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1.  A positive-margin resection model recreates the postsurgical tumor microenvironment and is a reliable model for adjuvant therapy evaluation 
Cancer Biology & Therapy  2012;13(9):745-755.
Up to 30% of cancer patients undergoing curative surgery develop local recurrences due to positive margins. Patients typically receive adjuvant chemotherapy, immunotherapy and/or radiation to prevent such relapses. Interestingly, evidence supporting these therapies is traditionally derived in animal models of primary tumors, thus failing to consider surgically induced tumor microenvironment changes that may influence adjuvant therapy efficacy. To address this consideration, we characterized a murine model of local cancer recurrence. This model was reproducible and generated a postoperative inflammatory tumor microenvironment that resembles those observed following human cancer surgery. To further validate this model, antagonists of two pro-inflammatory mediators, TGFβ and COX-2, were tested and found to be effective in decreasing the growth of recurrent tumors. We appreciated that preoperative TGFβ inhibition led to wound dehiscence, while postoperative initiation of COX-2 inhibition resulted in a loss of efficacy. In summary, although not an exact replica of all human cancer surgeries, our proposed local recurrence approach provides a biologically relevant and reliable model useful for preclinical evaluation of novel adjuvant therapies. The use of this model yields results that may be overlooked using traditional preclinical cancer models that fail to incorporate a surgical component.
doi:10.4161/cbt.20557
PMCID: PMC3606205  PMID: 22617772
Surgical model; adjuvant therapy; cancer recurrence; immunotherapy; oncology; tumor microenvironment
2.  Activation of Mitogen-Activated Protein Kinases by 5, 6-Dimethylxanthenone-4-Acetic Acid (DMXAA) Plays an Important Role in Macrophage Stimulation 
Biochemical pharmacology  2011;82(9):1175-1185.
The small molecule anti-tumor agent, 5, 6-dimethylxanthenone-4-acetic acid (DMXAA, now called Vadimezan) is a potent macrophage and dendritic cell activating agent that, in the murine system, results in the release of large amounts of cytokines and chemokines. The mechanisms by which this release is mediated have not been fully elucidated. The mitogen-activated protein kinase (MAPK) pathways plays an important role in the regulation of proinflammatory cytokines, such as, TNFα, IL-1β, as well as the responses to extracellular stimuli, such as, lipopolysaccharide (LPS). The results of this study demonstrate that DMXAA activates three members of mitogen-activated protein kinase (MAPK) superfamily, namely p38 MAPK, extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), and c-Jun N-terminal kinases (JNKs) via a RIP2-independent mechanism in murine macrophages. By using selective inhibitors of MAPKs, this study confirms that both activated p38/MK2 pathways and ERK1/2 MAPK play a significant role in regulation of both TNF-α and IL-6 protein production induced by DMXAA at the post-transcriptional level. Our findings also show that Interferon-γ priming can dramatically augment TNF-α protein secretion induced by DMXAA through enhancing activation of multiple MAPKs pathways at the post-transcriptional level. This study expands current knowledge on mechanisms of how DMXAA acts as a potent anti-tumor agent in murine system and also provides useful information for further study on the mechanism of action of this potential anti-tumor compound in human macrophages.
doi:10.1016/j.bcp.2011.07.086
PMCID: PMC3191304  PMID: 21819972
MAPK; post-transcriptional regulation; TNFα; DMXAA; proinflammatory cytokines
3.  Pharmacologic Activation of the Innate Immune System to Prevent Respiratory Viral Infections 
Drugs that can rapidly inhibit respiratory infection from influenza or other respiratory pathogens are needed. One approach is to engage primary innate immune defenses against viral infection, such as activating the IFN pathway. In this study, we report that a small, cell-permeable compound called 5,6-di-methylxanthenone-4-acetic acid (DMXAA) can induce protection against vesicular stomatitis virus in vitro and H1N1 influenza A virus in vitro and in vivo through innate immune activation. Using the mouse C10 bronchial epithelial cell line and primary cultures of nasal epithelial cells, we demonstrate DMXAA activates the IFN regulatory factor-3 pathway leading to production of IFN-β and subsequent high-level induction of IFN-β–dependent proteins, such as myxovirus resistance 1 (Mx1) and 2′,5′-oligoadenylate synthetase 1 (OAS1). Mice treated with DMXAA intranasally elevate mRNA/protein expression of Mx1 and OAS1 in the nasal mucosa, trachea, and lung. When challenged intranasally with a lethal dose of H1N1 influenza A virus, DMXAA reduced viral titers in the lungs and protected 80% of mice from death, even when given at 24 hours before infection. These data show that agents, like DMXAA, that can directly activate innate immune pathways, such as the IFN regulatory factor-3/IFN-β system, in respiratory epithelial cells can be used to protect from influenza pneumonia and potentially in other respiratory viral infections. Development of this approach in humans could be valuable for protecting health care professionals and “first responders” in the early stages of viral pandemics or bioterror attacks.
doi:10.1165/rcmb.2010-0288OC
PMCID: PMC3265219  PMID: 21148741
innate immunity; interferon; influenza; pneumonia; bronchial epithelium
4.  Transcriptomic Analysis Comparing Tumor-Associated Neutrophils with Granulocytic Myeloid-Derived Suppressor Cells and Normal Neutrophils 
PLoS ONE  2012;7(2):e31524.
The role of myeloid cells in supporting cancer growth is well established. Most work has focused on myeloid-derived suppressor cells (MDSC) that accumulate in tumor-bearing animals, but tumor-associated neutrophils (TAN) are also known to be capable of augmenting tumor growth. However, little is known about their evolution, phenotype, and relationship to naïve neutrophils (NN) and to the granulocytic fraction of MDSC (G-MDSC).
In the current study, a transcriptomics approach was used in mice to compare these cell types. Our data show that the three populations of neutrophils are significantly different in their mRNA profiles with NN and G-MDSC being more closely related to each other than to TAN. Structural genes and genes related to cell-cytotoxicity (i.e. respiratory burst) were significantly down-regulated in TAN. In contrast, many immune-related genes and pathways, including genes related to the antigen presenting complex (e.g. all six MHC-II complex genes), and cytokines (e.g. TNF-α, IL-1-α/β), were up-regulated in G-MDSC, and further up-regulated in TAN. Thirteen of the 25 chemokines tested were markedly up-regulated in TAN compared to NN, including striking up-regulation of chemoattractants for T/B-cells, neutrophils and macrophages.
This study characterizes different populations of neutrophils related to cancer, pointing out the major differences between TAN and the other neutrophil populations.
doi:10.1371/journal.pone.0031524
PMCID: PMC3279406  PMID: 22348096
5.  Monocyte Chemoattractant Protein–1 Blockade Inhibits Lung Cancer Tumor Growth by Altering Macrophage Phenotype and Activating CD8+ Cells 
The role of chemokines in the pathogenesis of lung cancer has been increasingly appreciated. Monocyte chemoattractant protein–1 (MCP-1, also known as CCL2) is secreted from tumor cells and associated tumor stromal cells. The blockade of CCL2, as mediated by neutralizing antibodies, was shown to reduce tumorigenesis in several solid tumors, but the role of CCL2 in lung cancer remains controversial, with evidence of both protumorigenic and antitumorigenic effects. We evaluated the effects and mechanisms of CCL2 blockade in several animal models of non–small-cell lung cancer (NSCLC). Anti-murine–CCL2 monoclonal antibodies were administered in syngeneic flank and orthotopic models of NSCLC. CCL2 blockade significantly slowed the growth of primary tumors in all models studied, and inhibited lung metastases in a model of spontaneous lung metastases of NSCLC. In contrast to expectations, no significant effect of treatment was evident in the number of tumor-associated macrophages recruited into the tumor after CCL2 blockade. However, a change occurred in the polarization of tumor-associated macrophages to a more antitumor phenotype after CCL2 blockade. This was associated with the activation of cytotoxic CD8+ T lymphocytes (CTLs). The antitumor effects of CCL2 blockade were completely lost in CB-17 severe combined immunodeficient mice or after CD8 T-cell depletion. Our data from NSCLC models show that CCL2 blockade can inhibit the tumor growth of primary and metastatic disease. The mechanisms of CCL2 blockade include an alteration of the tumor macrophage phenotype and the activation of CTLs. Our work supports further evaluation of CCL2 blockade in thoracic malignancies.
doi:10.1165/rcmb.2010-0080OC
PMCID: PMC3049234  PMID: 20395632
tumor immunology; CCL2; lung cancer; mesothelioma; tumor-associated macrophages
6.  CCL2 Blockade Augments Cancer Immunotherapy 
Cancer research  2009;70(1):109.
Since an immuno-inhibitory environment exists within tumors, successful vaccines will likely require additional approaches to alter the tumor microenvironment. Monocyte chemoattractant proteins (such as CCL2) are produced by many tumors and have both direct and indirect immuno-inhibitory effects. We hypothesized that CCL2 blockade would reduce immunosuppression and augment vaccine immunotherapy. Anti-murine-CCL2/CCL12 monoclonal antibodies were administered in three immunotherapy models: one aimed at the HPV-E7 antigen expressed by a non-small cell lung cancer line, one targeted to mesothelin expressed by a mesothelioma cell line, and one using an adenovirus expressing Interferon-α to treat a non-immunogenic, non-small cell lung cancer line. We evaluated the effect of the combination treatment on tumor growth and assessed the mechanism of these changes by evaluating cytotoxic T cells, immunosuppressive cells, and the tumor microenvironment. Administration of anti-CCL2/CCL12 antibodies along with the vaccines markedly augmented efficacy with enhanced reduction in tumor volume and cures of approximately half of the tumors. The combined treatment generated more total intra-tumoral CD8+ T-cells that were more activated and more anti-tumor antigen specific, as measured by tetramer evaluation. Another important potential mechanism was reduction in intratumoral T-regulatory (T-reg) cells. CCL2 appears to be a key proximal cytokine mediating immunosuppression in tumors. Its blockade augments CD8+ T cell immune response to tumors elicited by vaccines via multifactorial mechanisms. These observations suggest that combining CCL2 neutralization with vaccines should be considered in future immunotherapy trials.
doi:10.1158/0008-5472.CAN-09-2326
PMCID: PMC2821565  PMID: 20028856
CCL2; Cancer immunotherapy; Lung Cancer; Mesothelioma; T-lymphocytes
7.  Evaluation of an Attenuated Vesicular Stomatitis Virus Vector Expressing Interferon-β for Use in Malignant Pleural Mesothelioma: Heterogeneity in Interferon Responsiveness Defines Potential Efficacy 
Human Gene Therapy  2009;21(1):51-64.
Abstract
Vesicular stomatitis virus (VSV) has shown promise as an oncolytic agent, although unmodified VSV can be neurotoxic. To avoid toxicity, a vector was created by introducing the interferon-β (IFN-β) gene (VSV.IFN-β). We conducted this study to determine the ability of VSV.IFN-β to lyse human cancer (mesothelioma) cells and to evaluate the potential of this recombinant virus for clinical translation. Four normal human mesothelial and 12 mesothelioma cell lines were tested for their susceptibility to VSV vectors in vitro. VSV.hIFN-β did not cause cytotoxicity in any normal lines. Only 4 of 12 lines were effectively lysed by VSV.hIFN-β. In the eight resistant lines, pretreatment with IFN-β prevented lysis of cells by VSV.GFP, and VSV infection or addition of IFN-β protein resulted in the upregulation of double-stranded RNA-dependent protein kinase (PKR), myxovirus resistance A (MxA), and 2′,5′-oligo-adenylate-synthetase (2′5′-OAS) mRNA. In the susceptible lines, there was no protection by pretreatment with IFN-β protein and no IFN- or VSV-induced changes in PKR, MxA, and 2′5′-OAS mRNA. This complete lack of IFN responsiveness could be explained by marked downregulation of interferon alpha receptors (IFNARs), p48, and PKR in both the mesothelioma cell lines and primary tumor biopsies screened. Presence of p48 in three tumor samples predicted responsiveness to IFN. Our data indicate that many mesothelioma tumors have partially intact IFN pathways that may affect the efficacy of oncolytic virotherapy. However, it may be feasible to prescreen individual susceptibility to VSV.IFN-β by immunostaining for the presence of p48 protein.
doi:10.1089/hum.2009.088
PMCID: PMC2829454  PMID: 19715403
8.  Polarization of Tumor-Associated Neutrophil (TAN) Phenotype by TGF-β: “N1” versus “N2” TAN 
Cancer cell  2009;16(3):183-194.
Summary
TGF-β blockade significantly slows tumor growth through many mechanisms, including activation of CD8+ T-cells and macrophages. Here, we show that TGF-β blockade also increases neutrophil-attracting chemokines resulting in an influx of CD11b+/Ly6G+ tumor-associated neutrophils (TAN) that are hypersegmented, more cytotoxic to tumor cells, and express higher levels of pro-inflammatory cytokines. Accordingly, following TGF-β blockade, depletion of these neutrophils significantly blunts anti-tumor effects of treatment and reduces CD8+ T-cell activation. In contrast, in control tumors, neutrophil depletion decreases tumor growth and results in more activated CD8+ T-cells intra-tumorally. Together, these data suggest that TGF-β within the tumor microenvironment induces a population of TAN with a pro-tumor phenotype. TGF-β blockade results in the recruitment and activation of TAN with an anti-tumor phenotype.
doi:10.1016/j.ccr.2009.06.017
PMCID: PMC2754404  PMID: 19732719
tumor immunology; immunosuppression; TGFβ; tumor associated macrophages; Tumor associated neutrophils; lung cancer; mesothelioma
9.  Systemic Blockade of Transforming Growth Factor-β (TGF-β) Signaling Augments the Efficacy of Immunogene Therapy 
Cancer research  2008;68(24):10247-10256.
Locally-produced TGF-β promotes tumor-induced immunosuppression and contributes to resistance to immunotherapy. This paper explores the potential for increased efficacy when combining immunotherapies with TGF-β suppression using the TGF-β type I receptor kinase inhibitor, SM16. Adenovirus expressing IFNβ (Ad.IFNβ) was injected intratumorally once in established subcutaneous AB12 (mesothelioma) and LKR (lung cancer) tumors or intratracheally in a K-ras orthotopic lung tumor model. Mice bearing TC1 (lung cancer) tumors were vaccinated with two injections of adenovirus expressing HPV-E7 (Ad.E7). SM16 was administered orally in formulated chow. Tumor growth was assessed and cytokine-expression and cell populations were measured in tumors and spleens by real time-PCR and flow cytometry. SM16 potentiated the efficacy of both immunotherapies in each of the models and caused changes in the tumor microenvironment. The combination of SM16 and Ad.INFβ increased the number of intratumoral leukocytes (including macrophages, NK cells, and CD8+ cells) and increased the percentage of T-cells expressing the activation marker CD25. SM16 also augmented the anti-tumor effects of Ad.E7 in the TC1 flank tumor model. The combination did not increase HPV-E7 tetramer-positive CD8+ T cells in the spleens, but did induce a marked increase in the tumors. Tumors from SM16-treated mice showed increased mRNA and protein for immunostimulatory cytokines and chemokines, as well as endothelial adhesion molecules, suggesting a mechanism for the increased intratumoral leukocyte trafficking. Blockade of the TGF-β signaling pathway augments the anti-tumor effects of Ad.INFβ immune-activating or Ad.E7 vaccination therapy. The addition of TGF-β blocking agents in clinical trials of immunotherapies may increase efficacy.
doi:10.1158/0008-5472.CAN-08-1494
PMCID: PMC2637471  PMID: 19074893
tumor immunology; immunosuppression; TGFβ; tumor associated macrophages; cytokines; lung cancer; mesothelioma; tumor vaccine; interferon-β

Results 1-9 (9)