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1.  NAFLD causes selective CD4+ T lymphocyte loss and promotes hepatocarcinogenesis 
Nature  2016;531(7593):253-257.
Hepatocellular carcinoma (HCC) is the second most common cause of cancer related death. Non-alcoholic fatty liver disease (NAFLD) affects a large proportion of the US population and is considered a metabolic predisposition to liver cancer 1-5. However, the role of adaptive immune responses in NAFLD-promoted HCC is largely unknown. Here, we show that dysregulation of lipid metabolism in NAFLD causes a selective loss of intrahepatic CD4+ but not CD8+ T lymphocytes leading to accelerated hepatocarcinogenesis. We also found that CD4+ T lymphocytes have greater mitochondrial mass than CD8+ T lymphocytes and generate higher levels of mitochondrially-derived reactive oxygen species (ROS). Disruption of mitochondrial function by linoleic acid, a fatty acid accumulated in NAFLD, causes more oxidative damage than other free fatty acids such as palmitic acid, and mediates selective loss of intrahepatic CD4+ T lymphocytes. In vivo blockade of ROS reversed NAFLD-induced hepatic CD4+ T lymphocyte decrease and delayed NAFLD-promoted HCC. Our results provide an unexpected link between lipid dysregulation and impaired anti-tumor surveillance.
PMCID: PMC4786464  PMID: 26934227
2.  CD40 dependent exacerbation of immune mediated hepatitis by hepatic CD11b+ Gr-1+ myeloid derived suppressor cells in tumor bearing mice 
European journal of immunology  2015;45(4):1148-1158.
Immunosuppressive CD11b+Gr-1+ myeloid-derived suppressor cells (MDSC) accumulate in the livers of tumor-bearing mice. We studied hepatic MDSC in two murine models of immune mediated hepatitis. Unexpectedly, treatment of tumor bearing mice with Concanavalin A or α-Galactosylceramide resulted in increased ALT and AST serum levels in comparison to tumor free mice. Adoptive transfer of hepatic MDSC into naïve mice exacerbated Concanavalin A induced liver damage. Hepatic CD11b+Gr-1+ cells revealed a polarized pro-inflammatory gene signature after Concanavalin A treatment. An interferon gamma- dependent up-regulation of CD40 on hepatic CD11b+Gr-1+ cells along with an up-regulation of CD80, CD86, and CD1d after Concanavalin A treatment was observed. Concanavalin A treatment resulted in a loss of suppressor function by tumor-induced CD11b+Gr-1+ MDSC as well as enhanced reactive oxygen species-mediated hepatotoxicity. CD40 knockdown in hepatic MDSC led to increased arginase activity upon Concanavalin A treatment and lower ALT/AST serum levels. Finally, blockade of arginase activity in Cd40−/− tumor-induced myeloid cells resulted in exacerbation of hepatitis and increased reactive oxygen species production in vivo. Our findings indicate that in a setting of acute hepatitis, tumor-induced hepatic MDSC act as pro-inflammatory immune effector cells capable of killing hepatocytes in a CD40-dependent manner.
PMCID: PMC4425346  PMID: 25616156
immune mediated hepatitis; myeloid derived suppressor cells; CD40; Reactive Oxygen Species; Concanavalin A; α-Galactosylceramide
3.  Valproic acid improves second-line regimen of small cell lung carcinoma in preclinical models 
ERJ Open Research  2015;1(2):00028-2015.
With 5-year survival rates below 5%, small cell lung carcinoma (SCLC) has very poor prognosis and requires improved therapies. Despite an excellent overall response to first-line therapy, relapses are frequent and further treatments are disappointing. The goal of the study was to improve second-line therapy of SCLC.
The effect of chemotherapeutic agents was evaluated in cell lines (apoptosis, reactive oxygen species, and RNA and protein expression) and in mouse models (tumour development).
We demonstrate here that valproic acid, a histone deacetylase inhibitor, improves the efficacy of a second-line regimen (vindesine, doxorubicin and cyclophosphamide) in SCLC cells and in mouse models.
Transcriptomic profiling integrating microRNA and mRNA data identifies key signalling pathways in the response of SCLC cells to valproic acid, opening new prospects for improved therapies.
Valproic acid improves second-line regimen of SCLC response in preclinical models
PMCID: PMC5005116  PMID: 27730151
4.  Adenoviral Based Immunotherapy Provides Local Disease Control in an Orthotopic Murine Model of Esophageal Cancer 
Despite recent advances in the development of novel therapies, esophageal carcinoma remains an aggressive cancer associated with a poor prognosis. The lack a high throughput, reproducible syngeneic animal model that replicates human disease is partly responsible for the paucity of novel therapeutic approaches. In this report, we present the first successful syngeneic, orthotopic model for esophageal cancer. This model was used to test an established adenoviral-based tumor vaccine. We utilized a murine esophageal cancer cell line established from the EDL2-cyclin D1;p53−/− mouse that was transduced to express a viral tumor antigen, the Human Papilloma Virus (HPV) E7 protein. The tumor was established in its natural microenvironment at the gastroesophageal (GE) junction. Tumor growth was consistent and reproducible. An adenoviral vaccine to E7 (Ad.E7) induced an E7-specific population of functionally active CD8+ T cells which trafficked into the tumors and retained cytotoxicity. Ad.E7 vaccination reduced local tumor growth and prolonged overall survival. These findings suggest that orthotopic tumor growth is a reasonable preclinical model to validate novel therapies.
PMCID: PMC4552447  PMID: 24810640
Surgery; Esophageal Carcinoma; Immunotherapy; Animal Models; Orthotopic Implantation; CD8+ T Lymphocytes
5.  Multifactorial T cell Hypofunction that is Reversible Can Limit the Efficacy of Chimeric Antibody Receptor-transduced Human T cells in Solid Tumors 
Immunotherapy using vaccines or adoptively transferred tumor infiltrating lymphocytes (TILs) is limited by T cell functional inactivation within the solid tumor microenvironment. The purpose of this study was to determine if a similar tumor-induced inhibition occurred with genetically-modified cytotoxic T cells expressing chimeric antibody receptors (CARs) targeting tumor-associated-antigens.
Human T cells expressing CAR targeting mesothelin or fibroblast activation protein and containing CD3ζ and 4-1BB cytoplasmic domains were intravenously injected into immunodeficient mice bearing large, established human mesothelin- expressing flank tumors. CAR TILs were isolated from tumors at various time points and evaluated for effector functions and status of inhibitory pathways.
CAR T cells were able to traffic into tumors with varying efficiency and proliferate. They were able to slow tumor growth, but did not cause regressions or cures. The CAR TILs underwent rapid loss of functional activity that limited their therapeutic efficacy. This hypofunction was reversible when the T cells were isolated away from the tumor. The cause of the hypofunction appeared to be multifactorial and was associated with upregulation of intrinsic T cell inhibitory enzymes (diacylglycerol kinase and SHP-1) and the expression of surface inhibitory receptors (PD-1, LAG3, TIM3, 2B4).
Advanced generation human CAR T cells are reversibly inactivated within the solid tumor microenvironment of some tumors by multiple mechanisms. The model described here will be an important tool for testing T cell-based strategies or systemic approaches to overcome this tumor-induced inhibition. Our results suggest that PD-1 pathway antagonism may augment human CAR T cell function.
PMCID: PMC4134701  PMID: 24919573
6.  IFN-γ regulates survival and function of tumor-induced CD11b+Gr-1high myeloid derived suppressor cells by modulating the anti-apoptotic molecule Bcl2a1 
European journal of immunology  2014;44(8):2457-2467.
Myeloid derived suppressor cells (MDSCs) play a critical role in suppression of immune responses in cancer and inflammation. Here, we describe how regulation of Bcl2a1 by cytokines controls the suppressor function of CD11b+Gr-1high granulocytic MDSCs. Co-culture of CD11b+Gr-1high granulocytic MDSCs with antigen-stimulated T cells and simultaneous blockade of IFN-γ by the use of anti-IFN-γ blocking antibody, IFN-γ−/− effector T cells, IFN-γR−/− MDSCs or STAT1−/− MDSCs led to up-regulation of Bcl2a1 in CD11b+Gr-1high cells, improved survival and enhanced their suppressor function. Molecular studies revealed that GM-CSF released by antigen-stimulated CD8+ T cells induced Bcl2a1 up-regulation, which was repressed in the presence of IFN-γ by a direct interaction of phosphorylated STAT-1 with the Bcl2a1 promotor. Bcl2a1 overexpressing granulocytic MDSCs demonstrated prolonged survival and enhanced suppressor function in vitro. Our data suggest that IFN-γ/ STAT1-dependent regulation of Bcl2a1 regulates survival and thereby suppressor function of granulocytic MDSCs.
PMCID: PMC4140991  PMID: 24810636
G-MDSC; IFN-γ; GM-CSF; Bcl2a1; vaccine; immunotherapy
7.  CD11b+Ly6G+ cells inhibit tumor growth by suppressing IL-17 production at early stages of tumorigenesis 
Oncoimmunology  2015;5(1):e1061175.
Neutrophils are important innate immune cells involved in microbial clearance at the sites of infection. However, their role in cancer development is unclear. We hypothesized that neutrophils mediate antitumor effects in early tumorigenesis. To test this, we first studied the cytotoxic effects of neutrophils in vitro. Neutrophils were cytotoxic against tumor cells, with neutrophils isolated from tumor-bearing mice trending to have increased cytotoxic activities. We then injected an ELR+ CXC chemokine-producing tumor cell line into C57BL/6 and Cxcr2−/− mice, the latter lacking the receptors for neutrophil chemokines. We observed increased tumor growth in Cxcr2−/− mice. As expected, tumors from Cxcr2−/− mice contained fewer neutrophils. Surprisingly, these tumors also contained fewer CD8+ T cells, but more IL-17-producing cells. Replenishment of functional neutrophils was correlated with decreased IL-17-producing cells, increased CD8+ T cells, and decreased tumor size in Cxcr2−/− mice, while depletion of neutrophils in C57BL/6 mice showed the opposite effects. Results from a non-ELR+ CXC chemokine producing tumor further supported that functional neutrophils indirectly mediate tumor control by suppressing IL-17A production. We further studied the correlation of IL-17A and CD8+ T cells in vitro. IL-17A suppressed proliferation and IFNγ production of CD8+ T cells, while CD11b+Ly6G+ neutrophils did not suppress CD8+ T cell function. Taken together, these data demonstrate that, while neutrophils could control tumor growth by direct cytotoxic effects, the primary mechanism by which neutrophils exert antitumor effects is to regulate IL-17 production, through which they indirectly promote CD8+ T cell responses.
PMCID: PMC4760327  PMID: 26942073
CD11b+Ly6G+Cells, Neutrophils, Tumor, IL-17 and CD8+T cells
8.  Regulation of accumulation and function of myeloid derived suppressor cells in different murine models of hepatocellular carcinoma 
Journal of hepatology  2013;59(5):1007-1013.
Background and aims
Myeloid derived suppressor cells (MDSC) are immature myeloid cells with immunosuppressive activity. They accumulate in tumor-bearing mice and humans with different types of cancer, including hepatocellular carcinoma (HCC). The aim of this study was to examine the biology of MDSC in murine HCC models and to identify a model, which mimics the human disease.
The comparative analysis of MDSC was performed in mice, bearing transplantable, diethylnitrosoamine (DEN)-induced and MYC-expressing HCC at different ages.
An accumulation of MDSC was found in mice with HCC irrespectively of the model tested. Transplantable tumors rapidly induced systemic recruitment of MDSC, in contrast to slow-growing DEN-induced or MYC-expressing HCC, where MDSC numbers only increased intra-hepatically in mice with advanced tumors. MDSC derived from mice with subcutaneous tumors were more suppressive than those from mice with DEN-induced HCC. Enhanced expression of genes associated with MDSC generation (GM-CSF, VEGF, IL-6, IL-1β) and migration (MCP-1, KC, S100A8, S100A9) was observed in mice with subcutaneous tumors. In contrast, only KC levels increased in mice with DEN-induced HCC. Both KC and GM-CSF over-expression or anti-KC and anti-GM-CSF treatment controlled MDSC frequency in mice with HCC. Finally, the frequency of MDSC decreased upon successful anti-tumor treatment with sorafenib.
Our data indicate that MDSC accumulation is a late event during hepatocarcinogenesis and differs significantly depending on the tumor model studied.
PMCID: PMC3805787  PMID: 23796475
9.  A rapid and sensitive GC-MS/MS method to measure deuterium labeled deoxyadenosine in DNA from limited mouse cell populations 
Analytical chemistry  2013;85(9):4613-4620.
A rapid and sensitive GC-MS/MS method was developed to quantitatively measure low levels of DNA base deoxyadenosine (dA) and its isotopologues (e.g. dA M+1) from limited mouse cell populations. Mice undergoing allogeneic hematopoietic transplantation (AHSCT) received deuterated water at biologically relevant time intervals post AHSCT, allowing labeling of DNA upon cell division, which was detected as the dA M+1 isotopologue. Targeted mouse cell populations were isolated from lymphoid organs and purified by multi-parameter fluorescence activated cell sorting. Cell lysis, DNA extraction and hydrolysis were accomplished using available commercial procedures. The novel analytical method utilized a hydrophilic-lipophilic balanced sample preparation, rapid on-line hot GC inlet gas phase sample derivatization, fast GC low thermal mass technology, and a recently marketed GC-MS/MS system. Calibration standards containing dA and fortified with relevant levels of dA M+1 (0.25–20%) and dA M+5 (internal standard) were used for sample quantitation. The method employed a quadratic fit for calibration of dA M+1 (0.25–20%) and dA, demonstrated excellent accuracy and precision, and had limits of detection of 100 fg on-column for the dA isotopologues. The method was validated and required only 20,000 cells to characterize population dynamics of cells involved in the biology of chronic graft-versus-host disease, the main cause of late morbidity and non-relapse-mortality following AHSCT. The high sensitivity and specificity of the method makes it useful for investigating in vivo kinetics on limited and important cell populations (e.g. T regulatory cells) from disease conditions or in disease models that are immune-mediated, such as diabetes, HIV/AIDS, arthritis, inflammatory bowel disease, and multiple sclerosis.
PMCID: PMC3696408  PMID: 23541182
10.  Inhibition of p300 impairs Foxp3+ T-regulatory cell function and promotes anti-tumor immunity 
Nature medicine  2013;19(9):1173-1177.
Foxp3+ T-regulatory (Treg) cells maintain immune homeostasis and limit autoimmunity, but can also curtail host immune responses to various types of tumors1,2. Foxp3+ Tregs are therefore considered promising targets to enhance anti-tumor immunity, and efforts are underway to develop approaches for their therapeutic modulation. However, while studies showing that Foxp3+ Treg depletion experimentally can enhance anti-tumor responses provide proof-of-principle, they lack clear translational potential and have various shortcomings. Histone/protein acetyltransferases (HATs) promote chromatin accessibility, gene transcription and the function of multiple transcription factors and non-histone proteins3,4. We now report that conditional deletion or pharmacologic inhibition of one HAT, p300 (Ep300, KAT3B), in Foxp3+ Tregs, increased TCR-induced apoptosis in Tregs, impaired Treg suppressive function and peripheral Treg induction, and limited tumor growth in immunocompetent, but not in immunodeficient, hosts. Our data thereby demonstrate that p300 is important for Foxp3+ Treg function and homeostasis in vivo and in vitro, and identify novel mechanisms by which appropriate small molecule inhibitors can diminish Treg function without overtly impairing T-effector (Teff) cell responses or inducing autoimmunity. Collectively, these data suggest a new approach for cancer immunotherapy.
PMCID: PMC3793393  PMID: 23955711
12.  4-1BB Is Superior to CD28 Costimulation for Generating CD8+ Cytotoxic Lymphocytes for Adoptive Immunotherapy1 
Artificial APCs (aAPCs) genetically modified to express selective costimulatory molecules provide a reproducible, cost-effective, and convenient method for polyclonal and Ag-specific expansion of human T cells for adoptive immunotherapy. Among the variety of aAPCs that have been studied, acellular beads expressing anti-CD3/anti-CD28 efficiently expand CD4+ cells, but not CD8+ T cells. Cell-based aAPCs can effectively expand cytolytic CD8+ cells, but optimal costimulatory signals have not been defined. 4-1BB, a costimulatory molecule expressed by a minority of resting CD8+ T cells, is transiently up-regulated by all CD8+ T cells following activation. We compared expansion of human cytolytic CD8+ T cells using cell-based aAPCs providing costimulation via 4-1BB vs CD28. Whereas anti-CD3/anti-CD28 aAPCs mostly expand naive cells, anti-CD3/4-1BBL aAPCs preferentially expand memory cells, resulting in superior enrichment of Ag-reactive T cells which recognize previously primed Ags and efficient expansion of electronically sorted CD8+ populations reactive toward viral or self-Ags. Using HLA-A2-Fc fusion proteins linked to 4-1BBL aAPCs, 3-log expansion of Ag-specific CD8+ CTL was induced over 14 days, whereas similar Ag-specific CD8+ T cell expansion did not occur using HLA-A2-Fc/anti-CD28 aAPCs. Furthermore, when compared with cytolytic T cells expanded using CD28 costimulation, CTL expanded using 4-1BB costimulation mediate enhanced cytolytic capacity due, in part, to NKG2D up-regulation. These results demonstrate that 4-1BB costimulation is essential for expanding memory CD8+ T cells ex vivo and is superior to CD28 costimulation for generating Ag-specific products for adoptive cell therapy.
PMCID: PMC3809056  PMID: 17878391
13.  Intraoperative Near-Infrared Imaging of Surgical Wounds after Tumor Resections Can Detect Residual Disease 
Surgical resection remains the most effective therapy for solid tumors worldwide. The most important prognostic indicator for cure following cancer surgery is a complete resection with no residual disease. However, intraoperative detection of retained cancer cells after surgery is challenging, and residual disease continues to be the most common cause of local failure. We hypothesized visual enhancement of tumors using near-infrared imaging could potentially identify tumor deposits in the wound after resection.
A small animal model of surgery and retained disease was developed. Residual tumor deposits in the wound were targeted using an FDA approved imaging agent, indocyanine green, by the enhanced permeability and retention (EPR) effect. A novel hand-held spectrometer was used to optically visualize retained disease after surgery.
We found residual disease using near-infrared imaging during surgery that was not visible to the naked eye or microCT. Furthermore, examination of tumor nodules was remarkably precise in delineating margins from normal surrounding tissues. This approach was most successful for tumors with increased neovasculature.
The results suggest that near-infrared examination of the surgical wound after curative resection can potentially enable the surgeon to locate residual disease. The data in this study is the basis of an ongoing Phase I/II clinical trial in patients who undergo resection for lung and breast cancer.
PMCID: PMC3482441  PMID: 22932668
intraoperative imaging; surgical oncology; infrared; margins; indocyanine green
14.  High levels of IL-7 cause dysregulation of thymocyte development 
International Immunology  2012;24(10):661-671.
IL-7 signaling is required for thymocyte development and its loss has a severe deleterious effect on thymus function. Thymocyte–stromal cell interactions and other mechanisms tightly regulate IL-7 expression. We show that disruption of that regulation by over-expression of IL-7 inhibits T-cell development and promotes extensive B-cell lymphopoiesis in the thymus. Our data reveal that high levels of IL-7 negate Notch-1 function in thymocytes found in IL-7 transgenic mice and in co-culture with OP9-DL1 cells. While high levels of IL-7R are present on thymocytes, increased suppressor of cytokine signaling-1 expression blunts IL-7 downstream signaling, resulting in hypo-phosphorylation of proteins in the PI3K-Akt pathway. Consequently, GSK3β remains active and inhibits Notch-1 signaling as observed by decreased Hes-1 and Deltex expression in thymic progenitors. This is the first demonstration that high levels of IL-7 antagonize Notch-1 signaling and suggest that IL-7 may affect T- versus B-lineage choice in the thymus.
PMCID: PMC3530313  PMID: 22899673
CD127; IL-7; IL-7R; Notch-1; thymus
15.  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.
PMCID: PMC3606205  PMID: 22617772
Surgical model; adjuvant therapy; cancer recurrence; immunotherapy; oncology; tumor microenvironment
16.  Expression of a Functional CCR2 Receptor Enhances Tumor Localization and Tumor Eradication by Retargeted Human T Cells Expressing a Mesothelin - Specific Chimeric Antibody Receptor 
Adoptive T cell immunotherapy (ACT) with tumor infiltrating lymphocytes or genetically-modified T cells has yielded dramatic results in some cancers. However, T cells need to traffic properly into tumors in order to adequately exert therapeutic effects.
Experimental Design
The chemokine CCL2 was highly secreted by malignant pleural mesotheliomas (MPM) (a planned tumor target), but the corresponding chemokine receptor (CCR2) was minimally expressed on activated human T cells transduced with a chimeric antibody receptor (CAR) directed to the MPM tumor antigen mesothelin (mesoCAR T cells). The chemokine receptor CCR2b was thus transduced into mesoCAR T cells using a lentiviral vector and the modified T cells were used to treat established mesothelin-expressing tumors.
CCR2b transduction led to CCL2-induced calcium flux and increased transmigration, as well as augmentation of in vitro T cell killing ability. A single intravenous injection of 20 million mesoCAR + CCR2b T cells into immunodeficient mice bearing large, established tumors (without any adjunct therapy) resulted in a 12.5-fold increase in T cell tumor infiltration by Day 5 compared to mesoCAR T cells. This was associated with significantly increased anti-tumor activity.
CAR T cells bearing a functional chemokine receptor can overcome the inadequate tumor localization that limits conventional CAR targeting strategies and can significantly improve anti-tumor efficacy in vivo.
PMCID: PMC3612507  PMID: 21610146
17.  The HDAC inhibitor panobinostat (LBH589) inhibits mesothelioma and lung cancer cells in vitro and in vivo with particular efficacy for small cell lung cancer 
Molecular cancer therapeutics  2009;8(8):2221-2231.
Lung cancer is the leading cause of cancer deaths in the United States. Current therapies are inadequate. Histone deacetylase inhibitors (HDACi) are a recently developed class of anticancer agents that cause increased acetylation of core histones and nonhistone proteins leading to modulation of gene expression and protein activity involved in cancer cell growth and survival pathways. We examined the efficacy of the HDACi panobinostat (LBH589) in a wide range of lung cancers and mesotheliomas. Panobinostat was cytotoxic in almost all 37 cancer cell lines tested. IC50 and LD50 values were in the low nmol/L range (4–470 nmol/L; median, 20 nmol/L). Small cell lung cancer (SCLC) cell lines were among the most sensitive lines, with LD50 values consistently <25 nmol/L. In lung cancer and mesothelioma animal models, panobinostat significantly decreased tumor growth by an average of 62% when compared with vehicle control. Panobinostat was equally effective in immunocompetent and severe combined immunodeficiency mice, indicating that the inhibition of tumor growth by panobinostat was not due to direct immunologic effects. Panobinostat was, however, particularly effective in SCLC xenografts, and the addition of the chemotherapy agent etoposide augmented antitumor effects. Protein analysis of treated tumor biopsies revealed elevated amounts of cell cycle regulators such as p21 and proapoptosis factors, such as caspase 3 and 7 and cleaved poly[ADP-ribose] polymerase, coupled with decreased levels of antiapoptotic factors such as Bcl-2 and Bcl-XL. These studies together suggest that panobinostat may be a useful adjunct in the treatment of thoracic malignancies, especially SCLC.
PMCID: PMC3605895  PMID: 19671764
18.  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.
PMCID: PMC3191304  PMID: 21819972
MAPK; post-transcriptional regulation; TNFα; DMXAA; proinflammatory cytokines
19.  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.
PMCID: PMC3265219  PMID: 21148741
innate immunity; interferon; influenza; pneumonia; bronchial epithelium
20.  Cytoreduction surgery reduces systemic myeloid suppressor cell populations and restores intratumoral immunotherapy effectiveness 
Multiple immunotherapy approaches have improved adaptive anti-tumor immune responses in patients with early stage disease; however, results have been less dramatic when treating patients with late stage disease. These blunted responses are likely due to a host of factors, including changes in the tumor microenvironment and systemic immunosuppressive features, which accompany advanced tumor states. We hypothesized that cytoreductive surgery could control these immunosuppressive networks and restore the potency of immunotherapy in advanced disease scenarios.
To test these hypotheses, two representative intratumoral immunotherapies (an adenoviral vector encoding a suicide gene, AdV-tk, or a type-I interferon, Ad.IFNα) were tested in murine models of lung cancer. Cytoreductive surgery was performed following treatment of advanced tumors. Mechanistic underpinnings were investigated using flow cytometry, in vivo leukocyte depletion methods and in vivo tumor neutralization assays.
AdV-tk and Ad.IFNα were effective in treating early lung cancers, but had little anti-tumor effects in late stage cancers. Interestingly, in late stage scenarios, surgical cytoreduction unmasked the anti-tumor potency of both immunotherapeutic approaches. Immune mechanisms that explained restoration in anti-tumor immune responses included increased CD8 T-cell trafficking and reduced myeloid derived suppressor cell populations.
This study demonstrates that surgical resection combined with immunotherapy may be a rational therapeutic option for patients with advanced stage cancer.
PMCID: PMC3418164  PMID: 22742411
Surgical oncology; Immunotherapy; Cancer; Animal model
21.  Characterization of surgical models of postoperative tumor recurrence for preclinical adjuvant therapy assessment 
Nearly 30% of cancer patients undergoing curative surgery succumb to distant recurrent disease. Despite large implications and known differences between primary and recurrent tumors, preclinical adjuvant therapy evaluation frequently occurs only in primary tumors and not recurrent tumors. We hypothesized that well characterized and reproducible models of postoperative systemic recurrences should be used for preclinical evaluation of adjuvant approaches.
Experimental Design
We examined traditional animal models of cancer surgery that generate systemic cancer recurrences. We also investigated models of systemic cancer recurrences that incorporate spontaneously metastatic cell lines and surgical resection. For each model, we critiqued feasibility, reproducibility and similarity to human recurrence biology. Using our novel model, we then tested the adjuvant use of a novel systemic inhibitor of TGF-β, 1D11.
Traditional surgical models are confounded by immunologic factors including concomitant immunity and perioperative immunosuppression. A superior preclinical model of postoperative systemic recurrences incorporates spontaneously metastatic cell lines and primary tumor excision. This approach is biologically relevant and readily feasible. Using this model, we discovered that “perioperative” TGF-β blockade has strong anti-tumor effects in the setting of advanced disease that would not be appreciated in primary tumor cell lines or other surgical models.
There are multiple immunologic effects that rendered previous models of postoperative cancer recurrences inadequate. Use of spontaneously metastatic cell lines followed by surgical resection eliminates these confounders, and best resembles the clinical scenario. This preclinical model provides more reliable preclinical information when evaluating new adjuvant therapies.
PMCID: PMC3353530  PMID: 22611473
Surgery; recurrence; models; surgical oncology; concomitant immunity; perioperative immunosuppression; TGF-β
22.  Vascular Endothelial-Targeted Therapy Combined with Cytotoxic Chemotherapy Induces Inflammatory Intratumoral Infiltrates and Inhibits Tumor Relapses after Surgery1 
Neoplasia (New York, N.Y.)  2012;14(4):352-359.
Surgery is the most effective therapy for cancer in the United States, but disease still recurs in more than 40% of patients within 5 years after resection. Chemotherapy is given postoperatively to prevent relapses; however, this approach has had marginal success. After surgery, recurrent tumors depend on rapid neovascular proliferation to deliver nutrients and oxygen. Phosphatidylserine (PS) is exposed on the vascular endothelial cells in the tumor microenvironment but is notably absent on blood vessels in normal tissues. Thus, PS is an attractive target for cancer therapy after surgery. Syngeneic mice bearing TC1 lung cancer tumors were treated with mch1N11 (a novel mouse chimeric monoclonal antibody that targets PS), cisplatin (cis), or combination after surgery. Tumor relapses and disease progression were decreased 90% by combination therapy compared with a 50% response rate for cis alone (P = .02). Mice receiving postoperative mch1N11 had no wound-related complications or added systemic toxicity in comparison to control animals. Mechanistic studies demonstrated that the effects of mch1N11 were associated with a dense infiltration of inflammatory cells, particularly granulocytes. This strategy was independent of the adaptive immune system. Together, these data suggest that vascular-targeted strategies directed against exposed PS may be a powerful adjunct to postoperative chemotherapy in preventing relapses after cancer surgery.
PMCID: PMC3349261  PMID: 22577350
23.  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.
PMCID: PMC3279406  PMID: 22348096
24.  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.
PMCID: PMC3049234  PMID: 20395632
tumor immunology; CCL2; lung cancer; mesothelioma; tumor-associated macrophages
25.  The PDL1-PD1 Axis Converts Human Th1 Cells Into Regulatory T Cells 
Science translational medicine  2011;3(111):111ra120.
Immune surveillance by T helper type 1 (Th1) cells is critical for the host response to tumors and infection, but also contributes to autoimmunity and graft-versus-host disease (GvHD) after transplantation. The inhibitory molecule programmed death ligand-1 (PDL1) has been shown to anergize human Th1 cells, but other mechanisms of PDL1-mediated Th1 inhibition such as the conversion of Th1 cells to a regulatory phenotype have not been well characterized. We hypothesized that PDL1 may cause Th1 cells to manifest differentiation plasticity. Conventional T cells or irradiated K562 myeloid tumor cells overexpressing PDL1 converted TBET+ Th1 cells into FOXP3+ regulatory T cells (TREGS) in vivo, thereby preventing human-into-mouse xenogeneic GvHD (xGvHD). Either blocking PD1 expression on Th1 cells by siRNA targeting or abrogation of PD1 signaling by SHP1/2 pharmacologic inhibition stabilized Th1 cell differentiation during PDL1 challenge and restored the capacity of Th1 cells to mediate lethal xGVHD. PD1 signaling therefore induces human Th1 cells to manifest in vivo plasticity, resulting in a TREG phenotype that severely impairs cell-mediated immunity. Converting human Th1 cells to a regulatory phenotype with PD1 signaling provides a potential way to block GvHD after transplantation. Moreover, because this conversion can be prevented by blocking PD1 expression or pharmacologically inhibiting SHP1/2, this pathway provides a new therapeutic direction for enhancing T cell immunity to cancer and infection.
PMCID: PMC3235958  PMID: 22133721

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