Cigarette smoke-induced airspace disease in mice develops independently of HIF-1α signaling in leukocytes.

The pathogenesis of chronic obstructive pulmonary disease (COPD), a prevalent disease primarily caused by cigarette smoke exposure, is incompletely elucidated. Studies in humans and mice have suggested that hypoxia-inducible factor-1α (HIF-1α) may play a role. Reduced lung levels of HIF-1α are associated with decreased vascular density, whereas increased leukocyte HIF-1α may be responsible for increased inflammation. To elucidate the specific role of leukocyte HIF-1α in COPD, we exposed transgenic mice with conditional deletion or overexpression of HIF-1α in leukocytes to cigarette smoke for 7 mo. Outcomes included pulmonary physiology, aerated lung volumes via microcomputed tomography, lung morphometry and histology, and cardiopulmonary hemodynamics. On aggregate, cigarette smoke increased the aerated lung volume, quasi-static lung compliance, inspiratory capacity of all strains while reducing the total alveolar septal volume. Independent of smoke exposure, mice with leukocyte-specific HIF-1α overexpression had increased quasi-static compliance, inspiratory capacity, and alveolar septal volume compared with mice with leukocyte-specific HIF-1α deletion. However, the overall development of cigarette smoke-induced lung disease did not vary relative to control mice for either of the conditional strains. This suggests that the development of murine cigarette smoke-induced airspace disease occurs independently of leukocyte HIF-1α signaling.

Upregulation of complement proteins in lung cancer cells mediates tumor progression.

Introduction: In vivo, cancer cells respond to signals from the tumor microenvironment resulting in changes in expression of proteins that promote tumor progression and suppress anti-tumor immunity. This study employed an orthotopic immunocompetent model of lung cancer to define pathways that are altered in cancer cells recovered from tumors compared to cells grown in culture. Methods: Studies used four murine cell lines implanted into the lungs of syngeneic mice. Cancer cells were recovered using FACS, and transcriptional changes compared to cells grown in culture were determined by RNA-seq. Results: Changes in interferon response, antigen presentation and cytokine signaling were observed in all tumors. In addition, we observed induction of the complement pathway. We previously demonstrated that activation of complement is critical for tumor progression in this model. Complement can play both a pro-tumorigenic role through production of anaphylatoxins, and an anti-tumorigenic role by promoting complement-mediated cell killing of cancer cells. While complement proteins are produced by the liver, expression of complement proteins by cancer cells has been described. Silencing cancer cell-specific C3 inhibited tumor growth In vivo. We hypothesized that induction of complement regulatory proteins was critical for blocking the anti-tumor effects of complement activation. Silencing complement regulatory proteins also inhibited tumor growth, with different regulatory proteins acting in a cell-specific manner. Discussion: Based on these data we propose that localized induction of complement in cancer cells is a common feature of lung tumors that promotes tumor progression, with induction of complement regulatory proteins protecting cells from complement mediated-cell killing.

Microenvironmental Regulation of Macrophage Transcriptomic and Metabolomic Profiles in Pulmonary Hypertension.

The recruitment and subsequent polarization of inflammatory monocytes/macrophages in the perivascular regions of pulmonary arteries is a key feature of pulmonary hypertension (PH). However, the mechanisms driving macrophage polarization within the adventitial microenvironment during PH progression remain unclear. We previously established that reciprocal interactions between fibroblasts and macrophages are essential in driving the activated phenotype of both cell types although the signals involved in these interactions remain undefined. We sought to test the hypothesis that adventitial fibroblasts produce a complex array of metabolites and proteins that coordinately direct metabolomic and transcriptomic re-programming of naïve macrophages to recapitulate the pathophysiologic phenotype observed in PH. Media conditioned by pulmonary artery adventitial fibroblasts isolated from pulmonary hypertensive (PH-CM) or age-matched control (CO-CM) calves were used to activate bone marrow derived macrophages. RNA-Seq and mass spectrometry-based metabolomics analyses were performed. Fibroblast conditioned medium from patients with idiopathic pulmonary arterial hypertension or controls were used to validate transcriptional findings. The microenvironment was targeted in vitro using a fibroblast-macrophage co-culture system and in vivo in a mouse model of hypoxia-induced PH. Both CO-CM and PH-CM actively, yet distinctly regulated macrophage transcriptomic and metabolomic profiles. Network integration revealed coordinated rewiring of pro-inflammatory and pro-remodeling gene regulation in concert with altered mitochondrial and intermediary metabolism in response to PH-CM. Pro-inflammation and metabolism are key regulators of macrophage phenotype in vitro, and are closely related to in vivo flow sorted lung interstitial/perivascular macrophages from hypoxic mice. Metabolic changes are accompanied by increased free NADH levels and increased expression of a metabolic sensor and transcriptional co-repressor, C-terminal binding protein 1 (CtBP1), a mechanism shared with adventitial PH-fibroblasts. Targeting the microenvironment created by both cell types with the CtBP1 inhibitor MTOB, inhibited macrophage pro-inflammatory and metabolic re-programming both in vitro and in vivo. In conclusion, coordinated transcriptional and metabolic reprogramming is a critical mechanism regulating macrophage polarization in response to the complex adventitial microenvironment in PH. Targeting the adventitial microenvironment can return activated macrophages toward quiescence and attenuate pathological remodeling that drives PH progression.

Altered Macrophage Function Associated with Crystalline Lung Inflammation in Acid Sphingomyelinase Deficiency.

Deficiency of ASM (acid sphingomyelinase) causes the lysosomal storage Niemann-Pick disease (NPD). Patients with NPD type B may develop progressive interstitial lung disease with frequent respiratory infections. Although several investigations using the ASM-deficient (ASMKO) mouse NPD model revealed inflammation and foamy macrophages, there is little insight into the pathogenesis of NPD-associated lung disease. Using ASMKO mice, we report that ASM deficiency is associated with a complex inflammatory phenotype characterized by marked accumulation of monocyte-derived CD11b+ macrophages and expansion of airspace/alveolar CD11c+ CD11b- macrophages, both with increased size, granularity, and foaminess. Both the alternative and classical pathways were activated, with decreased in situ phagocytosis of opsonized (Fc-coated) targets, preserved clearance of apoptotic cells (efferocytosis), secretion of Th2 cytokines, increased CD11c+/CD11b+ cells, and more than a twofold increase in lung and plasma proinflammatory cytokines. Macrophages, neutrophils, eosinophils, and noninflammatory lung cells of ASMKO lungs also exhibited marked accumulation of chitinase-like protein Ym1/2, which formed large eosinophilic polygonal Charcot-Leyden-like crystals. In addition to providing insight into novel features of lung inflammation that may be associated with NPD, our report provides a novel connection between ASM and the development of crystal-associated lung inflammation with alterations in macrophage biology.

Cancer Cell-Intrinsic Expression of MHC Class II Regulates the Immune Microenvironment and Response to Anti-PD-1 Therapy in Lung Adenocarcinoma.

MHC class II (MHCII) expression is usually restricted to APC but can be expressed by cancer cells. We examined the effect of cancer cell-specific MHCII (csMHCII) expression in lung adenocarcinoma on T cell recruitment to tumors and response to anti-PD-1 therapy using two orthotopic immunocompetent murine models of non-small cell lung cancer: CMT167 (CMT) and Lewis lung carcinoma (LLC). We previously showed that CMT167 tumors are eradicated by anti-PD1 therapy, whereas LLC tumors are resistant. RNA sequencing analysis of cancer cells recovered from tumors revealed that csMHCII correlated with response to anti-PD1 therapy, with immunotherapy-sensitive CMT167 cells being csMHCII positive, whereas resistant LLC cells were csMHCII negative. To test the functional effects of csMHCII, MHCII expression was altered on the cancer cells through loss- and gain-of-function of CIITA, a master regulator of the MHCII pathway. Loss of CIITA in CMT167 decreased csMHCII and converted tumors from anti-PD-1 sensitive to anti-PD-1 resistant. This was associated with lower levels of Th1 cytokines, decreased T cell infiltration, increased B cell numbers, and decreased macrophage recruitment. Conversely, overexpression of CIITA in LLC cells resulted in csMHCII in vitro and in vivo. Enforced expression of CIITA increased T cell infiltration and sensitized tumors to anti-PD-1 therapy. csMHCII expression was also examined in a subset of surgically resected human lung adenocarcinomas by multispectral imaging, which provided a survival benefit and positively correlated with T cell infiltration. These studies demonstrate a functional role for csMHCII in regulating T cell infiltration and sensitivity to anti-PD-1.

Activation of PPARγ in Myeloid Cells Promotes Progression of Epithelial Lung Tumors through TGFß1.

Lung cancer is a heterogeneous disease in which patient-specific treatments are desirable and the development of targeted therapies has been effective. Although mutations in KRAS are frequent in lung adenocarcinoma, there are currently no targeted agents against KRAS. Using a mouse lung adenocarcinoma cell line with a Kras mutation (CMT167), we previously showed that PPARγ activation in lung cancer cells inhibits cell growth in vitro yet promotes tumor progression when activated in myeloid cells of the tumor microenvironment. Here, we report that PPARγ activation in myeloid cells promotes the production of TGFß1, which, in turn, acts on CMT167 cancer cells to increase migration and induce an epithelial-mesenchymal transition (EMT). Targeting TGFß1 signaling in CMT167 cells prevented their growth and metastasis in vivo. Similarly, another mouse lung adenocarcinoma cell line with a Kras mutation, LLC, induced TGFß1 in myeloid cells through PPARγ activation. However, LLC cells are more mesenchymal and did not undergo EMT in response to TGFß1, nor did LLC require TGFß1 signaling for metastasis in vivo. Converting CMT167 cells to a mesenchymal phenotype through overexpression of ZEB1 made them unresponsive to TGFß1 receptor inhibition. The ability of TGFß1 to induce EMT in lung tumors may represent a critical process in cancer progression. We propose that TGFß receptor inhibition could provide an additional treatment option for KRAS-mutant epithelial lung tumors.Implications: This study suggests that TGFß receptor inhibitors may be an effective therapy in a subset of KRAS-mutant patients with non-small cell lung cancer, which show an epithelial phenotype.

Tumor-intrinsic response to IFNγ shapes the tumor microenvironment and anti-PD-1 response in NSCLC.

Targeting PD-1/PD-L1 is only effective in ∼20% of lung cancer patients, but determinants of this response are poorly defined. We previously observed differential responses of two murine K-Ras-mutant lung cancer cell lines to anti-PD-1 therapy: CMT167 tumors were eliminated, whereas Lewis Lung Carcinoma (LLC) tumors were resistant. The goal of this study was to define mechanism(s) mediating this difference. RNA sequencing analysis of cancer cells recovered from lung tumors revealed that CMT167 cells induced an IFNγ signature that was blunted in LLC cells. Silencing Ifngr1 in CMT167 resulted in tumors resistant to IFNγ and anti-PD-1 therapy. Conversely, LLC cells had high basal expression of SOCS1, an inhibitor of IFNγ. Silencing Socs1 increased response to IFNγ in vitro and sensitized tumors to anti-PD-1. This was associated with a reshaped tumor microenvironment, characterized by enhanced T cell infiltration and enrichment of PD-L1hi myeloid cells. These studies demonstrate that targeted enhancement of tumor-intrinsic IFNγ signaling can induce a cascade of changes associated with increased therapeutic vulnerability.

Adenoviral vectors transduce alveolar macrophages in lung cancer models.

Adenoviral vectors expressing Cre recombinase are commonly used to initiate tumor formation in murine lung cancer models. While these vectors are designed to target genetic recombination to lung epithelial cells, adenoviruses can infect additional cell types that potentially influence tumor development. Our goal was to explore the consequences of adenoviral-mediated alveolar macrophage (AM) transduction in a Kras-initiated lung tumor model. As expected, treatment of animals harboring the KrasLSL-G12D allele and an inducible green fluorescence protein (GFP) tracking allele with an adenoviral vector expressing Cre recombinase under the control of the cytomegalovirus (CMV) promoter (Ad5-CMV-Cre), caused GFP-positive lung adenocarcinomas. Surprisingly, however, up to 70% of the total GFP+ cells were AM, and GFP+ AM could be detected 6 months after tumor initiation, and transduced AM demonstrated Kras activation and increased proliferation. In contrast, recombination was not detected in other immune cell populations and AM recombination could be eliminated by tumor initiation with an adenovirus expressing Cre recombinase under the control of the surfactant protein C (SPC) promoter. In addition, AM isolated from KrasLSL-G12D animals and transduced by Ad5-CMV-Cre ex vivo displayed prolonged survival in vitro and increased the growth of murine lung adenocarcinoma CMT/167 cells when co-injected in an orthotopic flank model. Given the importance of the immune system in tumor development and progression, inadvertent AM transduction by Ad5-CMV-Cre merits careful consideration during lung cancer model selection particularly if studies evaluating the tumor-immune interactions are planned.

Targeted overexpression of prostacyclin synthase inhibits lung tumor progression by recruiting CD4+ T lymphocytes in tumors that express MHC class II.

Lung-specific overexpression of prostacyclin synthase (PGIS) decreases tumor initiation in murine lung cancer models. Prostacyclin analogs prevent lung tumor formation in mice and reverse bronchial dysplasia in former smokers. However, the effect of prostacyclin on lung cancer progression has not been well studied. We investigated the effects of pulmonary PGIS overexpression in an orthotopic immunocompetent mouse model of lung cancer using two murine lung cancer cell lines. Pulmonary PGIS overexpression significantly inhibited CMT167 lung tumor growth, increased CXCL9 expression, and increased CD4+ tumor-infiltrating lymphocytes. Immunodepletion of CD4+ T cells abolished the inhibitory effect of pulmonary PGIS overexpression on CMT167 lung tumor growth. In contrast, pulmonary PGIS overexpression failed to inhibit growth of a second murine lung cancer cell line, Lewis Lung Carcinoma (LLC) cells, and failed to increase CXCL9 expression or CD4+ T lymphocytes in LLC lung tumors. Transcriptome profiling of CMT167 cells and LLC cells recovered from tumor-bearing mice demonstrated that in vivo, CMT167 cells but not LLC cells express MHC class II genes and cofactors necessary for MHC class II processing and presentation. These data demonstrate that prostacyclin can inhibit lung cancer progression and suggest that prostacyclin analogs may serve as novel immunomodulatory agents in a subset of lung cancer patients. Moreover, expression of MHC Class II by lung cancer cells may represent a biomarker for response to prostacyclin.

Bone marrow-derived cPLA2α contributes to renal fibrosis progression.

The group IVA calcium-dependent cytosolic phospholipase A2 (cPLA2α) enzyme directs a complex "eicosanoid storm" that accompanies the tissue response to injury. cPLA2α and its downstream eicosanoid mediators are also implicated in the pathogenesis of fibrosis in many organs, including the kidney. We aimed to determine the role of cPLA2α in bone marrow-derived cells in a murine model of renal fibrosis, unilateral ureteral obstruction (UUO). WT C57BL/6J mice were irradiated and engrafted with donor bone marrow from either WT mice [WT-bone marrow transplant (BMT)] or mice deficient in cPLA2α (KO-BMT). After full engraftment, mice underwent UUO and kidneys were collected 3, 7, and 14 days after injury. Using picrosirius red, collagen-3, and smooth muscle α actin staining, we determined that renal fibrosis was significantly attenuated in KO-BMT animals as compared with WT-BMT animals. Lipidomic analysis of homogenized kidneys demonstrated a time-dependent upregulation of pro-inflammatory eicosanoids after UUO; KO-BMT animals had lower levels of many of these eicosanoids. KO-BMT animals also had fewer infiltrating pro-inflammatory CD45+CD11b+Ly6Chi macrophages and reduced message levels of pro-inflammatory cytokines. Our results indicate that cPLA2α and/or its downstream mediators, produced by bone marrow-derived cells, play a major role in eicosanoid production after renal injury and in renal fibrinogenesis.

Complement Activation via a C3a Receptor Pathway Alters CD4+ T Lymphocytes and Mediates Lung Cancer Progression.

The complement cascade is a part of the innate immune system that acts primarily to remove pathogens and injured cells. However, complement activation is also peculiarly associated with tumor progression. Here we report mechanistic insights into this association in multiple immunocompetent orthotopic models of lung cancer. After tumor engraftment, we observed systemic activation of the complement cascade as reflected by elevated levels of the key regulator C3a. Notably, growth of primary tumors and metastases was both strongly inhibited in C3-deficient mice (C3-/- mice), with tumors undetectable in many subjects. Growth inhibition was associated with increased numbers of IFNγ+/TNFα+/IL10+ CD4+ and CD8+ T cells. Immunodepletion of CD4+ but not CD8+ T cells in tumor-bearing subjects reversed the inhibitory effects of C3 deletion. Similarly, antagonists of the C3a or C5a receptors inhibited tumor growth. Investigations using multiple tumor cell lines in the orthotopic model suggested the involvement of a C3/C3 receptor autocrine signaling loop in regulating tumor growth. Overall, our findings offer functional evidence that complement activation serves as a critical immunomodulator in lung cancer progression, acting to drive immune escape via a C3/C5-dependent pathway.Significance: This provocative study suggests that inhibiting complement activation may heighten immunotherapeutic responses in lung cancer, offering findings with immediate implications, given the existing clinical availability of complement antagonists. Cancer Res; 78(1); 143-56. ©2017 AACR.

The Tumor Microenvironment Regulates Sensitivity of Murine Lung Tumors to PD-1/PD-L1 Antibody Blockade.

Immune checkpoint inhibitors targeting the interaction between programmed cell death-1 (PD-1) and its ligand PD-L1 induce tumor regression in a subset of non-small cell lung cancer patients. However, clinical response rates are less than 25%. Evaluation of combinations of immunotherapy with existing therapies requires appropriate preclinical animal models. In this study, murine lung cancer cells (CMT167 and LLC) were implanted either orthotopically in the lung or subcutaneously in syngeneic mice, and response to anti-PD-1/PD-L1 therapy was determined. Anti-PD-1/PD-L1 therapy inhibited CMT167 orthotopic lung tumors by 95%. The same treatments inhibited CMT167 subcutaneous tumors by only 30% and LLC orthotopic lung tumors by 35%. CMT167 subcutaneous tumors had more Foxp3+ CD4+ T cells and fewer PD-1+ CD4+ T cells compared with CMT167 orthotopic tumors. Flow cytometric analysis also demonstrated increased abundance of PD-L1high cells in the tumor microenvironment in CMT167 tumor-bearing lungs compared with CMT167 subcutaneous tumors or LLC tumor-bearing lungs. Silencing PD-L1 expression in CMT167 cells resulted in smaller orthotopic tumors that remained sensitive to anti-PD-L1 therapy, whereas implantation of CMT167 cells into PD-L1- mice blocked orthotopic tumor growth, indicating a role for PD-L1 in both the cancer cell and the microenvironment. These findings indicate that the response of cancer cells to immunotherapy will be determined by both intrinsic properties of the cancer cells and specific interactions with the microenvironment. Experimental models that accurately recapitulate the lung tumor microenvironment are useful for evaluation of immunotherapeutic agents. Cancer Immunol Res; 5(9); 767-77. ©2017 AACR.

Differentiated Smooth Muscle Cells Generate a Subpopulation of Resident Vascular Progenitor Cells in the Adventitia Regulated by Klf4.

RATIONALE: The vascular adventitia is a complex layer of the vessel wall consisting of vasa vasorum microvessels, nerves, fibroblasts, immune cells, and resident progenitor cells. Adventitial progenitors express the stem cell markers, Sca1 and CD34 (adventitial sca1-positive progenitor cells [AdvSca1]), have the potential to differentiate in vitro into multiple lineages, and potentially contribute to intimal lesions in vivo. OBJECTIVE: Although emerging data support the existence of AdvSca1 cells, the goal of this study was to determine their origin, degree of multipotency and heterogeneity, and contribution to vessel remodeling. METHODS AND RESULTS: Using 2 in vivo fate-mapping approaches combined with a smooth muscle cell (SMC) epigenetic lineage mark, we report that a subpopulation of AdvSca1 cells is generated in situ from differentiated SMCs. Our data establish that the vascular adventitia contains phenotypically distinct subpopulations of progenitor cells expressing SMC, myeloid, and hematopoietic progenitor-like properties and that differentiated SMCs are a source to varying degrees of each subpopulation. SMC-derived AdvSca1 cells exhibit a multipotent phenotype capable of differentiating in vivo into mature SMCs, resident macrophages, and endothelial-like cells. After vascular injury, SMC-derived AdvSca1 cells expand in number and are major contributors to adventitial remodeling. Induction of the transcription factor Klf4 in differentiated SMCs is essential for SMC reprogramming in vivo, whereas in vitro approaches demonstrate that Klf4 is essential for the maintenance of the AdvSca1 progenitor phenotype. CONCLUSIONS: We propose that generation of resident vascular progenitor cells from differentiated SMCs is a normal physiological process that contributes to the vascular stem cell pool and plays important roles in arterial homeostasis and disease.

Expression Profiling of Macrophages Reveals Multiple Populations with Distinct Biological Roles in an Immunocompetent Orthotopic Model of Lung Cancer.

Macrophages represent an important component of the tumor microenvironment and play a complex role in cancer progression. These cells are characterized by a high degree of plasticity, and they alter their phenotype in response to local environmental cues. Whereas the M1/M2 classification of macrophages has been widely used, the complexity of macrophage phenotypes has not been well studied, particularly in lung cancer. In this study we employed an orthotopic immunocompetent model of lung adenocarcinoma in which murine lung cancer cells are directly implanted into the left lobe of syngeneic mice. Using multimarker flow cytometry, we defined and recovered several distinct populations of monocytes/macrophages from tumors at different stages of progression. We used RNA-seq transcriptional profiling to define distinct features of each population and determine how they change during tumor progression. We defined an alveolar resident macrophage population that does not change in number and expresses multiple genes related to lipid metabolism and lipid signaling. We also defined a population of tumor-associated macrophages that increase dramatically with tumor and selectively expresses a panel of chemokine genes. A third population, which resembles tumor-associated monocytes, expresses a large number of genes involved in matrix remodeling. By correlating transcriptional profiles with clinically prognostic genes, we show that specific monocyte/macrophage populations are enriched in genes that predict outcomes in lung adenocarcinoma, implicating these subpopulations as critical determinants of patient survival. Our data underscore the complexity of monocytes/macrophages in the tumor microenvironment, and they suggest that distinct populations play specific roles in tumor progression.

Deletion of 5-Lipoxygenase in the Tumor Microenvironment Promotes Lung Cancer Progression and Metastasis through Regulating T Cell Recruitment.

Eicosanoids, including PGs, produced by cyclooxygenases (COX), and leukotrienes, produced by 5-lipoxygenase (5-LO) have been implicated in cancer progression. These molecules are produced by both cancer cells and the tumor microenvironment (TME). We previously reported that both COX and 5-LO metabolites increase during progression in an orthotopic immunocompetent model of lung cancer. Although PGs in the TME have been well studied, less is known regarding 5-LO products produced by the TME. We examined the role of 5-LO in the TME using a model in which Lewis lung carcinoma cells are directly implanted into the lungs of syngeneic WT mice or mice globally deficient in 5-LO (5-LO-KO). Unexpectedly, primary tumor volume and liver metastases were increased in 5-LO-KO mice. This was associated with an ablation of leukotriene (LT) production, consistent with production mainly mediated by the microenvironment. Increased tumor progression was partially reproduced in global LTC4 synthase KO or mice transplanted with LTA4 hydrolase-deficient bone marrow. Tumor-bearing lungs of 5-LO-KO had decreased numbers of CD4 and CD8 T cells compared with WT controls, as well as fewer dendritic cells. This was associated with lower levels of CCL20 and CXL9, which have been implicated in dendritic and T cell recruitment. Depletion of CD8 cells increased tumor growth and eliminated the differences between WT and 5-LO mice. These data reveal an antitumorigenic role for 5-LO products in the microenvironment during lung cancer progression through regulation of T cells and suggest that caution should be used in targeting this pathway in lung cancer.

Vascular smooth muscle cell-derived transforming growth factor-ß promotes maturation of activated, neointima lesion-like macrophages.

OBJECTIVE: To define the contribution of vascular smooth muscle cell (SMC)-derived factors to macrophage phenotypic modulation in the setting of vascular injury. APPROACH AND RESULTS: By flow cytometry, macrophages (M4) were the predominant myeloid cell type recruited to wire-injured femoral arteries, in mouse, compared with neutrophils or eosinophils. Recruited macrophages from injured vessels exhibited a distinct expression profile relative to circulating mononuclear cells (peripheral blood monocytes; increased: interleukin-6, interleukin-10, interleukin-12b, CC chemokine receptor [CCR]3, CCR7, tumor necrosis factor-α, inducible nitric oxide synthase, arginase 1; decreased: interleukin-12a, matrix metalloproteinase [MMP]9). This phenotype was recapitulated in vitro by maturing rat bone marrow cells in the presence of macrophage-colony stimulating factor and 20% conditioned media from cultured rat SMC (sMϕ) compared with maturation in macrophage-colony stimulating factor alone (M0). Recombinant transforming growth factor (TGF)-ß1 recapitulated the effect of SMC conditioned media. Macrophage maturation studies performed in the presence of a pan-TGF-ß neutralizing antibody, a TGF-ß receptor inhibitor, or conditioned media from TGF-ß-depleted SMCs confirmed that the SMC-derived factor responsible for macrophage activation was TGF-ß. Finally, the effect of SMC-mediated macrophage activation on SMC biology was assessed. SMCs cocultured with sMϕ exhibited increased rates of proliferation relative to SMCs cultured alone or with M0 macrophages. CONCLUSIONS: SMC-derived TGF-ß modulates the phenotype of maturing macrophages in vitro, recapitulating the phenotype found in vascular lesions in vivo. SMC-modulated macrophages induce SMC activation to a greater extent than control macrophages.

Eicosanoid profiling in an orthotopic model of lung cancer progression by mass spectrometry demonstrates selective production of leukotrienes by inflammatory cells of the microenvironment.

Eicosanoids are bioactive lipid mediators derived from arachidonic acid(1) (AA), which is released by cytosolic phospholipase A2 (cPLA2). AA is metabolized through three major pathways, cyclooxygenase (COX), lipoxygenase (LO) and cytochrome P450, to produce a family of eicosanoids, which individually have been shown to have pro- or anti-tumorigenic activities in cancer. However, cancer progression likely depends on complex changes in multiple eicosanoids produced by cancer cells and by tumor microenvironment and a systematic examination of the spectrum of eicosanoids in cancer has not been performed. We used liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to quantitate eicosanoids produced during lung tumor progression in an orthotopic immunocompetent mouse model of lung cancer, in which Lewis lung carcinoma (LLC) cells are injected into lungs of syngeneic mice. The presence of tumor increased products of both the cyclooxygenase and the lipoxygenase pathways in a time-dependent fashion. Comparing tumors grown in cPLA2 knockout vs wild-type mice, we demonstrated that prostaglandins (PGE2, PGD2 and PGF2a) were produced by both cancer cells and the tumor microenvironment (TME), but leukotriene (LTB4, LTC4, LTD4, LTE4) production required cPLA2 expression in the TME. Using flow cytometry, we recovered tumor-associated neutrophils and 2 types of tumor-associated macrophages from tumor-bearing lungs and we defined their distinct eicosanoid profiles by LC/MS/MS. The combination of flow cytometry and LC/MS/MS unravels the complexity of eicosanoid production in lung cancer and provides a rationale to develop therapeutic strategies that target select cell populations to inhibit specific classes of eicosanoids.

Activation of PPARγ in myeloid cells promotes lung cancer progression and metastasis.

Activation of peroxisome proliferator-activated receptor-γ (PPARγ) inhibits growth of cancer cells including non-small cell lung cancer (NSCLC). Clinically, use of thiazolidinediones, which are pharmacological activators of PPARγ is associated with a lower risk of developing lung cancer. However, the role of this pathway in lung cancer metastasis has not been examined well. The systemic effect of pioglitazone was examined in two models of lung cancer metastasis in immune-competent mice. In an orthotopic model, murine lung cancer cells implanted into the lungs of syngeneic mice metastasized to the liver and brain. As a second model, cancer cells injected subcutaneously metastasized to the lung. In both models systemic administration of pioglitazone increased the rate of metastasis. Examination of tissues from the orthotopic model demonstrated increased numbers of arginase I-positive macrophages in tumors from pioglitazone-treated animals. In co-culture experiments of cancer cells with bone marrow-derived macrophages, pioglitazone promoted arginase I expression in macrophages and this was dependent on the expression of PPARγ in the macrophages. To assess the contribution of PPARγ in macrophages to cancer progression, experiments were performed in bone marrow-transplanted animals receiving bone marrow from Lys-M-Cre+/PPARγ(flox/flox) mice, in which PPARγ is deleted specifically in myeloid cells (PPARγ-Mac(neg)), or control PPARγ(flox/flox) mice. In both models, mice receiving PPARγ-Mac(neg) bone marrow had a marked decrease in secondary tumors which was not significantly altered by treatment with pioglitazone. This was associated with decreased numbers of arginase I-positive cells in the lung. These data support a model in which activation of PPARγ may have opposing effects on tumor progression, with anti-tumorigenic effects on cancer cells, but pro-tumorigenic effects on cells of the microenvironment, specifically myeloid cells.

Mapping of ESE-1 subdomains required to initiate mammary epithelial cell transformation via a cytoplasmic mechanism.

BACKGROUND: The ETS family transcription factor ESE-1 is often overexpressed in human breast cancer. ESE-1 initiates transformation of MCF-12A cells via a non-transcriptional, cytoplasmic process that is mediated by a unique 40-amino acid serine and aspartic acid rich (SAR) subdomain, whereas, ESE-1's nuclear transcriptional property is required to maintain the transformed phenotype of MCF7, ZR-75-1 and T47D breast cancer cells. RESULTS: To map the minimal functional nuclear localization (NLS) and nuclear export (NES) signals, we fused in-frame putative NLS and NES motifs between GFP and the SAR domain. Using these GFP constructs as reporters of subcellular localization, we mapped a single NLS to six basic amino acids (242 HGKRRR 247) in the AT-hook and two CRM1-dependent NES motifs, one to the pointed domain (NES1: 102 LCNCALEELRL 112) and another to the DNA binding domain (DBD), (NES2: 275 LWEFIRDILI 284). Moreover, analysis of a putative NLS located in the DBD (316 GQKKKNSN 323) by a similar GFP-SAR reporter or by internal deletion of the DBD, revealed this sequence to lack NLS activity. To assess the role of NES2 in regulating ESE-1 subcellular localization and subsequent transformation potency, we site-specifically mutagenized NES2, within full-length GFP-ESE-1 and GFP-NES2-SAR reporter constructs. These studies show that site-specific mutation of NES2 completely abrogates ESE-1 transforming activity. Furthermore, we show that exclusive cytoplasmic targeting of the SAR domain is sufficient to initiate transformation, and we report that an intact SAR domain is required, since block mutagenesis reveals that an intact SAR domain is necessary to maintain its full transforming potency. Finally, using a monoclonal antibody targeting the SAR domain, we demonstrate that the SAR domain contains a region accessible for protein - protein interactions. CONCLUSIONS: These data highlight that ESE-1 contains NLS and NES signals that play a critical role in regulating its subcellular localization and function, and that an intact SAR domain mediates MEC transformation exclusively in the cytoplasm, via a novel nontranscriptional mechanism, whereby the SAR motif is accessible for ligand and/or protein interactions. These findings are significant, since they provide novel molecular insights into the functions of ETS transcription factors in mammary cell transformation.