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Although the lung is the most common metastatic site for cancer cells, biologic mechanisms regulating lung metastasis are not fully understood. Using heterotopic and intravenous injection models of lung metastasis in mice, we found that IL5, a cytokine involved in allergic and infectious diseases, facilitates metastatic colonization through recruitment of sentinel eosinophils and regulation of other inflammatory/immune cells in the microenvironment of the distal lung. Genetic IL5 deficiency offered marked protection of the lungs from metastasis of different types of tumor cells, including lung cancer, melanoma, and colon cancer. IL5 neutralization protected subjects from metastasis, whereas IL5 reconstitution or adoptive transfer of eosinophils into IL5-deficient mice exerted prometastatic effects. However, IL5 deficiency did not affect the growth of the primary tumor or the size of metastatic lesions. Mechanistic investigations revealed that eosinophils produce CCL22, which recruits regulatory T cells to the lungs. During early stages of metastasis, Treg created a protumorigenic microenvironment, potentially by suppressing IFNγ-producing natural killer cells and M1-polarized macrophages. Together, our results establish a network of allergic inflammatory circuitry that can be co-opted by metastatic cancer cells to facilitate lung colonization, suggesting interventions to target this pathway may offer therapeutic benefits to prevent or treat lung metastasis.
©2015 American Association for Cancer Research.
There is growing evidence that generation of adenosine from ATP, which is mediated by the CD39/CD73 enzyme pair, predetermines immunosuppressive and proangiogenic properties of myeloid cells. We have previously shown that the deletion of the TGF-β type II receptor gene (Tgfbr2) expression in myeloid cells is associated with decreased tumor growth, suggesting protumorigenic effect of TGF-β signaling. In this study, we tested the hypothesis that TGF-β drives differentiation of myeloid-derived suppressor cells into protumorigenic terminally differentiated myeloid mononuclear cells (TDMMCs) characterized by high levels of cell-surface CD39/CD73 expression. We found that TDMMCs represent a major cell subpopulation expressing high levels of both CD39 and CD73 in the tumor microenvironment. In tumors isolated from mice with spontaneous tumor formation of mammary gland and conditional deletion of the type II TGF-β receptor in mammary epithelium, an increased level of TGF-β protein was associated with further increase in number of CD39(+)CD73(+) TDMMCs compared with MMTV-PyMT/TGFβRII(WT) control tumors with intact TGF-β signaling. Using genetic and pharmacological approaches, we demonstrated that the TGF-β signaling mediates maturation of myeloid-derived suppressor cells into TDMMCs with high levels of cell surface CD39/CD73 expression and adenosine-generating capacity. Disruption of TGF-β signaling in myeloid cells resulted in decreased accumulation of TDMMCs, expressing CD39 and CD73, and was accompanied by increased infiltration of T lymphocytes, reduced density of blood vessels, and diminished progression of both Lewis lung carcinoma and spontaneous mammary carcinomas. We propose that TGF-β signaling can directly induce the generation of CD39(+)CD73(+) TDMMCs, thus contributing to the immunosuppressive, proangiogenic, and tumor-promoting effects of this pleiotropic effector in the tumor microenvironment.
Copyright © 2014 by The American Association of Immunologists, Inc.
The applicability of a HVGGSSV peptide targeted "nanosponge" drug delivery system for sequential administration of a microtubule inhibitor (paclitaxel) and topoisomerase I inhibitor (camptothecin) was investigated in a lung cancer model. Schedule-dependent combination treatment with nanoparticle paclitaxel (NP PTX) and camptothecin (NP CPT) was studied in vitro using flow cytometry and confocal imaging to analyze changes in cell cycle, microtubule morphology, apoptosis, and cell proliferation. Results showed significant G2/M phase cell cycle arrest, changes in microtubule dynamics that produced increased apoptotic cell death and decreased proliferation with initial exposure to NP PTX, followed by NP CPT in lung cancer cells. In vivo molecular imaging and TEM studies validated HVGGSSV-NP tumor binding at 24 h and confirmed the presence of Nanogold labeled HVGGSSV-NPs in tumor microvascular endothelial cells. Therapeutic efficacy studies conducted with sequential HVGGSSV targeted NP PTX and NP CPT showed 2-fold greater tumor growth delay in combination versus monotherapy treated groups, and 4-fold greater delay compared to untargeted and systemic drug controls. Analytical HPLC/MS methods were used to quantify drug content in tumor tissues at various time points, with significant paclitaxel and camptothecin levels in tumors 2 days postinjection and continued presence of both drugs up to 23 days postinjection. The efficacy of the NP delivery system in sequential treatments was corroborated in both in vitro and in vivo lung cancer models showing increased G2/M phase arrest and microtubule disruption, resulting in enhanced apoptotic cell death, decreased cell proliferation and vascular density.
TNF-α, produced by most malignant cells, orchestrates the interplay between malignant cells and myeloid cells, which have been linked to tumor growth and metastasis. Although TNF-α can exist as one of two isoforms, a 26-kDa membrane tethered form (mTNF-α) or a soluble 17-kDa cytokine (sTNF-α), the vast majority of published studies have only investigated the biologic effects of the soluble form. We show for the first time that membrane and soluble isoforms have diametrically opposing effects on both tumor growth and myeloid content. Mouse lung and melanoma tumor lines expressing mTNF-α generated small tumors devoid of monocytes versus respective control lines or lines expressing sTNF-α. The lack of myeloid cells was due to a direct effect of mTNF-α on myeloid survival via induction of cell necrosis by increasing reactive oxygen species. Human non-small cell lung carcinoma expressed varying levels of both soluble and membrane TNF-α, and gene expression patterns favoring mTNF-α were predictive of improved lung cancer survival. These data suggest that there are significant differences in the role of different TNF-α isoforms in tumor progression and the bioavailability of each isoform may distinctly regulate tumor progression. This insight is critical for effective intervention in cancer therapy with the available TNF-α inhibitors, which can block both TNF-α isoforms.
Extracellular adenosine and purine nucleotides are elevated in many pathological situations associated with the expansion of CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSCs). Therefore, we tested whether adenosinergic pathways play a role in MDSC expansion and functions. We found that A(2B) adenosine receptors on hematopoietic cells play an important role in accumulation of intratumoral CD11b(+)Gr1(high) cells in a mouse Lewis lung carcinoma model in vivo and demonstrated that these receptors promote preferential expansion of the granulocytic CD11b(+)Gr1(high) subset of MDSCs in vitro. Flow cytometry analysis of MDSCs generated from mouse hematopoietic progenitor cells revealed that the CD11b(+)Gr-1(high) subset had the highest levels of CD73 (ecto-5'-nucleotidase) expression (Δmean fluorescence intensity [MFI] of 118.5 ± 16.8), followed by CD11b(+)Gr-1(int) (ΔMFI of 57.9 ± 6.8) and CD11b(+)Gr-1(-/low) (ΔMFI of 12.4 ± 1.0) subsets. Even lower levels of CD73 expression were found on Lewis lung carcinoma tumor cells (ΔMFI of 3.2 ± 0.2). The high levels of CD73 expression in granulocytic CD11b(+)Gr-1(high) cells correlated with high levels of ecto-5'-nucleotidase enzymatic activity. We further demonstrated that the ability of granulocytic MDSCs to suppress CD3/CD28-induced T cell proliferation was significantly facilitated in the presence of the ecto-5'-nucleotidase substrate 5'-AMP. We propose that generation of adenosine by CD73 expressed at high levels on granulocytic MDSCs may promote their expansion and facilitate their immunosuppressive activity.
BACKGROUND - Lung cancer and glioblastoma multiforme are highly angiogenic and, despite advances in treatment, remain resistant to therapy. Cytosolic phospholipase A2 (cPLA(2)) activation contributes to treatment resistance through transduction of prosurvival signals. We investigated cPLA(2) as a novel molecular target for antiangiogenesis therapy.
METHODS - Glioblastoma (GL261) and Lewis lung carcinoma (LLC) heterotopic tumor models were used to study the effects of cPLA(2) expression on tumor growth and vascularity in C57/BL6 mice wild type for (cPLA(2)α(+/+)) or deficient in (cPLA(2)α(-/-)) cPLA(2)α, the predominant isoform in endothelium (n = 6-7 mice per group). The effect of inhibiting cPLA(2) activity on GL261 and LLC tumor growth was studied in mice treated with the chemical cPLA(2) inhibitor 4-[2-[5-chloro-1-(diphenylmethyl)-2-methyl-1H-indol-3-yl]-ethoxy]benzoic acid (CDIBA). Endothelial cell proliferation and function were evaluated by Ki-67 immunofluorescence and migration assays in primary cultures of murine pulmonary microvascular endothelial cells (MPMEC) isolated from cPLA(2)α(+/+) and cPLA(2)α(-/-) mice. Proliferation, invasive migration, and tubule formation were assayed in mouse vascular endothelial 3B-11 cells treated with CDIBA. Effects of lysophosphatidylcholine, arachidonic acid, and lysophosphatidic acid (lipid mediators of tumorigenesis and angiogenesis) on proliferation and migration were examined in 3B-11 cells and cPLA(2)α(-/-) MPMEC. All statistical tests were two-sided.
RESULTS - GL261 tumor progression proceeded normally in cPLA(2)α(+/+) mice, whereas no GL261 tumors formed in cPLA(2)α(-/-) mice. In the LLC tumor model, spontaneous tumor regression was observed in 50% of cPLA(2)α(-/-) mice. Immunohistochemical examination of the remaining tumors from cPLA(2)α(-/-) mice revealed attenuated vascularity (P ≤ .001) compared with tumors from cPLA(2)α(+/+) mice. Inhibition of cPLA(2) activity by CDIBA resulted in a delay in tumor growth (eg, LLC model: average number of days to reach tumor volume of 700 mm(3), CDIBA vs vehicle: 16.8 vs 11.8, difference = 5, 95% confidence interval = 3.6 to 6.4, P = .04) and a decrease in tumor size (eg, GL261 model: mean volume on day 21, CDIBA vs vehicle: 40.1 vs 247.4 mm(3), difference = 207.3 mm(3), 95% confidence interval = 20.9 to 293.7 mm(3), P = .021). cPLA(2) deficiency statistically significantly reduced MPMEC proliferation and invasive migration (P = .002 and P = .004, respectively). Compared with untreated cells, cPLA(2)α(-/-) MPMEC treated with lysophosphatidylcholine and lysophosphatidic acid displayed increased cell proliferation (P = .011) and invasive migration (P < .001).
CONCLUSIONS - In these mouse models of brain and lung cancer, cPLA(2) and lysophospholipids have key regulatory roles in tumor angiogenesis. cPLA(2) inhibition may be a novel effective antiangiogenic therapy.
RATIONALE - 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.
OBJECTIVES - To determine the role of IL-5 in MPE formation.
METHODS - 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.
MEASUREMENTS AND MAIN RESULTS - 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.
CONCLUSIONS - Host-derived IL-5 promotes experimental MPE and may be involved in the pathogenesis of human MPE.
Endorepellin, the C-terminal domain of perlecan, is a powerful angiogenesis inhibitor. To dissect the mechanism of endorepellin-mediated endothelial silencing, we used an antibody array against multiple tyrosine kinase receptors. Endorepellin caused a widespread reduction in phosphorylation of key receptors involved in angiogenesis and a concurrent increase in phosphatase activity in endothelial cells and tumor xenografts. These effects were efficiently hampered by function-blocking antibodies against integrin alpha2beta1, the functional endorepellin receptor. The Src homology-2 protein phosphatase-1 (SHP-1) coprecipitated with integrin alpha2 and was phosphorylated in a dynamic fashion after endorepellin stimulation. Genetic evidence was provided by lack of an endorepellin-evoked phosphatase response in microvascular endothelial cells derived from integrin alpha2beta1(-/-) mice and by response to endorepellin in cells genetically engineered to express the alpha2beta1 integrin, but not in cells either lacking this receptor or expressing a chimera harboring the integrin alpha2 ectodomain fused to the alpha1 intracellular domain. siRNA-mediated knockdown of integrin alpha2 caused a dose-dependent reduction of SHP-1. Finally, the levels of SHP-1 and its enzymatic activity were substantially reduced in multiple organs from alpha2beta1(-/-) mice. Our results show that SHP-1 is an essential mediator of endorepellin activity and discover a novel functional interaction between the integrin alpha2 subunit and SHP-1.
Vav guanine nucleotide exchange factors modulate changes in cytoskeletal organization through activation of Rho, Rac, and Cdc42 small GTPases. Although Vav1 expression is restricted to the immune system, Vav2 and Vav3 are expressed in several tissues, including highly vascularized organs. Here, we provide the first evidence that Vav2 and Vav3 function within the tumor microenvironment to promote tumor growth, survival, and neovascularization. Host Vav2/3 deficiency reduced microvascular density, as well as tumor growth and/or survival, in transplanted B16 melanoma and Lewis lung carcinoma models in vivo. These defects were due in part to Vav2/3 deficiency in endothelial cells. Vav2/3-deficient endothelial cells displayed reduced migration in response to tumor cells in coculture migration assays, and failed to incorporate into tumor vessels and enhance tumor volume in tumor-endothelial cotransplantation experiments. These data suggest that Vav2/3 guanine nucleotide exchange factors play a critical role in host-mediated tumor progression and angiogenesis, particularly in tumor endothelium.
PURPOSE - In vascular endothelial cells, low doses of ionizing radiation trigger the immediate activation of cytosolic phospholipase A2 (cPLA2). This event initiates prosurvival signaling that could be responsible for radioresistance of tumor vasculature. Thus, the development of radiosensitizers targeting these survival pathways may enhance tumor response to radiation therapy. Arachidonyltrifluoromethyl Ketone (AACOCF3), a specific cPLA2 inhibitor, was studied as a potential radiosensitizer.
EXPERIMENTAL DESIGN - Vascular endothelial cells (3B11 and MPMEC) and lung tumor cells (LLC and H460) were treated with 1 micromol/L AACOCF3 for 30 minutes prior to irradiation. Treatment response was evaluated by clonogenic survival, activation of extracellular signal-regulated kinase 1/2 (ERK1/2), tubule formation, and migration assays. For in vivo experiments, mice with LLC or H460 tumors in the hind limbs were treated for 5 consecutive days with 10 mg/kg AACOCF3 administered daily 30 minutes prior to irradiation. Treatment response was assessed by tumor growth delay, Power Doppler Sonography, and immunohistochemistry.
RESULTS - In cell culture experiments, inhibition of cPLA2 with AACOCF3 prevented radiation-induced activation of ERK1/2 and decreased clonogenic survival of irradiated vascular endothelial cells but not the lung tumor cells. Treatment with AACOCF3 also attenuated tubule formation and migration in irradiated vascular endothelial cells. In both tumor mouse models, treatment with AACOCF3 prior to irradiation significantly suppressed tumor growth and decreased overall tumor blood flow and vascularity. Increased apoptosis in both tumor cells and tumor vascular endothelium was determined as a possible mechanism of the observed effect.
CONCLUSION - These findings identify cPLA2 as a novel molecular target for tumor sensitization to radiation therapy through the tumor vasculature.