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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.

IL-5 is a T helper 2 cytokine important in the trafficking and survival of eosinophils. Because eosinophils can be found in malignant pleural effusions (MPE) from mice and humans, we asked whether IL-5 is involved in the pathogenesis of MPE.To determine the role of IL-5 in MPE formation.The effects of IL-5 on experimental MPE induced in C57BL/6 mice by intrapleural injection of syngeneic lung (Lewis lung cancer [LLC]) or colon (MC38) adenocarcinoma cells were determined using wild-type (il5(+/+)) and IL-5-deficient (il5⁻(/)⁻) mice, exogenous administration of recombinant mouse (rm) IL-5, and in vivo antibody-mediated neutralization of endogenous IL-5. The direct effects of rmIL-5 on LLC cell proliferation and gene expression in vitro were determined by substrate reduction and microarray.Eosinophils and IL-5 were present in human and mouse MPE, but the cytokine was not detected in mouse (LLC) or human (A549) lung and mouse colon (MC38) adenocarcinoma-conditioned medium, suggesting production by host cells in MPE. Compared with il5(+/+) mice, il5⁻(/)⁻ mice showed markedly diminished MPE formation in response to both LLC and MC38 cells. Exogenous IL-5 promoted MPE formation in il5(+/+) and il5⁻(/)⁻ mice, whereas anti-IL-5 antibody treatment limited experimental MPE in il5(+/+) mice. Exogenous IL-5 had no effects on LLC cell proliferation and gene expression; however, IL-5 was found to be responsible for recruitment of eosinophils and tumor-promoting myeloid suppressor cells to MPE in vivo.Host-derived IL-5 promotes experimental MPE and may be involved in the pathogenesis of human MPE.

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.

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

Since recent evidence indicates a requirement for epithelial nuclear factor (NF)-κB signaling in lung tumorigenesis, we investigated the impact of the NF-κB inhibitor bortezomib on lung tumor promotion and growth. We used an experimental model in which wild-type mice or mice expressing an NF-κB reporter received intraperitoneal urethane (1 g/kg) followed by twice weekly bortezomib (1 mg/kg) during distinct periods of tumor initiation/progression. Mice were serially assessed for lung NF-κB activation, inflammation and carcinogenesis. Short-term proteasome inhibition with bortezomib did not impact tumor formation but retarded the growth of established lung tumors in mice via effects on cell proliferation. In contrast, long-term treatment with bortezomib resulted in significantly increased lung tumor number and size. This tumor-promoting effect of prolonged bortezomib treatment was associated with perpetuation of urethane-induced inflammation and chronic upregulation of interleukin-1β and proinflammatory C-X-C motif chemokine ligands (CXCL) 1 and 2 in the lungs. In addition to airway epithelium, bortezomib inhibited NF-κB in pulmonary macrophages in vivo, presenting a possible mechanism of tumor amplification. In this regard, RAW264.7 macrophages exposed to bortezomib showed increased expression of interleukin-1β, CXCL1 and CXCL2. In conclusion, although short-term bortezomib may exert some beneficial effects, prolonged NF-κB inhibition accelerates chemical lung carcinogenesis by perpetuating carcinogen-induced inflammation. Inhibition of NF-κB in pulmonary macrophages appears to play an important role in this adverse process.

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 Although the relationship between allergic inflammation and lung carcinogenesis is not clearly defined, several reports suggest an increased incidence of lung cancer in patients with asthma. We aimed at determining the functional impact of allergic inflammation on chemical carcinogenesis in the lungs of mice. Methods Balb/c mice received single-dose urethane (1 g/kg at day 0) and two-stage ovalbumin during tumor initiation (sensitization: days -14 and 0; challenge: daily at days 6-12), tumor progression (sensitization: days 70 and 84; challenge: daily at days 90-96), or chronically (sensitization: days -14 and 0; challenge: daily at days 6-12 and thrice weekly thereafter). In addition, interleukin (IL)-5 deficient and wild-type C57BL/6 mice received ten weekly urethane injections. All mice were sacrificed after four months. Primary end-points were number, size, and histology of lung tumors. Secondary end-points were inflammatory cells and mediators in the airspace compartment. Results Ovalbumin provoked acute allergic inflammation and chronic remodeling of murine airways, evident by airspace eosinophilia, IL-5 up-regulation, and airspace enlargement. Urethane resulted in formation of atypical alveolar hyperplasias, adenomas, and adenocarcinomas in mouse lungs. Ovalbumin-induced allergic inflammation during tumor initiation, progression, or continuously did not impact the number, size, or histologic distribution of urethane-induced pulmonary neoplastic lesions. In addition, genetic deficiency in IL-5 had no effect on urethane-induced lung tumorigenesis. Conclusions Allergic inflammation does not impact chemical-induced carcinogenesis of the airways. These findings suggest that not all types of airway inflammation influence lung carcinogenesis and cast doubt on the idea of a mechanistic link between asthma and lung cancer.

Since recent evidence indicates a requirement for epithelial NF-κB signaling in lung tumorigenesis, we investigated the impact of the NF-κB inhibitor bortezomib on lung tumor promotion and growth. We used an experimental model in which wild-type mice or mice expressing an NF-κB reporter received intraperitoneal urethane (1 g/kg) followed by twice-weekly bortezomib (1 mg/kg) during distinct periods of tumor initiation/progression. Mice were serially assessed for lung NF-κB activation, inflammation, and carcinogenesis. Short-term proteasome inhibition with bortezomib did not impact tumor formation, but retarded the growth of established lung tumors in mice via effects on cell proliferation. In contrast, long-term treatment with bortezomib resulted in significantly increased lung tumor number and size. This tumor-promoting effect of prolonged bortezomib treatment was associated with perpetuation of urethane-induced inflammation and chronic up-regulation of interleukin-1α and proinflammatory C-X-C motif chemokine ligands (CXCL) 1 and 2 in the lungs. In addition to airway epithelium, bortezomib inhibited NF-κB in pulmonary macrophages in vivo, presenting a possible mechanism of tumor amplification. In this regard, RAW264.7 macrophages exposed to bortezomib showed increased expression of interleukin-1α, CXCL1, and CXCL2. In conclusion, although short-term bortezomib may exert some beneficial effects, prolonged NF-κB inhibition accelerates chemical lung carcinogenesis by perpetuating carcinogen-induced inflammation. Inhibition of NF-κB in pulmonary macrophages appears to play an important role in this adverse process. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2873. doi:1538-7445.AM2012-2873

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.