophysiology and pave the way for the clinical development of novel anti-angiogenic strategies.

Malignant pleural mesothelioma is resistant to currently used treatment. Angiopoieitn-1 directly promotes mesothelioma cell growth in a Tie-2-dependent fashion. Angiopoietin/Tie-2 axis may thus be valid targets for therapeutic interventions against mesothelioma. We hypothesized that a soluble angiopoietin inhibitor (Murine Tek-deltaFc) would halt mesothelioma progression in vivo by enhancing mesothelioma cell proliferation and inhibiting tumor angiogenesis. Our hypothesis was challenged on two syngeneic mesothelioma in vivo models (AB1 cells- Balb/c mice and AE17 cells-C57BL/6 mice. Even though both mesothelioma cell lines express the Angiopoietin-1/-2 and Tie-2, murine Tek-deltaFc hampered AB1 but not AE17 mesothelioma growth in vivo by enhancing tumor cell apoptosis and limiting tumor angiogenesis. Neither angiopoietins (Angs)-1 and -2 nor the inhibitor affected mesothelioma cell growth in vitro. AB1 (responding) tumors were more vascularized and displayed higher endothelial Tie-2 and lower tumor Ang-1 expression than the (non-responding) AE17 tumors. Angiopoietins-1 and -2 are expressed in tumors and pleural cavity of mesothelioma patients demonstrating the clinical relevance of our experimental observations. In conclusion, disrupting Ang-Tie-2 signaling limits mesothelioma angiogenesis and halts tumor progression. Tumor vascularity, endothelial Tie-2 expression and tumor Ang-1 expression may predict mesothelioma response to Tek-deltaFc.

Oxidative stress and inadequate redox homeostasis is crucial for tumor initiation and progression. MTH1 (NUDT1) enzyme prevents incorporation of oxidized dNTPs by sanitizing the deoxynucleoside triphosphate (dNTP) pool and is therefore vital for the survival of tumor cells. MTH1 inhibition has been found to inhibit the growth of several experimental tumors, but its role in mesothelioma progression remained elusive. Moreover, although MTH1 is nonessential to normal cells, its role in survival of host cells in tumor milieu, especially tumor endothelium, is unclear. We validated a clinically relevant MTH1 inhibitor (Karonudib) in mesothelioma treatment using human xenografts and syngeneic murine models. We show that MTH1 inhibition impedes mesothelioma progression and that inherent tumoral MTH1 levels are associated with a tumor's response. We also identified tumor endothelial cells as selective targets of Karonudib and propose a model of intercellular signaling among tumor cells and bystander tumor endothelium. We finally determined the major biological processes associated with elevated MTH1 gene expression in human mesotheliomas.

Osteopontin (secreted phosphoprotein 1; SPP1) expression has been identified in human lung cancer and has been linked with enhanced tumour progression. To examine its functional role, we induced lung tumours by repetitive urethane or MCA/BHT lung carcinogens in C57BL/6 mice lacking both ( Spp1-/- ), one ( Spp1+/- ), or no ( Spp1+/+ ) copy of the endogenous Spp1 gene. Primary end-points were lung tumour number and size; secondary end-points were SPP1 expression, epithelial cell survival, carcinogen-induced inflammation, and angiogenesis. Data are presented as mean ± SD. Compared with Spp1+/+ mice (n = 22), Spp1-/- mice (n = 25) developed dramatically fewer and significantly smaller lung tumours in response to urethane, while Spp1± mice (n = 12) behaved similar to Spp1-/- mice (number/diameter of lung tumours in Spp1+/+ , Spp1+/- , and Spp1-/- mice, respectively: 16.1 ± 12.7/1.2 ± 0.3 mm, 2.4 ± 2.3/0.9 ± 0.2 mm, and 1.3 ± 1.6/0.7 ± 0.2 mm; P Spp1+/+ with Spp1-/- and Spp1+/- mice). Spp1-/- mice were also protected from two-hit MCA/BHT-oncogenesis compared with Spp1+/+ controls. Spp1-/- mice displayed decreased epithelial cell survival and reduced numbers of airspace macrophages early after urethane, and enhanced tumour cell apoptosis and limited tumour angiogenesis at late stages of lung tumour progression. SPP1 was expressed in the naive lung by non-ciliated airway epithelial cells and alveolar macrophages and was significantly up-regulated during multi-stage lung carcinogenesis. Our data indicate that SPP1 is functionally involved in airway epithelial carcinogenesis and may present a target for lung cancer treatment and prevention.

Increased expression of osteopontin (secreted phosphoprotein 1, SPP1) is associated with aggressive human lung adenocarcinoma (LADC), but its function remains unknown. Our aim was to determine the role of SPP1 in smoking-induced LADC. We combined mouse models of tobacco carcinogen-induced LADC, of deficiency of endogenous Spp1 alleles, and of adoptive pulmonary macrophage reconstitution to map the expression of SPP1 and its receptors and determine its impact during carcinogenesis. Co-expression of Spp1 and mutant KrasG12C in benign cells was employed to investigate SPP1/KRAS interactions in oncogenesis. Finally, intratracheal adenovirus encoding Cre recombinase was delivered to LSL.KRASG12D mice lacking endogenous or overexpressing transgenic Spp1 alleles. SPP1 was overexpressed in experimental and human LADC and portended poor survival. In response to two different smoke carcinogens, Spp1-deficient mice developed fewer and smaller LADC with decreased cellular survival and angiogenesis. Both lung epithelial- and macrophage-secreted SPP1 drove tumor-associated inflammation, while epithelial SPP1 promoted early tumorigenesis by fostering the survival of KRAS-mutated cells. Finally, loss and overexpression of Spp1 was, respectively, protective and deleterious for mice harboring KRASG12D-driven LADC. Our data support that SPP1 is functionally involved in early stages of airway epithelial carcinogenesis driven by smoking and mutant KRAS and may present an important therapeutic target.

No large study to date has ever evaluated the effectiveness, safety and tolerability of imipenem/clavulanateversusmeropenem/clavulanate to treat multidrug- and extensively drug-resistant tuberculosis (MDR- and XDR-TB). The aim of this observational study was to compare the therapeutic contribution of imipenem/clavulanateversusmeropenem/clavulanate added to background regimens to treat MDR- and XDR-TB cases.84 patients treated with imipenem/clavulanate-containing regimens showed a similar median number of antibiotic resistances (8versus8) but more fluoroquinolone resistance (79.0%versus48.9%, pversus49.0%, p=0.01) in comparison with 96 patients exposed to meropenem/clavulanate-containing regimens. Patients were treated with imipenem/clavulanate- and meropenem/clavulanate-containing regimens for a median (interquartile range) of 187 (60–428)versus85 (49–156) days, respectively.Statistically significant differences were observed on sputum smear and culture conversion rates (79.7%versus94.8%, p=0.02 and 71.9%versus94.8%, pversus77.5%, p=0.03). Adverse events to imipenem/clavulanate and meropenem/clavulanate were reported in 5.4% and 6.5% of cases only.Our study suggests that meropenem/clavulanate is more effective than imipenem/clavulanate in treating MDR/XDR-TB patients.

// Sophia Magkouta 1 , Apostolos Pappas 1 , Charalampos Moschos 1 , Maria-Eleni Vazakidou 1 , Katherina Psarra 2 , Ioannis Kalomenidis 1 1 “Marianthi Simou Laboratory”, 1st Department of Critical Care and Pulmonary Medicine, National and Kapodistrian University of Athens, School of Medicine, Evangelismos Hospital, Athens, Greece 2 Department of Immunology - Histocompatibility, Evangelismos Hospital, Athens, Greece Correspondence to: Sophia Magkouta, e-mail: smagkouta@med.uoa.gr Keywords: Icmt, malignant pleural effusion, adenocarcinoma, mesothelioma, GTPases Received: September 25, 2015 Accepted: January 14, 2016 Published: March 04, 2016 ABSTRACT Small GTPases are pivotal regulators of several aspects of tumor progression. Their implication in angiogenesis, vascular permeability and tumor-associated inflammatory responses is relevant to the pathobiology of Malignant Pleural Effusion (MPE). Inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt) abrogates small GTPase activation. We therefore hypothesized that cysmethynil, an Icmt inhibitor would limit pleural fluid accumulation in two models, a lung-adenocarcinoma and a mesothelioma-induced MPE. Cysmethynil significantly reduced MPE volume in both models and tumor burden in the adenocarcinoma model. It inhibited pleural vascular permeability and tumor angiogenesis in vivo and reduced endothelial cell proliferation, migration and tube formation in vitro . Cysmethynil also promoted M1 anti-tumor macrophage homing in the pleural space in vivo , and inhibited tumor-induced polarization of macrophages towards a M2 phenotype in vitro . In addition, the inhibitor promoted adenocarcinoma cell apoptosis in vivo . Inhibition of small GTPase might thus represent a valuable strategy for pharmacotherapy of MPE.

Versican promotes experimental tumor growth through cell- and non cell-autonomous mechanisms. Its role in mesothelioma progression has not been investigated so far. In this study we investigated the impact of tumor-derived versican in mesothelioma progression and the underlying mechanism of its action. For this purpose, versican-silenced or control ΑE17 and ΑΒ1 murine mesothelioma cells were intrapleuraly injected into syngeneic mice, in order to create pleural mesotheliomas and pleural effusions. Intratumoral and pleural immune subsets were assessed using flow cytometry. Mesothelioma cells were co-cultured with syngeneic macrophages to examine versican's impact on their interaction and endothelial cells to assess the effect of versican in endothelial permeability. Versican expression was assessed in human mesotheliomas and mesothelioma-related pleural effusions and benign pleural tissue and effusions. We observed that, versican silencing reduced mesothelioma mass and pleural fluid volume by affecting tumor cell proliferation and apoptosis in vivo, while tumor cell growth remained intact in vitro, and limited pleural vascular permeability. Mice harboring versican-deficient tumors presented fewer tumor/pleural macrophages and neutrophils, and fewer pleural T-regulatory cells, compared to the control animals. Macrophages co-cultured with versican-deficient mesothelioma cells were polarized towards M1 anti-tumor phenotype and demonstrated increased tumor cell phagocytic capacity, compared to macrophages co-cultured with control tumor cells. In co-culture, endothelial monolayer permeability was less effectively stimulated by versican-deficient cells than control cells. Versican was over-expressed in human mesothelioma tissue and mesothelioma-associated effusion. In conclusion, tumor cell-derived versican stimulates mesothelioma progression by shaping a tumor friendly inflammatory milieu, mainly by blunting macrophage anti-tumor activities.

Background and objective The mechanistic target of rapamycin (mTOR) promotes cancer cell proliferation and survival, transduces pro-angiogenic signals and regulates immune cell differentiation and function. We hypothesized that temsirolimus, an mTOR inhibitor, would curtail experimental mesothelioma progression in vivo by limiting tumour cell growth, abrogating tumour angiogenesis and modulating immune/inflammatory tumour milieu. Methods We produced flank and pleural syngeneic murine mesotheliomas by delivering AE17 and AB1 murine mesothelioma cells into the right flank or the pleural space of C57BL/6 and BALB/c mice, respectively. Animals were given five times/week intraperitoneal injections of 20 mg/kg temsirolimus or vehicle and were sacrificed on day 26 (flank) or on day 15 (pleural) post-tumour cell propagation. Results Temsirolimus limited mesothelioma growth in vivo by stimulating tumour cell apoptosis, inhibiting tumour angiogenesis, enhancing tumour lymphocyte abundance and blocking pro-tumour myeloid cell recruitment. Pleural fluid accumulation was significantly mitigated in AE17 but not in AB1 mesotheliomas. In vitro, temsirolimus hindered mesothelioma cell growth, NF-kappaB activation and macrophage migration. Conclusions In conclusion, temsirolimus apart from inducing tumour cell apoptosis, targets tumour angiogenesis and influences inflammatory tumour microenvironment to halt experimental mesothelioma growth in vivo.

The World Health Organization (WHO) recommends that countries implement pharmacovigilance and collect information on active drug safety monitoring (aDSM) and management of adverse events.The aim of this prospective study was to evaluate the frequency and severity of adverse events to anti-tuberculosis (TB) drugs in a cohort of consecutive TB patients treated with new (i.e. bedaquiline, delamanid) and repurposed (i.e. clofazimine, linezolid) drugs, based on the WHO aDSM project. Adverse events were collected prospectively after attribution to a specific drug together with demographic, bacteriological, radiological and clinical information at diagnosis and during therapy. This interim analysis included patients who completed or were still on treatment at time of data collection.Globally, 45 centres from 26 countries/regions reported 658 patients (68.7% male, 4.4% HIV co-infected) treated as follows: 87.7% with bedaquiline, 18.4% with delamanid (6.1% with both), 81.5% with linezolid and 32.4% with clofazimine. Overall, 504 adverse event episodes were reported: 447 (88.7%) were classified as minor (grade 1–2) and 57 (11.3%) as serious (grade 3–5). The majority of the 57 serious adverse events reported by 55 patients (51 out of 57, 89.5%) ultimately resolved. Among patients reporting serious adverse events, some drugs held responsible were discontinued: bedaquiline in 0.35% (two out of 577), delamanid in 0.8% (one out of 121), linezolid in 1.9% (10 out of 536) and clofazimine in 1.4% (three out of 213) of patients. Serious adverse events were reported in 6.9% (nine out of 131) of patients treated with amikacin, 0.4% (one out of 221) with ethionamide/prothionamide, 2.8% (15 out of 536) with linezolid and 1.8% (eight out of 498) with cycloserine/terizidone.The aDSM study provided valuable information, but implementation needs scaling-up to support patient-centred care.

The aim of this study was to investigate the pleural and systemic expression of interleukin-18 (IL-18) in patients with pleural effusions (PEs), and the effects of the cytokine in mouse pleural space. One hundred and sixty patients, 23 with pleural effusions (PEs) due to heart failure, 60 malignant, 25 parapneumonic/empyemas, 15 tuberculous and 37 with exudates of miscellaneous etiologies were included in the study. Pleural fluid (PF) and serum IL-18 content was determined using ELISA. IL-18 was injected intrapleurally in mice and pleural inflammation was assessed using pleural lavage. The highest PF IL-18 levels were observed in parapneumonic PEs and the lowest PF IL-18 levels in patients with exudates of miscellaneous aetiologies and transudates. PF IL-18 levels were significantly higher in patients with empyemas compared to those with uncomplicated ( p = 0.009) or complicated ( p = 0.028) parapneumonic effusions, while serum levels did not differ significantly among the three groups. Pleural IL-18 content was higher than that of blood only in patients with empyemas. In patients with pleural exudates of all etiologies and in those with parapneumonic PEs/empyema, PF IL-18 levels were correlated with markers of acute pleural inflammation such as the percentage of PF neutrophils, PF LDH and PF/serum LDH ratio, low PF glucose and PF/serum glucose ratio and low PF pH. In mice, intrapleural IL-18 caused neutrophil-predominant pleural inflammation. In conclusion, IL-18 is linked to the intensity of neutrophilic pleural inflammation in patients with PEs, it is up-regulated in the pleural space of patients with empyema and it stimulates the accumulation of neutrophils in mouse pleura.

Background and objective The role of pro-inflammatory interleukin-17A (IL-17A), in pleural diseases is unknown. We sought to investigate IL-17A expression and its clinical implications in patients with pleural effusion (PE) and IL-17A involvement in the pathobiology of pleural inflammation elicited by bacterial products. Methods Pleural and blood IL-17A content was examined in 84 patients with PE of different aetiologies, and the diagnostic value of pleural IL-17A was explored in 92 patients with neutrophil-predominant PE. IL-17A contribution in pleural inflammation was evaluated in mice injected intrapleurally with either IL-17A or bacterial products with or without IL-17A-neutralizing antibodies. Results IL-17A was upregulated in the pleural space of patients with parapneumonic PE. It was detected in a minority of patients with tuberculous PE and very uncommonly in patients with malignant or other pleural exudates. Pleural fluid (PF) IL-17A levels were correlated with markers of acute pleural inflammation, as well as vascular endothelial growth factor and IL-8 levels. Among patients with neutrophil-predominant PE, PF IL-17A was detected only in those with parapneumonic PE, although the sensitivity of the test was low (

Background: Meropenem/clavulanate (MC) has been prescribed to treat MDR/XDR-TB cases, although limited scientific evidence exists. Aim: To retrospectively evaluate the effectiveness, safety, and tolerability of MC added to build an optimal regimen when treating MDR/XDR-TB patients. Methods: An observational, retrospective, cohort study was performed in 5 countries across Europe and South America, where consecutive patients with bacteriologically proven MDR-TB were recruited and divided by MC exposure. Information on demographic, epidemiological, microbiological, and clinical variables were collected from the clinical files using standardized e-forms. Results: A cohort of 264 MDR-TB patients was established: 96 patients were treated with MC-containing regimens and 168 without. Only thirteen (5.2%) were HIV-infected. The proportion of XDR-TB was higher in those exposed to MC-containing regimens (49.0% vs. 6.0%), as was the median (IQR) number of antibiotic resistances (8[6-9] vs 5[4-6]). Sputum smear and culture conversion rates were not statistically different in MDR-TB cases exposed and not exposed to MC-containing regimens, including the XDR-TB subgroup (88.0% vs. 100.0%, P=1.00, and 88.0% vs. 100.0%, P=1.00, respectively). Only 6 MC-related adverse events were reported. In 4 cases, MC was re-started after interruption Conclusion: MC-containing regimens can help achieve sputum culture conversion in MDR/XDR-TB cases with no significant toxicity. This study suggests a potential added value of MC in severe forms of drug-resistant TB when an optimal regimen can not be built with available drugs.

Background: Carbapenems are used to treat MDR/XDR-TB cases in order to build a robust regimen, however limited evidence of their effectiveness exists. Aim: To compare the therapeutic contribution of imipenem and amoxicillin/clavulanate (IC) vs. meropenem and amoxicillin/clavulanate (MC) added to background regimens to treat MDR/XDR-TB cases. Methods: An observational, retrospective, cohort study was performed in 5 centres in Europe and South America, consecutive adult patients with bacteriologically proven MDR-TB were recruited and divided by MC and IC exposure. Demographic, epidemiological, microbiological, and clinical variables were collected using standardized e-forms. Results: A cohort of 180 MDR-TB patients were enrolled: 84 and 96 were treated with IC and MC respectively. Both groups had similar median number of antibiotic resistances (8 vs. 8) but more fluoroquinolone resistance (79.0% vs. 48.9% ; p-value: Conclusion: Our study suggests that MC is safe and more effective than IC in treating M/XDR-TB patients.

Osteopontin (SPP1) expression has been identified in human lung cancer and has been linked with enhanced tumor progression. To examine its functional role, we induced lung tumors by repetitive urethane or MCA/BHT lung carcinogens in C57BL/6 mice lacking both ( Spp1-/- ), one ( Spp1+/- ), or no ( Spp1+/+ ) copy of the endogenous Spp1 gene. Primary end-points were lung tumor number and size; secondary end-points were SPP1 expression, epithelial cell survival, carcinogen-induced inflammation, and angiogenesis. Data are presented as mean±SD. Compared with Spp1+/+ mice (n=22), Spp1-/- mice (n=25) developed dramatically fewer and significantly smaller lung tumors in response to urethane, while Spp1+/- mice (n=12) behaved similar to Spp1-/- mice (number/diameter of lung tumors in Spp1+/+ , Spp1+/- , and Spp1-/- mice, respectively: 16.1±12.7/1.2±0.3 mm, 2.4±2.3/0.9±0.2 mm, and 1.3±1.6/0.7±0.2 mm; P Spp1+/+ with Spp1-/- and Spp1+/- mice). Spp1-/- mice were also protected from two-hit MCA/BHT-oncogenesis compared with Spp1+/+ controls. Spp1-/- mice displayed decreased epithelial cell survival and reduced numbers of airspace macrophages early after urethane, and enhanced tumor cell apoptosis and limited tumor angiogenesis at late stages of lung tumor progression. SPP1 was expressed in the naive lung by non-ciliated airway epithelial cells and alveolar macrophages and was significantly up-regulated during multi-stage lung carcinogenesis. SPP1 also initiated the survival of mutant Kras cells. Our data indicate that SPP1 is functionally involved in airway epithelial carcinogenesis via survival of mutant Kras cells and may present a target for lung cancer treatment and prevention.

No large study has ever evaluated the efficacy, safety and tolerability of meropenem/clavulanate to treat multidrug- and extensively drug-resistant tuberculosis (MDR- and XDR-TB). The aim of this observational study was to evaluate the therapeutic contribution, effectiveness, safety and tolerability profile of meropenem/clavulanate added to a background regimen when treating MDR- and XDR-TB cases.Patients treated with a meropenem/clavulanate-containing regimen (n=96) showed a greater drug resistance profile than those exposed to a meropenem/clavulanate-sparing regimen (n=168): in the former group XDR-TB was more frequent (49% versus 6.0%, pversus 5 (4–6)). Patients were treated with a meropenem/clavulanate-containing regimen for a median (IQR) of 85 (49–156) days.No statistically significant differences were observed in the overall MDR-TB cohort and in the subgroups with and without the XDR-TB patients; in particular, sputum smear and culture conversion rates were similar in XDR-TB patients exposed to meropenem/clavulanate-containing regimens (88.0% versus 100.0%, p=1.00 and 88.0% versus 100.0%, p=1.00, respectively). Only six cases reported adverse events attributable to meropenem/clavulanate (four of them then restarting treatment).The nondifferent outcomes and bacteriological conversion rate observed in cases who were more severe than controls might imply that meropenem/clavulanate could be active in treating MDR- and XDR-TB cases.

Simon Tiberi, Giovanni Sotgiu, Lia D’Ambrosio, Rosella Centis, Marcos Abdo Arbex, Edith Alarcon Arrascue, Jan Willem Alffenaar, Jose A. Caminero, Mina Gaga, Gina Gualano, Alena Skrahina, Ivan Solovic, Giorgia Sulis, Marina Tadolini, Valentina Alarcon Guizado, Saverio De Lorenzo, Aurora Jazmin Roby Arias, Anna Scardigli, Onno W. Akkerman, Alena Aleksa, Janina Artsukevich, Vera Avchinko, Eduardo Henrique Bonini, Felix Antonio Chong Marin, Lorena Collahuazo Lopez, Gerard de Vries, Simone Dore, Heinke Kunst, Alberto Matteelli, Charalampos Moschos, Fabrizio Palmieri, Apostolos Papavasileiou, Marie-Christine Payen, Andrea Piana, Antonio Spanevello, Dante Vargas Vasquez, Pietro Viggiani, Veronica White, Alimuddin Zumla and Giovanni Battista Migliori

Tumor necrosis factor (TNF) has been implicated in inflammation-associated tumor progression. Although multiple reports identified a role for TNF signaling in established cancers, few studies have assessed the impact of TNF blockade on early tumor formation promotion. We aimed at exploring the effects of TNF neutralization in a preclinical mouse model of lung carcinogenesis. For this, Balb/c mice (n = 42) received four weekly intraperitoneal urethane injections (1 g/kg) and twice-weekly intraperitoneal soluble TNF receptor (etanercept; 10 mg/kg) administered during tumor initiation/promotion, tumor progression, or continuously (months 1, 6, and 1-8 after urethane start, respectively). Lung oncogenesis was assessed after 8 months. In separate short-term studies, Balb/c mice (n = 21) received a single control or urethane injection followed by twice-weekly intraperitoneal control or sTNFR:Fc injections. Lung inflammation was assessed after 1 week. We found that sTNFR:Fc treatment during tumor initiation/promotion resulted in a significant reduction of tumor number but not dimensions. However, sTNFR:Fc administered during tumor progression did not impact tumor multiplicity but significantly decreased tumor diameter. Continued sTNFR:Fc administration was effective in halting both respiratory tumor formation and progression in response to urethane. This favorable impact was associated with impaired cellular proliferation and new vessel formation in lung tumors. In addition, TNF neutralization altered the lung inflammatory response to urethane, evidenced by reductions in TNF and macrophage and increases in interferon γ and interleukin 10 content of the air spaces. sTNFR:Fc treatment of RAW264.7 macrophages downregulated TNF and enhanced interferon γ and interleukin 10 expression. In conclusion, TNF neutralization is effective against urethane-induced lung oncogenesis in mice and could present a lung chemoprevention strategy worth testing clinically.

Background and objective: Osteopontin (OPN) is an important mediator of inflammation and cancer progression. In the present study, we asked whether pleural fluid (PF) and serum OPN concentrations differed between patients with pleural effusions of different aetiologies, and whether assessment of OPN levels was useful for diagnostic purposes. Methods: One hundred and nine consecutive patients with pleural effusions of different aetiologies were recruited prospectively during daily clinics. OPN levels were measured by ELISA. Results: PF OPN levels were 10-fold higher in exudates than in transudates and were significantly correlated with markers of pleural inflammation and vascular hyper-permeability, such as PF/serum LDH or protein ratios, PF protein and PF vascular endothelial growth factor levels. Patients with malignant pleural effusions had higher PF and lower serum OPN concentrations than those with benign disease. The diagnostic accuracies of PF and PF/serum OPN for malignancy were 71.5% (95% CI: 64–80) and 70.6% (95% CI: 62–80), respectively. Conclusions: OPN levels were elevated in exudative pleural effusions, as compared with the levels in blood or transudative pleural effusions. While PF and PF/serum OPN were higher in patients with malignancies, the diagnostic accuracy of the tests was not sufficient to permit routine use in clinical practice.

PURPOSE: Angiopoietins and their receptor, Tie2, participate in angiogenesis, regulation of vascular permeability, and inflammation, all central to the pathogenesis of malignant pleural effusions (MPEs). In the present study, we aimed to examine the role of the angiopoietin/Tie2 axis in MPE pathogenesis. EXPERIMENTAL DESIGN: MPE was induced by intrapleural injection of murine adenocarcinoma cells in C57BL/6 mice. Animals were given twice-weekly intraperitoneal injections of 40 mg/kg MuTekdeltaFc or vehicle. MuTekdeltaFc is a soluble Tie2 (sTie2) receptor that binds murine angiopoietins thereby disrupting their interaction with Tie2 receptors expressed on tissues. Animals were killed on day 14. RESULTS: Angiopoietin/Tie2 axis blockade significantly reduced pleural fluid volume and pleural tumor foci. The mean ± SEM pleural fluid volumes were 617 ± 48 μl and 316 ± 62 μl for the control and treated groups, respectively (P = .001), whereas the mean ± SEM tumor foci were 7.3 ± 1.0 and 3.0 ± 0.52 for the control and treated groups, respectively (P = .001). In addition, tumor-associated cachexia, tumor angiogenesis, pleural vascular permeability, recruitment of inflammatory cells to the pleural cavity, and local elaboration of vascular endothelial growth factor and interleukin 6 were also downregulated, and tumor cell apoptosis was induced in animals treated with the inhibitor. CONCLUSIONS: Our results indicate that the angiopoietin/Tie2 axis is an important component of MPE pathogenesis. Further studies are required to determine whether therapeutic interventions targeting this pathway could be beneficial for patients with MPE.

Tumor cells in malignant pleural effusions (MPEs) are an important source of monocyte chemoattractant protein (MCP)-1. However, the role of tumor-derived MCP-1 in the pathogenesis and progression of MPE has not been determined.B16 mouse skin melanoma cells, which are deficient in MCP-1 expression, and mouse Lewis lung cancer (LLC) cells, which express high levels of MCP-1, were engineered to stably express MCP-1 and short hairpin RNAs (shRNAs) targeting the MCP-1 transcript, respectively. Cells were injected into the pleural cavities of syngeneic immunocompetent mice, and MPE volume and pleural tumors were quantified at necropsy (day 14). MCP-1 and other mediators were determined by cytometric bead array and enzyme-linked immunosorbent assay, and mononuclear and endothelial cells were identified by immunolabeling of F4/80 and factor VIII-related antigen respectively. Mouse survival was assessed using Kaplan-Meier analysis. Vascular permeability in mice with MPE was assessed using albumin-binding Evans blue. Statistical tests were two-sided.LLC cells expressing shRNA against MCP-1 elaborated less than 5% of the MCP-1 level in cells expressing nonspecific shRNA (control cells), and intrapleural delivery of these cells resulted in less MPE (mean MPE volume = 86 and 585 muL, respectively; difference = 499 muL; 95% confidence interval [CI] = 331 to 669 muL; P.001), reduced MCP-1 levels in the pleural fluid, and lower mortality than when control cells were delivered. Overexpression of MCP-1 in intrapleurally injected B16 melanoma cells led to increased MPE and reduced survival. In mice with MPE, MCP-1 was a potent inducer of vascular permeability, mononuclear recruitment, and, in pleural tumors, of angiogenesis.MCP-1 produced by tumor cells is an important determinant of their capacity to induce the formation of MPE and may be a useful target for the treatment of malignant pleural disease.

Aminobiphosphonates, such as zoledronic acid (ZA), exert potent indirect antitumor effects and are currently being tested against human solid tumors. The antitumor actions of aminobiphosphonates, including angiostasis, are relevant to the pathogenesis of malignant pleural effusion (MPE), but no study has addressed the efficacy of these compounds against malignant pleural disease.Here we hypothesized that treatment of immunocompetent mice with ZA would halt tumor progression in a mouse model of adenocarcinoma-induced MPE.To induce MPE in mice, Lewis lung carcinoma cells were delivered directly into the pleural space. Subsequently, animals were treated with ZA in both a prevention and a regression protocol.ZA treatment resulted in significant reductions in pleural fluid accumulation and tumor dissemination, while it significantly prolonged survival. These effects of ZA were linked to enhanced apoptosis of pleural tumor cells, decreased formation of new vessels in pleural tumors, and reduced pleural vascular permeability. In addition, ZA was able to inhibit the recruitment of mononuclear cells to pleural tumors, with concomitant reductions in matrix metalloproteinase-9 release into the pleural space. Finally, ZA limited the expression of proinflammatory and angiogenic mediators, as well as the activity of small GTP proteins Ras and RhoA, in tumor cells in vivo and in vitro.ZA is effective against experimental MPE, suggesting that this intervention should be considered for testing in clinical trials.

Introduction: Chronic obstructive pulmonary disease (COPD) patients are at higher risk for pulmonary embolism (PE) due to limited mobility, smoking, inflammation or comorbidities. High D-dimer levels have been detected in many diseases including COPD exacerbation, even in the absence of detectable PE. Aims and Objectives: To determine the optimal D-dimer cutoff value for the diagnosis of PE, in patients hospitalized for COPD exacerbation. Methods: We retrospectively analyzed 180 consecutive patients, admitted to “KAA” and 5 th Respiratory Clinic of Athens Chest Hospital “Sotiria” for COPD exacerbation, according to Anthonisen criteria, during 2014. Fifty from the above patients were found to have been investigated with computed tomography angiography despite a low probability for PE (Wells score). All 50 patients had D-Dimer levels measured as well. Receiver operating characteristic (ROC) curve analysis was used to determine the accuracy of D-dimer measurements in diagnosing PE. Results: Fifty patients (72.1±12.0 years old) were included in the analysis. PE was detected in 10 patients (20%). D-dimer levels of COPD patients with PE (median value: 7655 mcg/L) were significantly higher than those of the patients without PE (median value: 940 mcg/L)(p Conclusions: Patients hospitalized for COPD exacerbation with low clinical probability for PE present D-dimer levels higher than normal, even without PE. The D-dimer cutoff value used to exclude PE should be adjusted, thus reducing the unnecessary use of diagnostic procedures.

The mechanistic target of rapamycin (mTOR) promotes cancer cell proliferation and survival, transduces pro-angiogenic signals and regulates immune cell differentiation and function. We hypothesized that temsirolimus, an mTOR inhibitor, would curtail experimental mesothelioma progression in vivo by limiting tumour cell growth, abrogating tumour angiogenesis and modulating immune/inflammatory tumour milieu.We produced flank and pleural syngeneic murine mesotheliomas by delivering AE17 and AB1 murine mesothelioma cells into the right flank or the pleural space of C57BL/6 and BALB/c mice, respectively. Animals were given five times/week intraperitoneal injections of 20 mg/kg temsirolimus or vehicle and were sacrificed on day 26 (flank) or on day 15 (pleural) post-tumour cell propagation.Temsirolimus limited mesothelioma growth in vivo by stimulating tumour cell apoptosis, inhibiting tumour angiogenesis, enhancing tumour lymphocyte abundance and blocking pro-tumour myeloid cell recruitment. Pleural fluid accumulation was significantly mitigated in AE17 but not in AB1 mesotheliomas. In vitro, temsirolimus hindered mesothelioma cell growth, NF-kappaB activation and macrophage migration.In conclusion, temsirolimus apart from inducing tumour cell apoptosis, targets tumour angiogenesis and influences inflammatory tumour microenvironment to halt experimental mesothelioma growth in vivo.

Rationale: Osteopontin (OPN) is overexpressed in mesothelioma tissue and has been associated with impaired patient survival. However, whether OPN is involved in mesothelioma growth and regulation of tumor-stimulated immune reaction is unknown. Aim: To examine the functional importance of OPN in experimental malignant pleural mesothelioma. Methods: AE17 murine mesothelioma cells which express high levels of OPN, engineered to stably express short hairpin RNAs (shRNAs) targeting the OPN transcript. To mimic pleural mesothelioma we intrapleurally injected syngeneic C57/Bl6 mice with AE17/OPNshRNA or AE17/vector cells. Tumor tissue and pleural fluid were harvested 15 days post-injection. The presence of pro-tumor cells including CD11b(+)/F4/80(+)/CD206(+) macrophages and CD11b(+)/Gr1(+) myeloid suppressor cells were evaluated using FACS. Results: Pleural tumors in mice injected with AE17/shRNA were significantly smaller than those developed in mice injected with AE17/vector (mean tumor weight 428.3±41.2mg versus 107.2±25.6mg, P

Secreted phosphoprotein-1 (SPP1) promotes cancer cell survival and regulates tumor-associated angiogenesis and inflammation, both central to the pathogenesis of malignant pleural effusion (MPE). Here, we examined the impact of tumor- and host-derived SPP1 in MPE formation and explored the mechanisms by which the cytokine exerts its effects. We used a syngeneic murine model of lung adenocarcinoma-induced MPE. To dissect the effects of tumor- versus host-derived SPP1, we intrapleurally injected wild-type and SPP1-knockout C57/BL/6 mice with either wild-type or SPP1-deficient syngeneic lung cancer cells. We demonstrated that both tumor- and host-derived SPP1 promoted pleural fluid accumulation and tumor dissemination in a synergistic manner (P

Background Despite its widespread use in pulmonary fibrosis research, the bleomycin mouse model has not been thoroughly validated from a pulmonary functional standpoint using new technologies. Purpose of this study was to systematically assess the functional alterations induced in murine lungs by fibrogenic agent bleomycin and to compare the forced oscillation technique with quasi-static pressure-volume curves in mice following bleomycin exposure. Methods Single intratracheal injections of saline (50 μL) or bleomycin (2 mg/Kg in 50 μL saline) were administered to C57BL/6 (n = 40) and Balb/c (n = 32) mice. Injury/fibrosis score, tissue volume density (TVD), collagen content, airway resistance (R N ), tissue damping (G) and elastance coefficient (H), hysteresivity (η), and area of pressure-volume curve (PV-A) were determined after 7 and 21 days (inflammation and fibrosis stage, respectively). Statistical hypothesis testing was performed using one-way ANOVA with LSD post hoc tests. Results Both C57BL/6 and Balb/c mice developed weight loss and lung inflammation after bleomycin. However, only C57BL/6 mice displayed cachexia and fibrosis, evidenced by increased fibrosis score, TVD, and collagen. At day 7, PV-A increased significantly and G and H non-significantly in bleomycin-exposed C57BL/6 mice compared to saline controls and further increase in all parameters was documented at day 21. G and H, but not PV-A, correlated well with the presence of fibrosis based on histology, TVD and collagen. In Balb/c mice, no change in collagen content, histology score, TVD, H and G was noted following bleomycin exposure, yet PV-A increased significantly compared to saline controls. Conclusions Lung dysfunction in the bleomycin model is more pronounced during the fibrosis stage rather than the inflammation stage. Forced oscillation mechanics are accurate indicators of experimental bleomycin-induced lung fibrosis. Quasi-static PV-curves may be more sensitive than forced oscillations at detecting inflammation and fibrosis.

Background and objectives: The pathogenesis and the optimal treatment of eosinophilic pleural effusions are unknown. We aimed to examine whether pneumothorax-associated pleural eosinophilia in mice is dependent on tumour necrosis factor (TNF)-alpha, and whether it is affected by systemic administration of corticosteroids. Methods: Mice were injected intrapleurally with 0.4 mL air to create pneumothoraces. Animals were sacrificed 24 or 48 h later, and pleural lavage (PL) was performed. In the first experiment, comparisons were made between wild-type and TNF-α knockout mice with pneumothorax. In the second experiment, wild-type mice were injected intraperitoneally with different doses of dexamethasone (0, 0.25, 0.5 and 1 mg/kg), 5 min before and 24 h after the induction of pneumothorax. Results: After induction of a pneumothorax, TNF-α knockout mice had significantly fewer total number of cells (P = 0.004), mononuclear cells (P = 0.01), neutrophils (P = 0.017) and eosinophils (P = 0.002) in their PL compared with wild-type animals. TNF-α was detected in the PL of most of the control mice but not in TNF-α knockouts. Dexamethasone induced a significant, dose-dependent reduction of PL total cells (P

Tumor necrosis factor (TNF)-α is present in the microenvironment of human tumors, including malignant pleural effusion (MPE). Although the cytokine is produced in the pleural cavity by both tumor and host cells, its effects on MPE formation are unknown. In these studies, we sought to determine the role of TNF-α in the pathogenesis of MPE and to assess the therapeutic effects of its neutralization in a preclinical model. For this, MPEs were generated in immunocompetent mice using intrapleural injection of mouse lung adenocarcinoma cells. The roles of tumor- and host-derived TNF-α were assessed using combined experimentation with TNF-α gene–deficient mice and in vivo TNF-α neutralization. To expand the scope of preclinical data, TNF-α and vascular endothelial growth factor (VEGF) expression were determined in human cancer cell lines and human MPE. In the MPE model, TNF-α of host and tumor origin was present. TNF-α neutralization significantly limited tumor dissemination, effusion formation, vascular hyperpermeability, TNF-α and VEGF expression, and angiogenesis, thereby improving survival. In contrast, these variables were not different between TNF-α gene–sufficient and TNF-α gene–deficient mice. In mouse cancer cells, TNF-α functioned via nuclear factor-κB– and neutral sphingomyelinase–dependent pathways to induce TNF-α and VEGF, respectively. These results were recapitulated in human cancer cells, and a correlation was detected between TNF-α and VEGF content of human MPE. We conclude that tumor-derived TNF-α is important in the development of MPE in mice, and provide preclinical evidence supporting the efficacy of TNF-α blockade against malignant pleural disease. [Cancer Res 2007;67(20):9825–34]

A 35-yr-old male was examined in the outpatient clinic of the present authors' department. He complained of a dry paroxysmal cough of indeterminate origin that he had noticed 3 months earlier. The cough was associated with a tickling feeling in the throat. There was no association between coughing flares and meals, nasal discharge, season, time of day or body posture. The patient did not recall having symptoms compatible with an upper airway viral infection in the past 3 months and reported no gastro-intestinal symptoms like daily heartburn and regurgitation. He lived in the city, was a lifetime nonsmoker, his previous medical history was unremarkable and he was not taking any medication. He worked as an electronic engineer in a smoke- and dust-free environment and exhibited a type A personality. Upon appearance of the cough 3 months earlier, he had been examined by a physician and had since received inhaled salmeterol (50 μg b.i.d. ) and fluticasone (250 μg b.i.d. ), without improvement. A thorough physical examination and routine laboratory tests, including white blood cell count and differential, red blood cell count, erythrocyte sedimentation rate, liver and renal function tests, serum C-reactive protein and an ECG were normal. Chest (fig. 1⇓) and paranasal sinus (image not shown) radiographs were taken. Ear, nose and throat assessment was unremarkable. After discontinuing treatment with inhaled salmeterol and fluticasone, simple spirometry pre- and post-bronchodilator (four puffs of 100 μg salbutamol) and bronchoprovocation challenge with methacholine were performed (fig. 2⇓). The patient was given a peak flow meter and during a monthly observation period his diurnal peak flow variation was below 5% (data not shown). Fig. 1— Chest radiograph showing a) posterior-anterior view and b) lateral view. Fig. 2— Pulmonary function tests of a) flow–volume plot and b) volume–time curve. …

Background We have previously shown that nuclear factor (NF)-κ B activation of mouse Lewis lung carcinoma (LLC) specifically promotes the induction of malignant pleural effusions (MPE) by these cells. In the present studies we hypothesized that treatment of immunocompetent mice with bortezomib tailored to inhibit cancer cell NF-κ B activation and not proliferation specifically inhibits MPE formation by LLC cells. Results Treatment of LLC cells with low concentrations of bortezomib (100 ng/ml) inhibited NF-κ B activation and NF-κ B-dependent transcription, but not cellular proliferation. Bortezomib treatment of immunocompetent C57BL/6 mice bearing LLC-induced subcutaneous tumors and MPEs significantly blocked tumor-specific NF-κ B activation. However, bortezomib treatment did not impair subcutaneous LLC tumor growth, but was effective in limiting LLC-induced MPE. This specific effect was evidenced by significant reductions in effusion accumulation and the associated mortality and was observed with both preventive (beginning before MPE formation) and therapeutic (beginning after MPE establishment) bortezomib treatment. The favorable impact of bortezomib on MPE was associated with suppression of cardinal MPE-associated phenomena, such as inflammation, vascular hyperpermeability, and angiogenesis. In this regard, therapeutic bortezomib treatment had identical favorable results on MPE compared with preventive treatment, indicating that the drug specifically counteracts effusion formation. Conclusions These studies indicate that proteasome inhibition tailored to block NF-κ B activation of lung adenocarcinoma specifically targets the effusion-inducing phenotype of this tumor. Although the drug has limited activity against advanced solid lung cancer, it may prove beneficial for patients with MPE.