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Lung cancer chemoprevention with the prostacyclin analogue iloprost is the most promising approach to date for intercepting progression of premalignant lung lesions in former smokers. Previous preclinical studies of iloprost used oral delivery, but a study modeling delivery directly to the target organ was needed. In vivo and in vitro studies have identified gene expression changes following iloprost treatment, including increased e-cadherin and Ppargγ and decreased COX2 and vimentin. We used tumor counts and gene expression to demonstrate the effectiveness of intranasal delivery of iloprost in a murine model of premalignant adenomas. Intranasal delivery of iloprost reduced adenoma multiplicity 14 weeks after urethane exposure in FVB/N mice compared with untreated urethane controls. Intranasal iloprost reversed urethane-induced gene expression changes in tumors and whole lung. These results correspond to previous studies of oral iloprost and in vitro treatment of human bronchial epithelial cells. This study demonstrates that intranasal delivery of iloprost in a mouse model of lung premalignant lesions is effective chemoprevention. This will be an essential tool for exploring mechanisms and outcomes of iloprost chemoprevention, along with supporting ongoing clinical trials of inhaled iloprost chemoprevention. Prevention Relevance: Iloprost is a promising chemoprevention agent for lung cancer and this work describes a new delivery approach in vivo.

Lung cancer is the leading cause of cancer death worldwide and global burden could be reduced through targeted application of chemoprevention. The development of squamous lung carcinoma has been linked with persistent, high-grade bronchial dysplasia. Bronchial histology improved in former smokers in a chemoprevention trial with the prostacyclin analogue iloprost. Prostacyclin acts through peroxisome proliferator-activated receptor gamma (PPARγ) to reverse epithelial to mesenchymal transition and promote anticancer signaling. We hypothesized that the prostacyclin signaling pathway and EMT could provide response markers for prostacyclin chemoprevention of lung cancer. Human bronchial epithelial cells were treated with cigarette smoke condensate (CSC) or iloprost for 2 weeks, CSC for 16 weeks, or CSC for 4 weeks followed by 4 weeks of CSC and/or iloprost, and RNA was extracted. Wild-type or prostacyclin synthase transgenic mice were exposed to 1 week of cigarette smoke or one injection of urethane, and RNA was extracted from the lungs. We measured potential markers of prostacyclin and iloprost efficacy in these models. We identified a panel of markers altered by tobacco carcinogens and inversely affected by prostacyclin, including PPARγ, 15PGDH, CES1, COX-2, ECADHERIN, SNAIL, VIMENTIN, CRB3, MIR34c, and MIR221. These data introduce a panel of potential markers for monitoring interception of bronchial dysplasia progression during chemoprevention with prostacyclin. Chemoprevention is a promising approach to reduce lung cancer mortality in a high-risk population. Identifying markers for targeted use is critical for success in future clinical trials of prostacyclin for lung cancer chemoprevention. Cancer Prev Res; 11(10); 643–54. ©2018 AACR.

Persistent bronchial dysplasia is associated with increased risk of developing invasive squamous cell carcinoma (SCC) of the lung. In this study, we hypothesized that differences in gene expression profiles between persistent and regressive bronchial dysplasia would identify cellular processes that underlie progression to SCC. RNA expression arrays comparing baseline biopsies from 32 bronchial sites that persisted/progressed to 31 regressive sites showed 395 differentially expressed genes [ANOVA, FDR ≤ 0.05). Thirty-one pathways showed significantly altered activity between the two groups, many of which were associated with cell-cycle control and proliferation, inflammation, or epithelial differentiation/cell–cell adhesion. Cultured persistent bronchial dysplasia cells exhibited increased expression of Polo-like kinase 1 (PLK1), which was associated with multiple cell-cycle pathways. Treatment with PLK1 inhibitor induced apoptosis and G2–M arrest and decreased proliferation compared with untreated cells; these effects were not seen in normal or regressive bronchial dysplasia cultures. Inflammatory pathway activity was decreased in persistent bronchial dysplasia, and the presence of an inflammatory infiltrate was more common in regressive bronchial dysplasia. Regressive bronchial dysplasia was also associated with trends toward overall increases in macrophages and T lymphocytes and altered polarization of these inflammatory cell subsets. Increased desmoglein 3 and plakoglobin expression was associated with higher grade and persistence of bronchial dysplasia. These results identify alterations in the persistent subset of bronchial dysplasia that are associated with high risk for progression to invasive SCC. These alterations may serve as strong markers of risk and as effective targets for lung cancer prevention. Significance: Gene expression profiling of high-risk persistent bronchial dysplasia reveals changes in cell-cycle control, inflammatory activity, and epithelial differentiation/cell–cell adhesion that may underlie progression to invasive SCC. Cancer Res; 78(17); 4971–83. ©2018 AACR.

Half of lung cancers are diagnosed in former smokers, leading to a significant treatment burden in this population. Chemoprevention in former smokers using the prostacyclin analogue iloprost reduces endobronchial dysplasia, a premalignant lung lesion. Iloprost requires the presence of the WNT receptor Frizzled 9 (Fzd9) for inhibition of transformed growth in vitro. To investigate the relationship between iloprost, cigarette smoke and Fzd9 expression, we used human samples, mouse models and in vitro studies. Fzd9 expression was low in human lung tumors and in progressive dysplasias. In mouse models and in vitro studies, tobacco smoke carcinogens reduced expression of Fzd9 while prostacyclin maintained or increased expression. Expression of miR-31 repressed Fzd9 expression, which was abrogated by prostacyclin. We propose a model where cigarette smoke exposure increases miR-31 expression, which leads to decreased Fzd9 expression and prevents response to iloprost. When smoke is removed miR-31 is reduced, prostacyclin can increase Fzd9 expression and progression of dysplasia is inhibited. Fzd9 and miR-31 are candidate biomarkers for precision application of iloprost and monitoring of treatment progress. As we continue to investigate the mechanisms of prostacyclin chemoprevention and identify biomarkers for its use, we will facilitate clinical trials and speed implementation of this valuable prevention approach.

In the original manuscript, Hao Yin’s name was misspelled. The correct name is Hao Yin. The incorrect spelling is Hao Lin. The original article has been updated.

The aim of this contribution is to investigate the effects of a weak vertical heterogeneity of the porous medium on the stability of the Darcy-Hadley flow. We will assume that the permeability and the thermal conductivity of the porous medium undergo a weak linear change in the vertical direction. The stability analysis will be carried out by considering linear perturbations of the basic state in the form of longitudinal rolls, under the hypothesis that the weak heterogeneity does not alter the result that these modes are the most unstable. The neutral stability conditions and the critical values of the vertical Darcy-Rayleigh number will be obtained on varying the horizontal Darcy-Rayleigh number as well as the parameters of heterogeneity relative both to the permeability and to the conductivity. The solution of the linear disturbance equations will be obtained by employing Galerkin’s method of weighted residuals.

A horizontal porous layer with an impermeable adiabatic bottom wall and an impermeable isothermal top wall is studied. An impressed concentration gradient across the layer is assumed. A linear stability analysis is carried out both numerically and by a first-order perturbation method. It is shown that the combined effects of viscous dissipation and mass diffusion may lead to the instability of a basic horizontal uniform flow through the layer.

Chronic inflammation is a risk factor for lung cancer, and low-dose aspirin intake reduces lung cancer risk. However, the roles that specific inflammatory cells and their products play in lung carcinogenesis have yet to be fully elucidated. In mice, alveolar macrophage numbers increase as lung tumors progress, and pulmonary macrophage programing changes within 2 weeks of carcinogen exposure. To examine how macrophages specifically affect lung tumor progression, they were depleted in mice bearing urethane-induced lung tumors using clodronate-encapsulated liposomes. Alveolar macrophage populations decreased to ≤50% of control levels after 4–6 weeks of liposomal clodronate treatment. Tumor burden decreased by 50% compared to vehicle treated mice, and tumor cell proliferation, as measured by Ki67 staining, was also attenuated. Pulmonary fluid levels of insulin-like growth factor-I, CXCL1, IL-6, and CCL2 diminished with clodronate liposome treatment. Tumor-associated macrophages expressed markers of both M1 and M2 programing in vehicle and clodronate liposome-treated mice. Mice lacking CCR2 (the receptor for macrophage chemotactic factor CCL2) had comparable numbers of alveolar macrophages and showed no difference in tumor growth rates when compared to similarly treated wild-type mice suggesting that while CCL2 may recruit macrophages to lung tumor microenvironments, redundant pathways can compensate when CCL2/CCR2 signaling is inactivated. Depletion of pulmonary macrophages rather than inhibition of their recruitment may be an advantageous strategy for attenuating lung cancer progression.

Background Worldwide, lung cancer kills more people than breast, colon and prostate cancer combined. Alterations in macrophage number and function during lung tumorigenesis suggest that these immune effector cells stimulate lung cancer growth. Evidence from cancer models in other tissues suggests that cancer cells actively recruit growth factor-producing macrophages through a reciprocal signaling pathway. While the levels of lung macrophages increase during tumor progression in mouse models of lung cancer, and high pulmonary macrophage content correlates with a poor prognosis in human non-small cell lung cancer, the specific role of alveolar macrophages in lung tumorigenesis is not clear. Methods After culturing either an immortalized lung macrophage cell line or primary murine alveolar macrophages from naïve and lung-tumor bearing mice with primary tumor isolates and immortalized cell lines, the effects on epithelial proliferation and cellular kinase activation were determined. Insulin-like growth factor-1 (IGF-1) was quantified by ELISA, and macrophage conditioned media IGF-1 levels manipulated by IL-4 treatment, immuno-depletion and siRNA transfection. Results Primary macrophages from both naïve and lung-tumor bearing mice stimulated epithelial cell proliferation. The lungs of tumor-bearing mice contained 3.5-times more IGF-1 than naïve littermates, and media conditioned by freshly isolated tumor-educated macrophages contained more IGF-1 than media conditioned by naïve macrophages; IL-4 stimulated IGF-1 production by both macrophage subsets. The ability of macrophage conditioned media to stimulate neoplastic proliferation correlated with media IGF-1 levels, and recombinant IGF-1 alone was sufficient to induce epithelial proliferation in all cell lines evaluated. Macrophage-conditioned media and IGF-1 stimulated lung tumor cell growth in an additive manner, while EGF had no effect. Macrophage-derived factors increased p-Erk1/2, p-Akt and cyclin D1 levels in neoplastic cells, and the combined inhibition of both MEK and PI3K ablated macrophage-mediated increases in epithelial growth. Conclusions Macrophages produce IGF-1 which directly stimulates neoplastic proliferation through Erk and Akt activation. This observation suggests that combining macrophage ablation therapy with IGF-1R, MEK and/or PI3K inhibition could improve therapeutic response in human lung cancer. Exploring macrophage-based intervention could be a fruitful avenue for future research.

Purpose: Sustained nitric oxide (NO) generation positively correlates with lung cancer development and progression. Herein, we genetically confirmed this role of iNOS and evaluated the chemopreventive efficacy of silibinin in carcinogen-treated B6/129 wild-type (WT) and iNOS−/− mice. Experimental Design: Male B6/129-Nos2tm1Lau (iNOS−/−) and B6/129PF2 WT mice were injected i.p. with 1 mg/g body weight urethane once weekly for 7 consecutive weeks, followed by silibinin gavage (742 mg/kg body weight) for 5 d/wk for 18 weeks. Results: Quantification of micro-CT data in real-time showed that silibinin significantly decreases urethane-induced tumor number and size in WT mice, consistent with measurements made ex vivo at study termination. Genetic ablation of iNOS decreased urethane-induced tumor multiplicity by 87% (P < 0.001) compared to WT mice. Silibinin decreased tumor multiplicity by 71% (P < 0.01) in WT mice, but did not show any such considerable effect in iNOS−/− mice. Tumors from WT mice expressed more iNOS (P < 0.01) but almost similar eNOS and nNOS than those in silibinin-treated mice. In these tumors, silibinin moderately (P < 0.01) inhibited cell proliferation but strongly (P < 0.01) reduced the number of newly formed nestin-positive microvessels. Silibinin decreased VEGFR2 level, and STAT3 and NF-κB activation in tumors. Conclusions: The lack of effect of silibinin in iNOS−/− mice suggests that silibinin exerts most of its chemopreventive and angiopreventive effects through its inhibition of iNOS expression in lung tumors. Our results support iNOS as a potential target for controlling lung cancer, and demonstrate the value of real-time noninvasive micro-CT imaging modality for evaluating the efficacy of lung cancer chemopreventive agents. Clin Cancer Res; 17(4); 1–9. ©2010 AACR.

Tumor-associated macrophages (TAMs) encourage and coordinate neoplastic growth. In late stage human lung adenocarcinoma, TAMs exhibited mixed M1 (classical; argI(low)iNOS(high)) and M2 (alternative; argI(high)iNOS(low)) polarization based on arginine metabolism. In several murine cancer models including chemically and genetically-induced primary lung tumors, prostate tumors, colon xenografts, and lung metastases, TAMs expressed argI(high)iNOS(low) early during tumor formation; argI(low)iNOS(high) polarization also occurred during malignancy in some models. In a chemically-induced lung tumor model, macrophages expressed argI(high)iNOS(low) within one week after carcinogen treatment, followed by similar polarization of bone marrow-derived monocytes (BDMCs) a few days later. TAMs surrounding murine prostate tumors also expressed argI(high)iNOS(low) early during tumorigenesis, indicating that this polarization is not unique to neoplastic lungs. In a human colon cancer xenograft model, the primary tumor was surrounded by argI(high)iNOS(low)-expressing TAMs, and BDMCs also expressed argI(high)iNOS(low), but pulmonary macrophages adopted argI(high)iNOS(low) polarization only after tumors metastasized to the lungs. Persistence of tumors is required to maintain TAM polarization. Indeed, in both conditional mutant Kras- and FGF10-driven models of lung cancer, mice expressing the transgene develop lung tumors that regress rapidly when the transgene is silenced. Furthermore, pulmonary macrophages expressed argI(high)iNOS(low) on tumor induction, but then returned to argI(low) iNOS(low) (no polarization) after tumors regressed. Manipulating TAM function or depleting TAMs may provide novel therapeutic strategies for preventing and treating many types of cancer.

Background Human lung cancer patients exhibit different KRAS mutations depending on smoking status. In a mouse model of human cancer, A/J and BALB/cBy mice treated with the tobacco carcinogen, 3-methylcholanthrene (MCA), followed by butylated hydroxytoluene (BHT)-elicited chronic inflammation develop a high multiplicity of lung tumors. Methods DNA was isolated from MCA-induced lung tumors in A/J and BALB/cByJ mice. Kras codon 12 sequences from these tumors were compared to those in human lung tumors from smokers and never-smokers. Results The distribution of Kras codon 12 mutations in MCA-induced A/J lung tumors is strikingly similar to those found in adenocarcinomas from human smokers. In contrast, codon 12 mutations in BALB/cBy mice contain predominantly G → D mutations, which is the most common mutation in never smokers. Conclusions A single lung carcinogen induces different tumor initiating mutations in different strains of mice. This may be useful for investigating the role of specific KRAS mutations in adenocarcinoma pathogenesis in smokers versus never smokers, identifying mechanisms that select for certain KRAS mutations and developing new drugs that specifically target cells with different KRAS mutations.

Cancer susceptibility results from interactions between sensitivity and resistance alleles. We employed murine chromosome substitution strains to study how resistance alleles affected sensitive alleles during chemically-induced lung carcinogenesis. The C57BL/6J-Chr#(A/J) strains, constructed by selectively breeding sensitive A/J and resistant C57BL/6J (B6) mice, each contain one pair of A/J chromosomes within an otherwise B6 genome. Pas1, the major locus responsible for this differential strain response to urethane carcinogenesis, resides on Chr 6, but C57BL/6J-Chr6(A/J) mice (hereafter CSS-6) developed few tumors following a single urethane injection, which demonstrates epistatic interactions with other B6 alleles. CSS6 mice developed dozens of lung tumors after chronic urethane exposure, however, indicating that these epistatic interactions could be overcome by repeated carcinogen administration. Unlike A/J, but similar to B6 mice, CSS6 mice were resistant to lung carcinogenesis induced by 3-methylcholanthrene (MCA). Tumor multiplicity increased if BHT administration followed urethane exposure, showing that a Chr 6 gene(s) regulates sensitivity to chemically-induced tumor promotion. Unlike A/J tumors (predominantly codon 61 A-->T transversions), Kras mutations in tumors induced by urethane in CSS-6 mice were similar to B6 tumors (codon 61 A-->G transitions). DNA repair genes not located on Chr 6 may determine the nature of Kras mutations. CSS-6 mice are a valuable resource for testing the ability of candidate genes to modulate lung carcinogenesis.

Human adenocarcinoma (AC) is the most frequently diagnosed human lung cancer, and its absolute incidence is increasing dramatically. Compared to human lung AC, the A/J mouse-urethane model exhibits similar histological appearance and molecular changes. We examined the gene expression profiles of human and murine lung tissues (normal or AC) and compared the two species' datasets after aligning approximately 7500 orthologous genes. A list of 409 gene classifiers (P value0.0001), common to both species (joint classifiers), showed significant, positive correlation in expression levels between the two species. A number of previously reported expression changes were recapitulated in both species, such as changes in glycolytic enzymes and cell-cycle proteins. Unexpectedly, joint classifiers in angiogenesis were uniformly down-regulated in tumor tissues. The eicosanoid pathway enzymes prostacyclin synthase (PGIS) and inducible prostaglandin E(2) synthase (PGES) were joint classifiers that showed opposite effects in lung AC (PGIS down-regulated; PGES up-regulated). Finally, tissue microarrays identified the same protein expression pattern for PGIS and PGES in 108 different non-small cell lung cancer biopsies, and the detection of PGIS had statistically significant prognostic value in patient survival. Thus, the A/J mouse-urethane model reflects significant molecular details of human lung AC, and comparison of changes in orthologous gene expression may provide novel insights into lung carcinogenesis.

The onset of the convective instability in the horizontal throughflow of a power-law fluid saturating a horizontal porous layer heated from below is studied. A linear stability analysis of the basic flow is carried out and the disturbance equations are solved analytically. The problem examined here is an extension of the classical Prats problem for Newtonian fluids. It is shown that the marginal stability condition, as well as the critical values of the wave number and of the Darcy–Rayleigh number, is affected by the value of the Peclet number associated with the basic flow, except for the special case of a Newtonian fluid. The limit of a vanishingly small Peclet number is considered leading to the special case of the Horton–Rogers–Lapwood (HRL) problem for a power-law fluid, i.e., the Prats problem with a vanishing basic throughflow. It is shown that the generalized HRL problem is always linearly stable for pseudoplastic fluids and always linearly unstable for dilatant fluids.

The combined effects of the double-diffusion and of the viscous dissipation on the convective instability in a fluid-saturated porous medium with a basic horizontal throughflow are investigated. A horizontal porous layer with an impermeable adiabatic lower wall and an impermeable isothermal upper wall is considered. The parallel boundary walls are assumed to have uniform, but unequal, concentrations of the solute. A linear stability analysis is carried out both numerically and by a first-order perturbation method. General disturbances having the form of oblique rolls are considered, reducing either to longitudinal rolls or to transverse rolls in the special cases of roll axes parallel or orthogonal to the basic flow direction, respectively. It is shown that the combined effects of viscous dissipation and mass diffusion may lead to the instability of the basic horizontal flow. Either the longitudinal rolls or the transverse rolls may be the preferred modes of instability depending on the value of the viscous dissipation parameter Xi. The longitudinal rolls are the most unstable when Xi < 61.86657.