Search

OBJECTIVE To evaluate the therapeutic efficacy of a novel modular polymer platform in the treatment of head and neck squamous cell carcinoma (HNSCC). DESIGN In vivo study. SETTING Academic research laboratory. SUBJECTS AND METHODS C3H/HeJ mice and SCID/beige mice were randomized to receive implantation of no polymer, plain polymer, plain polymer with local cisplatin injection, or cisplatin polymer. The 2 groups of mice implanted with cisplatin polymer or no polymer were further randomized to receive 4 Gy of external beam radiation for 4 days or no radiation. Tumor size was measured until the mice were humanely killed. At necropsy, the tumors were excised and weighed. RESULTS There was a significant reduction in tumor growth using this novel polymer platform. The cisplatin-secreting polymer effectively reduced human head and neck tumor growth in SCID mice by 17-fold and SCC VII/SF tumors in C3H/HeJ mice by more than 16-fold compared with the control, plain polymer, and plain polymer + intratumoral cisplatin injection groups (P = .01 for both). We also observed a statistically significant lower tumor weight in mice treated with cisplatin polymer and concomitant radiation compared with the radiation alone and control groups. CONCLUSIONS We demonstrate the efficacy of a novel polymer platform in delivering cisplatin to a partially resected SCC in a murine model. This polymer may represent a new therapeutic modality for patients with HNSCC. Once this polymer platform is optimized, we will plan for validation in the context of a prospective trial in patients with unresectable advanced or recurrent HNSCC.

Lung cancer is the most common cause of cancer mortality worldwide for both men and women, causing approximately 1.2 million deaths per year. With the existing therapeutic efforts, the long-term survival for lung cancer patients remains low with only 15% surviving for 5 years following diagnosis. Therefore, new therapeutic strategies are needed. One such approach is the development of immune therapy for lung cancer. Immune approaches for lung cancer remain attractive because although surgery, chemotherapy and radiotherapy alone or in combination have response rates in all histological types of lung cancer, relapse is frequent. Immunologic targeting of lung cancer has the potential for nontoxic and specific therapy. Strategies that harness the immune system to react against tumors can be integrated with existing forms of therapy for optimal responses toward this devastating disease.

Lung cancer cells elaborate the immunosuppressive and antiapoptotic mediator prostaglandin E2(PGE2), a product of cyclooxygenase-2 (COX-2) enzyme activity. Because peroxisome proliferator-activated receptor (PPAR)γ ligands, such as thiazolidinediones (TZDs), inhibit lung cancer cell growth, we examined the effect of the TZDs pioglitazone and rosiglitazone on PGE2levels in non–small-cell lung cancer (NSCLC) A427 and A549 cells. Both TZDs inhibited PGE2production in NSCLC cells via a COX-2 independent pathway. To define the mechanism underlying COX-2 independent suppression of PGE2production, we focused on other enzymes responsible for the synthesis and degradation of PGE2. The expression of none of the three prostaglandin synthases (microsomal PGES1, PGES2 and cystosolic PGES) was down-regulated by the TZDs. It is noteworthy that 15-hydroxyprostaglandin dehydrogenase (15-PGDH), an enzyme that produces biologically inactive 15-ketoprostaglandins from active PGE2, was induced by TZDs. The TZD-mediated suppression of PGE2concentration was significantly inhibited by small interfering RNA to 15-PGDH. Studies using dominant-negative PPARγ overexpression or 2-chloro-5-nitrobenzanilide (GW9662; a PPARγ antagonist) revealed that the suppressive effect of the TZDs on PGE2is PPARγ-independent. Together, these findings indicate that it is possible to use a clinically available pharmacological intervention to suppress tumor-derived PGE2by enhancing catabolism rather than blocking synthesis.

Oncogenic RAS expression occurs in up to 40% of multiple myeloma (MM) cases and correlates with aggressive disease. Since activated RAS induces cyclooxygenase-2 (cox-2) expression in other tumor models, we tested a role for cox-2 in mutant RAS–containing MM cells. We used the ANBL-6 isogenic MM cell lines in which the IL-6–dependent parental line becomes cytokine independent following transfection with mutated N-RASor K-RAS. Both mutated N-RAS–and K-RAS–expressing ANBL-6 cells demonstrated a selective up-regulation of cox-2 expression and enhanced secretion of PGE2, a product of cox-2. Furthermore, in 3 primary marrow specimens, which contained MM cells expressing mutated RAS, 15% to 40% of tumor cells were positive for cox-2 expression by immunohistochemistry. We used cox-2 inhibitors, NS398 and celecoxib, and neutralizing anti-PGE2 antibody to test whether cox-2/PGE2 was involved in the aggressive phenotype of MM ANBL-6 cells containing mutated RAS. Although these interventions had no effect on IL-6–independent growth or adhesion to marrow stromal cells, they significantly inhibited the enhanced binding of mutant RAS–containing MM cells to fibronectin and the enhanced resistance to melphalan. These results indicate a selective induction of cox-2 in MM cells containing RASmutations, which results in heightened binding to extracellular matrix protein and chemotherapeutic drug resistance.

Oncogenic RAS expression occurs in up to 40% of multiple myeloma (MM) cases and correlates with aggressive disease. Since activated RAS induces cyclooxygenase-2 (cox-2) expression in other tumor models, we tested a role for cox-2 in mutant RAS–containing MM cells. We used the ANBL-6 isogenic MM cell lines in which the IL-6–dependent parental line becomes cytokine independent following transfection with mutated N-RAS or K-RAS. Both mutated N-RAS– and K-RAS–expressing ANBL-6 cells demonstrated a selective up-regulation of cox-2 expression and enhanced secretion of PGE2, a product of cox-2. Furthermore, in 3 primary marrow specimens, which contained MM cells expressing mutated RAS, 15% to 40% of tumor cells were positive for cox-2 expression by immunohistochemistry. We used cox-2 inhibitors, NS398 and celecoxib, and neutralizing anti-PGE2 antibody to test whether cox-2/PGE2 was involved in the aggressive phenotype of MM ANBL-6 cells containing mutated RAS. Although these interventions had no effect on IL-6–independent growth or adhesion to marrow stromal cells, they significantly inhibited the enhanced binding of mutant RAS– containing MM cells to fibronectin and the enhanced resistance to melphalan. These results indicate a selective induction of cox-2 in MM cells containing RAS mutations, which results in heightened binding to extracellular matrix protein and chemotherapeutic drug resistance.

Cyclooxygenase-2 (COX-2) overexpression is seen in many malignancies including lung cancer. Elevated tumor prostaglandin E2 (PGE2), a major COX-2 metabolite, levels have been implicated in angiogenesis, tumor growth and invasion, apoptosis resistance and suppression of anti-tumor immunity. Recent studies also revealed that PGE2 signaling may confer cells resistant to targeted growth factor receptor therapy by cross-activation of the receptor signaling pathway downstream components. Pre-clinical studies in animal tumor models have shown tumor reduction when animals are treated with COX-2 inhibitors and have demonstrated promising results when COX-2 inhibitors were combined with chemotherapeutic drugs. Based on these observations several ongoing clinical trials are currently evaluating COX-2 inhibitors as adjuvants with chemotherapy or radiation therapy in patients with advanced non-small cell lung cancer. Further understanding of the mechanisms of COX-2 in tumorigenesis and its interaction with other cellular pathways may highlight the new diagnostic, prognostic and therapeutic markers and facilitate future development of targeted strategies for lung cancer treatment and prevention.

Control of apoptosis is fundamental for dendritic cell (DC) homeostasis. Numerous factors maintain DC viability throughout their lifespan, including inhibitor of apoptosis proteins. Among them, survivin is overexpressed in many human malignancies, but its physiological function in normal cells has not been fully delineated. Prostaglandin E2(PGE2), also overproduced in several malignancies, has shown to induce proapoptotic and antiapoptotic effects in different cell types, including immune cells. In DC, PGE2predominantly affects maturation and modulates immune functions. Here, we show that exposure of monocyte‐derived DC to PGE2(10−5M) for 72 h significantly increased DC survivin mRNA and protein expression. In contrast, DC, matured with lipopolysaccharide or tumor necrosis factor α, did not reveal survivin induction in response to PGE2. Following exposure to apoptotic stimuli, DC treated with PGE2exhibited an overall increased viability compared with control DC, and this effect was correlated inversely with caspase‐3 activation. Moreover, PGE2‐treated, survivin‐deficient DC demonstrated reduced viability in response to apoptotic stimuli. Further analysis indicated that PGE2induced DC survivin expression in an E prostanoid (EP)2/EP4receptor and phosphatidylinositol‐3 kinase‐dependent manner. These findings suggest that PGE2‐dependent regulation of survivin is important in modulating apoptosis resistance in human DC.

Clinical lung cancer is the ultimate event resulting from a series of genetic and epigenetic alterations in the respiratory epithelium at risk. According to the "field carcinogenesis" theory, these alterations can occur throughout the entire lung. In individuals with a genetic predisposition combined with a sufficient amount of procarcinogenic environmental influences, a few of these sites may eventually progress to malignancies. Recent advances in the understanding of tumor biology have identified new therapeutic targets for lung cancer chemoprevention, among which is cyclooxgygenase (COX)–2. Ample preclinical data suggest that the COX-2/prostaglandin E2(PGE2) signaling pathway plays a pivotal role in conferring the malignant phenotype. Produced primarily by the action of COX on the free arachidonic acid liberated from membrane phospholipids, overproduction of PGE2, which is predominantly generated by upregulation of COX-2, is associated with a variety of mechanisms known to facilitate tumorigenesis. These mechanisms include abnormal expression of epithelial growth factors, epithelial and microvascular proliferation, resistance to apoptosis, and suppression of antitumor immunity. The lung is one of the major sites of PGE2production, and previous studies have shown elevated PGE2levels in bronchoalveolar lavage fluid of patients with bronchogenic carcinoma. In animal models, inhibition of COX-2 and PGE2synthesis suppresses lung tumorigenesis. These preclinical data suggesting the antineoplastic effect of COX-2 inhibitors provide the basis for several ongoing pilot clinical trials to determine the feasibility of COX-2 inhibition in chemoprevention of bronchogenic carcinoma.

The antitumor efficiency of dendritic cells transduced with an adenovirus vector expressing interleukin (IL)-7 (DC-AdIL-7) was evaluated in a murine model of spontaneous bronchoalveolar cell carcinoma. These transgenic mice (CC-10 TAg), expressing the SV40 large T antigen under the Clara cell promoter, develop bilateral multifocal pulmonary adenocarcinomas and die at 4 months as a result of progressive pulmonary tumor burden. Injection of DC-AdIL-7 in the axillary lymph node region (ALNR) weekly for 3 weeks led to a marked reduction in tumor burden with extensive lymphocytic infiltration of the tumors and enhanced survival. The antitumor responses were accompanied by the enhanced elaboration of interferon (IFN)-γ and IL-12 as well as an increase in the antiangiogenic chemokines, IFN-γ-inducible protein 10 (IP-10/CXCL10) and monokine induced by IFN-γ (MIG/CXCL9). In contrast, production of the immunosuppressive mediators IL-10, transforming growth factor (TGF)-β, prostaglandin E2 (PGE2), and the proangiogenic modulator vascular endothelial growth factor (VEGF) decreased in response to DC-AdIL-7 treatment. Significant reduction in tumor burden in a model in which tumors develop in an organ-specific manner provides a strong rationale for further evaluation of DC-AdIL-7 in regulation of tumor immunity and its use in lung cancer genetic immunotherapy.

Tumor cyclooxygenase-2 (COX-2) expression is known to be associated with enhanced tumor invasiveness. In the present study, we evaluated the importance of the COX-2 product prostaglandin E2 (PGE2) and its signaling through the EP4 receptor in mediating non-small cell lung cancer (NSCLC) invasiveness. Genetic inhibition of tumor COX-2 led to diminished matrix metalloproteinase (MMP)-2, CD44, and EP4 receptor expression and invasion. Treatment of NSCLC cells with exogenous 16,16-dimethylprostaglandin E2 significantly increased EP4 receptor, CD44, and MMP-2 expression and matrigel invasion. In contrast, anti-PGE2 decreased EP4 receptor, CD44, and MMP-2 expression in NSCLC cells. EP4 receptor signaling was found to be central to this process, because antisense oligonucleotide-mediated inhibition of tumor cell EP4 receptors significantly decreased CD44 expression. In addition, agents that increased intracellular cAMP, as is typical of EP4 receptor signaling, markedly increased CD44 expression. Moreover, MMP-2-AS treatment decreased PGE2-mediated CD44 expression, and CD44-AS treatment decreased MMP-2 expression. Thus, PGE2-mediated effects through EP4 required the parallel induction of both CD44 and MMP-2 expression because genetic inhibition of either MMP-2 or CD44 expression effectively blocked PGE2-mediated invasion in NSCLC. These findings indicate that PGE2 regulates COX-2-dependent, CD44- and MMP-2-mediated invasion in NSCLC in an autocrine/paracrine manner via EP receptor signaling. Thus, blocking PGE2 production or activity by genetic or pharmacological interventions may prove to be beneficial in chemoprevention or treatment of NSCLC.

Dendritic cells (DCs) are rare antigen-presenting cells that play a central role in stimulating immune responses. The combination of recombinant granulocyte macrophage–colony-stimulating factor (rGM-CSF) and recombinant interleukin-4 (rIL-4) provides an important stimulus for generating DCs from murine bone marrow precursors in vitro. Using miniature osmotic pumps, we now demonstrate that continuous infusion of these cytokines for 7 days had a similar effect in vivo, increasing the number and function of splenic DCs. Administration of rGM-CSF/rIL-4 (10 μg/d each) increased the concentration of CD11+ DCs by 2.7-fold and the absolute number of splenic DCs by an average of 5.7-fold. DC number also increased in peripheral blood and lymph nodes. The resultant DCs exhibited a different phenotype and function than those in control mice or mice treated with rGM-CSF alone. rGM-CSF/IL-4 increased both the myeloid (CD11c+/CD11b+) and the lymphoid (CD11c+/CD8α+) subpopulations, whereas rGM-CSF increased only myeloid DCs. DCs were highly concentrated in the T-cell areas of white pulp after rGM-CSF/IL-4 administration, whereas they were diffusely distributed throughout white pulp, marginal zones, and red pulp in mice treated with rGM-CSF alone. rGM-CSF/rIL-4 also significantly increased the expression of major histocompatibility complex (MHC) class I and MHC class II on CD11c+ cells and increased their capacity to take up antigens by macropinocytosis and receptor-mediated endocytosis. Splenic DCs generated in response to rGM-CSF/rIL-4 were functionally immature in terms of allostimulatory activity, but this activity increased after short-term in vitro culture. Systemic treatment with rGM-CSF/rIL-4 enhanced the response to an adenoviral-based vaccine and led to antigen-specific retardation in the growth of established tumor. We conclude that systemic therapy with the combination of rGM-CSF/rIL-4 provides a new approach for generating DCs in vivo.

Dendritic cells (DCs) are rare antigen-presenting cells that play a central role in stimulating immune responses. The combination of recombinant granulocyte macrophage–colony-stimulating factor (rGM-CSF) and recombinant interleukin-4 (rIL-4) provides an important stimulus for generating DCs from murine bone marrow precursors in vitro. Using miniature osmotic pumps, we now demonstrate that continuous infusion of these cytokines for 7 days had a similar effect in vivo, increasing the number and function of splenic DCs. Administration of rGM-CSF/rIL-4 (10 µg/d each) increased the concentration of CD11+DCs by 2.7-fold and the absolute number of splenic DCs by an average of 5.7-fold. DC number also increased in peripheral blood and lymph nodes. The resultant DCs exhibited a different phenotype and function than those in control mice or mice treated with rGM-CSF alone. rGM-CSF/IL-4 increased both the myeloid (CD11c+/CD11b+) and the lymphoid (CD11c+/CD8α+) subpopulations, whereas rGM-CSF increased only myeloid DCs. DCs were highly concentrated in the T-cell areas of white pulp after rGM-CSF/IL-4 administration, whereas they were diffusely distributed throughout white pulp, marginal zones, and red pulp in mice treated with rGM-CSF alone. rGM-CSF/rIL-4 also significantly increased the expression of major histocompatibility complex (MHC) class I and MHC class II on CD11c+cells and increased their capacity to take up antigens by macropinocytosis and receptor-mediated endocytosis. Splenic DCs generated in response to rGM-CSF/rIL-4 were functionally immature in terms of allostimulatory activity, but this activity increased after short-term in vitro culture. Systemic treatment with rGM-CSF/rIL-4 enhanced the response to an adenoviral-based vaccine and led to antigen-specific retardation in the growth of established tumor. We conclude that systemic therapy with the combination of rGM-CSF/rIL-4 provides a new approach for generating DCs in vivo.

Elevated tumor cyclooxygenase (COX-2) expression is associated with increased angiogenesis, tumor invasion, and suppression of host immunity. We have previously shown that genetic inhibition of tumor COX-2 expression reverses the immunosuppression induced by non-small cell lung cancer (NSCLC). To assess the impact of COX-2 expression in lung cancer invasiveness, NSCLC cell lines were transduced with a retroviral vector expressing the human COX-2 cDNA in the sense (COX-2-S) and antisense (COX-2-AS) orientations. COX-2-S clones expressed significantly more COX-2 protein, produced 10-fold more prostaglandin E2, and demonstrated an enhanced invasive capacity compared with control vector-transduced or parental cells. CD44, the cell surface receptor for hyaluronate, was overexpressed in COX-2-S cells, and specific blockade of CD44 significantly decreased tumor cell invasion. In contrast, COX-2-AS clones had a very limited capacity for invasion and showed diminished expression of CD44. These findings suggest that a COX-2-mediated, CD44-dependent pathway is operative in NSCLC invasion. Because tumor COX-2 expression appears to have a multifaceted role in conferring the malignant phenotype, COX-2 may be an important target for gene or pharmacologic therapy in NSCLC.

In two murine lung cancer models adenoviral interleukin 7-transduced dendritic cells (DC-AdIL-7) were administered intratumorally, resulting in complete tumor regression. Intratumoral DC-AdIL-7 therapy was as effective as DCs pulsed with specific tumor peptide antigens. Comparison with other intratumoral therapies including recombinant IL-7, AdIL-7 vector alone, unmodified DCs, IL-7-transduced fibroblasts, or DCs pulsed with tumor lysates revealed DC-AdIL-7 therapy to be superior in achieving antitumor responses and augmenting immunogenicity. Mice with complete tumor eradication as a result of either DC-AdIL-7 or AdIL-7 therapy were rechallenged with parental tumor cells 30 days or more after complete tumor eradication. All the DC-AdIL-7-treated mice completely rejected a secondary rechallenge, whereas the AdIL-7-treated mice had sustained antitumor effects in only 20-25% of the mice. DC-AdIL-7 therapy was more effective than AdIL-7 in achieving systemic antitumor responses and enhancing immunogenicity. After complete tumor eradication, those mice treated with DC-AdIL-7 evidenced significantly greater release of splenocyte GM-CSF and IFN-gamma than did controls or AdIL-7-treated mice. After intratumoral injection, gene-modified DCs trafficked from the tumor to lymph node sites and spleen. DCs were detected in nodal tissues for up to 7 days after intratumoral injection. We report that intratumoral DC-AdIL-7 leads to significant systemic immune responses and potent antitumor effects in murine lung cancer models.

Cocaine has multiple immunomodulatory effects, including the ability to influence cytokine release in immunoeffector cells. Little is known, however, regarding the effects of cocaine on cytokine production by human peripheral blood lymphocytes (PBL). The effect of cocaine on PBL cytokine profiles and the molecular mechanisms responsible for the modulation of cytokine mRNA expression were investigated. To evaluate the effects of cocaine on cytokine production, conditioned supernatant from IL-2-stimulated PBL was evaluated by cytokine-specific ELISA (IL-4, IL-5, IL-8, IL-10, IFN-γ, and TGF-β) followingin vitrococaine exposure. Cocaine abrogated the IL-2-induced production of IFN-γ and IL-8 in a dose-responsive manner. Cocaine also decreased PBL IFN-γ and IL-8 mRNA expression as determined by Northern blot and slot blot analysis. Cocaine did not affect the stability of the IFN-γ and IL-8 mRNA. Nuclear run-on assays revealed that cocaine down-regulated the rate of IFN-γ and IL-8 transcription. These findings suggest that the immunomodulatory effects of cocaine may be mediated, in part, by modification of lymphocyte cytokine production.

Cocaine use is associated with modulation of a broad range of biological functions including the capacity to influence cytokine production in murine and human immunoeffector cells. Little is known, however, regarding the effects of cocaine on endothelial cell cytokine production. Because the vascular endothelium actively participates in acute and chronic inflammatory responses and interleukin-8 (IL-8) is one of the key cytokines involved in the inflammatory process, modification of the production of IL-8 by vascular endothelial cells may interfere with the host response to infection or tissue injury. We investigated the effect of cocaine on endothelial cell IL-8 production. Conditioned supernatant from EA.hy 926 cells were evaluated by ELISA followingin vitrococaine exposure. Cocaine decreased IL-8 production in a dose-responsive manner, and this reduction correlated with down-regulation of IL-8 mRNA expression. Cocaine also increased the production of TGF-β by EA.hy 926 cells and anti-TGF-β abrogated the cocaine-mediated decrement of IL-8 production, indicating that cocaine down-regulates endothelial IL-8 production by increasing TGF-β. Our findings suggest that the immunomodulatory effects of cocaine may be mediated, in part, by modification of endothelial-derived cytokine production.

Interleukin (IL)-10 is a pleiotropic cytokine which inhibits a broad array of immune parameters including T helper cell type 1 (Th1) cytokine production, antigen presentation, and antigenspecific T cell proliferation. To understand the consequences of altered expression of IL-10 in immune models of autoimmune disease, the response to infectious agents, and the response to tumors, we developed transgenic mice expressing IL-10 under the control of the IL-2 promoter. Upon in vitro stimulation, spleen cells from unimmunized transgenic mice secrete higher levels of IL-10 and lower amounts of IFN-γ than do controls, although no gross abnormalities were detected in lymphocyte populations or serum Ig levels. Transfer of normally pathogenic CD4+ CD45RBhigh splenic T cells from IL-10 transgenic mice did not cause colitis in recipient severe combined immunodeficiency mice. Furthermore, co-transfer of these transgenic cells with CD4+ CD45RBhigh T cells from control mice prevented disease. Transgenic mice retained their resistance to Leishmania major infection, indicating that their cell-mediated immune responses were not globally suppressed. Lastly, in comparison to controls, IL-10 transgenic mice were unable to limit the growth of immunogenic tumors. Administration of blocking IL-10 mAbs restored in vivo antitumor responses in the transgenic mice. These results demonstrate that a single alteration in the T cell cytokine profile can lead to dramatic changes in immune responses in a manner that is stimulus dependent. These mice will be useful in defining differences in inflammatory conditions and cellular immunity mediated by IL-10.

To assess the technical feasibility and safety of repeated percutaneous computed tomographic (CT)-guided transthoracic biopsies and intratumoral injections of gene-modified dendritic cells in metastatic non-small cell lung cancer.

Although tumor growth leads to inflammatory responses, the immune system develops tolerance to cancer. One way to break host tolerance to tumors is to activate key immune effector activities. Toward this end, various adjuvants are under investigation in an effort to harness the immune system to overcome tolerance to tumor-associated self-antigens. There is enthusiasm for the use of specific ligands for toll-like receptor 3 (TLR3) that play a key role in the innate immune system. TLR3 agonists serve as immune adjuvants because they potently induce innate immune responses by activating dendritic cell (DC) maturation and inflammatory cytokine secretion. These activities facilitate the bridge between the innate and adaptive immune systems promoting the expansion of cytotoxic T lymphocytes (CTL) that destroy cancer cells. TLR3 agonists either alone or in combination with tumor antigens have shown success in terms of enhancing immune responses and eliciting antitumor activity in preclinical models. However, TLR3 agonists can also impact regulatory cells that dampen immune responses. Thus, immune strategies that utilize TLR3 agonists should consider the relative induction of suppressive as well as beneficial antitumor immune activities. Herein, we summarize the TLR3 agonists that will hopefully come to clinical fruition.

Commentary to:Influence of E3 region on conditionally replicative adenovirus mediated cytotoxicity in hepatocellular carcinoma cellsQin Lu, De-Hong Yu, Chao Fang, Fang Liu, Xun Ye, Yi Zhao, Jie Qin, Xiao-Kui Guo, Min Liang, Fang Hu, Hong-zhuan Chen

Myeloid-derived suppressor cells (MDSCs) are important regulators of immune responses. These cells suppress the cytotoxic activities of natural killer (NK)-cell and T-cell effectors and promote tumor growth. We demonstrated that depleting MDSCs improve therapeutic responses to vaccination in a murine model of lung cancer. This approach may prove beneficial against tumors in which MDSC exert prominent immunosuppressive effects.

An immune tolerant tumor microenvironment promotes immune evasion of lung cancer. Agents that antagonize immune tolerance will thus aid the fight against this devastating disease. Members of the tumor necrosis factor receptor (TNFR) family modulate the magnitude, duration and phenotype of immune responsiveness to antigens. Among these, GITR expressed on immune cells functions as a key regulator in inflammatory and immune responses. Here, we evaluate the GITR agonistic antibody (DTA-1) as a mono-therapy and in combination with therapeutic vaccination in murine lung cancer models. We found that DTA-1 treatment of tumor-bearing mice increased: (i) the frequency and activation of intratumoral natural killer (NK) cells and T lymphocytes, (ii) the antigen presenting cell (APC) activity in the tumor, and (iii) systemic T-cell specific tumor cell cytolysis. DTA-1 treatment enhanced tumor cell apoptosis as quantified by cleaved caspase-3 staining in the tumors. DTA-1 treatment increased expression of IFNγ, TNFα and IL-12 but reduced IL-10 levels in tumors. Furthermore, increased anti-angiogenic chemokines corresponding with decreased pro-angiogenic chemokine levels correlated with reduced expression of the endothelial cell marker Meca 32 in the tumors of DTA-1 treated mice. In accordance, there was reduced tumor growth (8-fold by weight) in the DTA-1 treatment group. NK cell depletion markedly inhibited the antitumor response elicited by DTA-1. DTA-1 combined with therapeutic vaccination caused tumor rejection in 38% of mice and a 20-fold reduction in tumor burden in the remaining mice relative to control. Mice that rejected tumors following therapy developed immunological memory against subsequent re-challenge. Our data demonstrates GITR agonist antibody activated NK cell and T lymphocyte activity, and enhanced therapeutic vaccination responses against lung cancer.