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1.  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
2.  Impact of Tumor-Derived CCL2 on Macrophage Effector Function 
Monocyte chemoattractant protein-1 (MCP-1, CCL2) is produced by many different types of cells. In the current investigation, the effect of tumor-derived CCL2 on macrophages was evaluated to determine the extent to which this chemokine influenced the innate immune response to cancer. To do this, we used the 4T1 murine mammary carcinoma cell line that constitutively expresses CCL2 and generated 4T1 expressing an antisense CCL2 transcript. The antisense-CCL2-expressing 4T1 produced no detectable CCL2. Macrophages from female BALB/c mice were exposed to supernatants from these tumor cells. The results showed that tumor-derived CCL2 was capable of modulating cytokine gene expression but not protein production in resting, activated, and tumor-associated macrophages. In addition, tumor-derived CCL2 did not affect phagocytic activity, nitric oxide production, or cytolytic activity of the macrophages. Overall, these data suggest that tumor-derived CCL2 does not directly influence macrophage-mediated antitumor activity.
doi:10.1155/S1110724304401041
PMCID: PMC1138266  PMID: 15689637
3.  Expression of a Functional CCR2 Receptor Enhances Tumor Localization and Tumor Eradication by Retargeted Human T Cells Expressing a Mesothelin - Specific Chimeric Antibody Receptor 
Purpose
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.
Results
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.
Conclusions
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.
doi:10.1158/1078-0432.CCR-11-0351
PMCID: PMC3612507  PMID: 21610146
4.  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
5.  Regulated Expression of CCL21 in the Prostate Tumor Microenvironment Inhibits Tumor Growth and Metastasis in an Orthotopic Model of Prostate Cancer 
Cancer Microenvironment  2009;2(1):59-67.
Currently there are no curative therapies available for patients with metastatic prostate cancer. Thus, novel therapies are needed to treat this patient population. Immunotherapy represents one promising approach for the elimination of occult metastatic tumors. However, the prostate tumor microenvironment (TME) represents a hostile environment capable of suppressing anti-tumor immunity and effector cell function. In view of this immunosuppressive activity, we engineered murine prostate cancer cells with regulated expression (tet-on) of CCL21. Prostate tumor cells implanted orthotopically produced primary prostate tumors with predictable metastatic disease in draining lymph nodes and distant organs. Expression of CCL21 in the prostate TME enhanced survival, inhibited tumor growth and decreased the frequency of local (draining lymph node) and distant metastasis. Therefore, these studies provide a strong rationale for further evaluation of CCL21 in tumor immunity and its use in cancer immunotherapy.
doi:10.1007/s12307-009-0021-z
PMCID: PMC2787929  PMID: 19418243
Tumor microenvironment; CCL21; Prostate cancer; TRAMP; T-REx system; Cancer immunosupression
6.  Regulated Expression of CCL21 in the Prostate Tumor Microenvironment Inhibits Tumor Growth and Metastasis in an Orthotopic Model of Prostate Cancer 
Cancer Microenvironment  2009;2(1):59-67.
Currently there are no curative therapies available for patients with metastatic prostate cancer. Thus, novel therapies are needed to treat this patient population. Immunotherapy represents one promising approach for the elimination of occult metastatic tumors. However, the prostate tumor microenvironment (TME) represents a hostile environment capable of suppressing anti-tumor immunity and effector cell function. In view of this immunosuppressive activity, we engineered murine prostate cancer cells with regulated expression (tet-on) of CCL21. Prostate tumor cells implanted orthotopically produced primary prostate tumors with predictable metastatic disease in draining lymph nodes and distant organs. Expression of CCL21 in the prostate TME enhanced survival, inhibited tumor growth and decreased the frequency of local (draining lymph node) and distant metastasis. Therefore, these studies provide a strong rationale for further evaluation of CCL21 in tumor immunity and its use in cancer immunotherapy.
doi:10.1007/s12307-009-0021-z
PMCID: PMC2787929  PMID: 19418243
Tumor microenvironment; CCL21; Prostate cancer; TRAMP; T-REx system; Cancer immunosupression
7.  Involvement of CCR6/CCL20/IL-17 Axis in NSCLC Disease Progression 
PLoS ONE  2011;6(9):e24856.
Objectives
Autocrine and paracrine chemokine/chemokine receptor-based interactions promote non-small-cell-lung-cancer (NSCLC) carcinogenesis. CCL20/CCR6 interactions are involved in prostatic and colonic malignancy pathogenesis. The expression and function of CCL20/CCR6 and its related Th-17 type immune response in NSCLC is not yet defined. We sought to characterize the role of the CCL20/CCR6/IL-17 axis in NSCLC tumor growth.
Methods
A specialized histopathologist blindly assessed CCL20/CCR6 expression levels in 49 tissue samples of NSCLC patients operated in our department. Results were correlated to disease progression. Colony assays, ERK signaling and chemokine production were measured to assess cancer cell responsiveness to CCL20 and IL-17 stimulation.
Results
CCL20 was highly expressed in the majority (38/49, 77.5%) of tumor samples. Only a minority of samples (8/49, 16.5%) showed high CCR6 expression. High CCR6 expression was associated with a shorter disease-free survival (P = 0.008) and conferred a disease stage-independent 4.87-fold increased risk for disease recurrence (P = 0.0076, CI 95% 1.52–15.563). Cancerous cell colony-forming capacity was increased by CCL20 stimulation; this effect was dependent in part on ERK phosphorylation and signaling. IL-17 expression was detected in NSCLC; IL-17 potentiated the production of CCL20 by cancerous cells.
Conclusion
Our findings suggest that the CCL20/CCR6 axis promotes NSCLC disease progression. CCR6 is identified as a potential new prognostic marker and the CCL20/CCR6/IL-17 axis as a potential new therapeutic target. Larger scale studies are required to consolidate these observations.
doi:10.1371/journal.pone.0024856
PMCID: PMC3174223  PMID: 21949768
8.  The Chemokine CCL2 Increases Prostate Tumor Growth and Bone Metastasis through Macrophage and Osteoclast Recruitment1 
Neoplasia (New York, N.Y.)  2009;11(11):1235-1242.
CC chemokine ligand 2 (CCL2, also known as monocyte chemoattractant protein-1) has been demonstrated to recruit monocytes to tumor sites. Monocytes are capable of being differentiated into tumor-associated macrophages (TAMs) and osteoclasts (OCs). TAMs have been shown to promote tumor growth in several cancer types. Osteoclasts have also been known to play an important role in cancer bone metastasis. To investigate the effects of CCL2 on tumorigenesis and its potential effects on bone metastasis of human prostate cancer, CCL2 was overexpressed into a luciferase-tagged human prostate cancer cell line PC-3. In vitro, the conditioned medium of CCL2 overexpressing PC-3luc cells (PC-3lucCCL2) was a potent chemoattractant for mouse monocytes in comparison to a conditioned medium from PC-3lucMock. In addition, CCL2 overexpression increased the growth of transplanted xenografts and increased the accumulation of macrophages in vivo. In a tumor dissemination model, PC-3lucCCL2 enhanced the growth of bone metastasis, which was associated with more functional OCs. Neutralizing antibodies targeting both human and mouse CCL2 inhibited the growth of PC-3luc, which was accompanied by a decrease in macrophage recruitment to the tumor. These findings suggest that CCL2 increases tumor growth and bone metastasis through recruitment of macrophages and OCs to the tumor site.
PMCID: PMC2767225  PMID: 19881959
9.  Novel CCL21-Vault Nanocapsule Intratumoral Delivery Inhibits Lung Cancer Growth 
PLoS ONE  2011;6(5):e18758.
Background
Based on our preclinical findings, we are assessing the efficacy of intratumoral injection of dendritic cells (DC) transduced with an adenoviral vector expressing the secondary lymphoid chemokine (CCL21) gene (Ad-CCL21-DC) in a phase I trial in advanced non-small cell lung cancer (NSCLC). While this approach shows immune enhancement, the preparation of autologous DC for CCL21 genetic modification is cumbersome, expensive and time consuming. We are evaluating a non-DC based approach which utilizes vault nanoparticles for intratumoral CCL21 delivery to mediate antitumor activity in lung cancer.
Principal Findings
Here we describe that vault nanocapsule platform for CCL21 delivery elicits antitumor activity with inhibition of lung cancer growth. Vault nanocapsule packaged CCL21 (CCL21-vaults) demonstrated functional activity in chemotactic and antigen presenting activity assays. Recombinant vaults impacted chemotactic migration of T cells and this effect was predominantly CCL21 dependent as CCL21 neutralization abrogated the CCL21 mediated enhancement in chemotaxis. Intratumoral administration of CCL21-vaults in mice bearing lung cancer enhanced leukocytic infiltrates (CXCR3+T, CCR7+T, IFNγ+T lymphocytes, DEC205+ DC), inhibited lung cancer tumor growth and reduced the frequencies of immune suppressive cells [myeloid derived suppressor cells (MDSC), T regulatory cells (Treg), IL-10 T cells]. CCL21-vaults induced systemic antitumor responses by augmenting splenic T cell lytic activity against parental tumor cells.
Significance
This study demonstrates that the vault nanocapsule can efficiently deliver CCL21 to sustain antitumor activity and inhibit lung cancer growth. The vault nanocapsule can serve as an “off the shelf” approach to deliver antitumor cytokines to treat a broad range of malignancies.
doi:10.1371/journal.pone.0018758
PMCID: PMC3086906  PMID: 21559281
10.  Control of human mesothelin-expressing tumors by DNA vaccines 
Gene therapy  2007;14(16):1189-1198.
Mesothelin has been implicated as a potential ideal target antigen for the development of antigen-specific cancer immunotherapy for the control of mesothelin-expressing cancers such as ovarian cancer, mesothelioma and pancreatic adenocarcinoma. In the current study, we utilized a DNA vaccine encoding human mesothelin (pcDNA3-Hmeso) to treat C57BL/6 mice challenged with luciferase-expressing, Hmeso-expressing ovarian cancer cell line, Defb29 Vegf-luc/Hmeso. The therapeutic effect of the tumor-challenged mice was followed by noninvasive bioluminescence imaging systems. The mechanism of the antitumor effect was characterized by depletion of subsets of lymphocytes as well as adopted transfer of serum from pcDNA3-Hmeso-vaccinated mice. We found that vaccination with pcDNA3-Hmeso DNA vaccine generates a significant antitumor effect and promotes survival in mice challenged with Defb29 Vegf-luc/Hmeso. Furthermore, we found CD4+ and CD8+ T-cell immune responses as well as the humoral immune responses are important for the observed antitumor effects in vaccinated mice. Our data indicated that vaccination with DNA vaccine targeting Hmeso could generate potent antitumor effects against mesothelin-expressing tumors through both T cell-mediated immunity as well as antibody-mediated immunity.
doi:10.1038/sj.gt.3302974
PMCID: PMC3182456  PMID: 17581599
ovarian cancer; adoptive serum transfer; human mesothelin-specific antibodies; DNA vaccine
11.  CCL2 is a Potent Regulator of Prostate Cancer Cell Migration and Proliferation1 
Neoplasia (New York, N.Y.)  2006;8(7):578-586.
Abstract
Tumor cells in the bone interact with the microenvironment to promote tumor cell survival and proliferation, resulting in a lethal phenotype for patients with advanced prostate cancer. Monocyte chemoattractant protein 1 (CCL2) is a member of the CC chemokine family and is known to promote monocyte chemotaxis to sites of inflammation. Here we have shown that human bone marrow endothelial (HBME) cells secrete significantly higher levels of CCL2 compared to human aortic endothelial cells and human dermal micro-vascular endothelial cells. Furthermore, we demonstrate that CCL2 is a potent chemoattractant of prostate cancer epithelial cells, and that stimulation of PC-3 and VCaP cells resulted in a dose-dependent activation of PI3 kinase/Akt signaling pathway. Activation of the PI3 kinase/Akt pathway was found to be vital to the proliferative effects of CCL2 stimulation of both PC-3 and VCaP cells. Additionally, CCL2 stimulated the phosphorylation of p70-S6 kinase (a downstream target of Akt) and induced actin rearrangement, resulting in a dynamic morphologic change indicative of microspike formation. These data suggest that bone marrow endothelial cells are a major source of CCL2, and that an elevated secretion of CCL2 recruits prostate cancer epithelial cells to the bone microenvironment and regulates their proliferation rate.
PMCID: PMC1601934  PMID: 16867220
CCL2; prostate cancer; migration; chemokine; metastasis
12.  Pre-clinical characterization of GMP grade CCL21-gene modified dendritic cells for application in a phase I trial in Non-Small Cell Lung Cancer 
Background
Our previous studies have demonstrated that transduction of human dendritic cells (DC) with adenovirus encoding secondary lymphoid chemokine, CCL21, led to secretion of biologically active CCL21 without altering DC phenotype or viability. In addition, intratumoral injections of CCL21-transduced DC into established murine lung tumors resulted in complete regression and protective anti-tumor immunity. These results have provided the rationale to generate a clinical grade adenoviral vector encoding CCL-21 for ex vivo transduction of human DC in order to assess intratumoral administration in late stage human lung cancer.
Methods
In the current study, human monocyte-derived DC were differentiated by exposure to GM-CSF and IL-4 from cryopreserved mononuclear cells obtained from healthy volunteers. Transduction with clinical grade adenoviral vector encoding CCL21 (1167 viral particles per cell) resulted in secretion of CCL21 protein.
Results
CCL21 protein production from transduced DC was detected in supernatants (24–72 hours, 3.5–6.7 ng/4–5 × 106 cells). DC transduced with the clinical grade adenoviral vector were > 88% viable (n = 16), conserved their phenotype and maintained integral biological activities including dextran uptake, production of immunostimulatory cytokines/chemokines and antigen presentation. Furthermore, supernatant from CCL21-DC induced the chemotaxis of T2 cells in vitro.
Conclusion
Viable and biologically active clinical grade CCL21 gene-modified DC can be generated from cryopreserved PBMC.
doi:10.1186/1479-5876-6-38
PMCID: PMC2507704  PMID: 18644162
13.  Mini-chaperones 
The immunogenic properties of heat shock proteins (HSPs) have prompted investigations into their application as immuno-modulatory agents. HSPs have been used as potent adjuvants in immunotherapy of cancer and infectious diseases. Some studies showed that immune activities reside within N- or C-terminal fragments of HSPs. These small fragments are sufficient to link peptides, to bind and be taken up by the receptors CD91 and scavenger receptor type A on antigen presenting cells (APCs). Thus, these mini-chaperones can be used in immunotherapy of tumors and vaccine development. The data clearly demonstrated the potential of using HSP fragments as a possible adjuvant to augment CTL response against infectious diseases. Some HSP domains have been shown to inhibit endothelial cell growth, angiogenesis or tumor growth. In this review, we describe the immuno-stimulatory activities of various mini-chaperones in development of different vaccine strategies (DNA-based vaccine and protein/peptide-based vaccines).
doi:10.4161/hv.22248
PMCID: PMC3667931  PMID: 23108356
heat shock protein; mini-chaperone; immuno-adjuvant; vaccine; cancer; infectious disease
14.  Lung tumor NF-κB signaling promotes T cell–mediated immune surveillance 
The Journal of Clinical Investigation  2013;123(6):2509-2522.
NF-κB is constitutively activated in many cancer types and is a potential key mediator of tumor-associated inflammation, tumor growth, and metastasis. We investigated the role of cancer cell NF-κB activity in T cell–mediated antitumor responses. In tumors rendered immunogenic by model antigen expression or following administration of antitumor vaccines, we found that high NF-κB activity leads to tumor rejection and/or growth suppression in mice. Using a global RNA expression microarray, we demonstrated that NF-κB enhanced expression of several T cell chemokines, including Ccl2, and decreased CCL2 expression was associated with enhanced tumor growth in a mouse lung cancer model. To investigate NF-κB function in human lung tumors, we identified a gene expression signature in human lung adenocarcinoma cell lines that was associated with NF-κB activity level. In patient tumor samples, overall lung tumor NF-κB activity was strongly associated with T cell infiltration but not with cancer cell proliferation. These results therefore indicate that NF-κB activity mediates immune surveillance and promotes antitumor T cell responses in both murine and human lung cancer.
doi:10.1172/JCI67250
PMCID: PMC3668836  PMID: 23635779
15.  CCL2 recruits inflammatory monocytes to facilitate breast tumor metastasis 
Nature  2011;475(7355):222-225.
Macrophages abundantly found in the tumor microenvironment enhance malignancy1. At metastatic sites a distinct population of metastasis associated macrophages (MAMs) promote tumor cell extravasation, seeding and persistent growth2. Our study has defined the origin of these macrophages by showing Gr1+ inflammatory monocytes (IMs) are preferentially recruited to pulmonary metastases but not primary mammary tumors, a process also found for human IMs in pulmonary metastases of human breast cancer cells. The recruitment of these CCR2 (receptor for chemokine CCL2) expressing IMs and subsequently MAMs and their interaction with metastasizing tumor cells is dependent on tumor and stromal synthesized CCL2 (FigS1). Inhibition of CCL2/CCR2 signaling using anti-CCL2 antibodies blocks IM recruitment and inhibits metastasis in vivo and prolongs the survival of tumor-bearing mice. Depletion of tumor cell-derived CCL2 also inhibits metastatic seeding. IMs promote tumor cell extravasation in a process that requires monocyte-derived VEGF. CCL2 expression and macrophage infiltration are correlated with poor prognosis and metastatic disease in human breast cancer (Fig S2)3-6. Our data provides the mechanistic link between these two clinical associations and indicates new therapeutic targets for treating metastatic breast disease.
doi:10.1038/nature10138
PMCID: PMC3208506  PMID: 21654748
16.  CCL2 as an Important Mediator of Prostate Cancer Growth In Vivo through the Regulation of Macrophage Infiltration1 
Neoplasia (New York, N.Y.)  2007;9(7):556-562.
The ability of CCL2 to influence prostate cancer tumorigenesis and metastasis may occur through two distinct mechanisms: 1) a direct effect on tumor cell growth and function, and 2) an indirect effect on the tumor microenvironment by the regulation of macrophage mobilization and infiltration into the tumor bed. We have previously demonstrated that CCL2 exerts a direct effect on prostate cancer epithelial cells by the regulation of their growth, invasion, and migration, resulting in enhanced tumorigenesis and metastasis. Here we describe an indirect effect of CCL2 on prostate cancer growth and metastasis by regulating monocyte/macrophage infiltration into the tumor microenvironment and by stimulating a phenotypic change within these immune cells to promote tumor growth (tumor-associated macrophages). VCaP prostate cancer cells were subcutaneously injected in male SCID mice and monitored for tumor volume, CD68+ macrophage infiltration, and microvascular density. Systemic administration of anti-CCL2 neutralizing antibodies (CNTO888 and C1142) significantly retarded tumor growth and attenuated CD68+ macrophage infiltration, which was accompanied by a significant decrease in microvascular density. These data suggest that CCL2 contributes to prostate cancer growth through the regulation of macrophage infiltration and enhanced angiogenesis within the tumor.
PMCID: PMC1939930  PMID: 17710158
Monocyte chemoattractant protein 1; prostate cancer; chemokine; tumor-associated macrophage; angiogenesis
17.  Oncogene MYCN regulates localization of NKT cells to the site of disease in neuroblastoma 
The Journal of Clinical Investigation  2007;117(9):2702-2712.
Vα24-invariant natural killer T (NKT) cells are potentially important for antitumor immunity. We and others have previously demonstrated positive associations between NKT cell presence in primary tumors and long-term survival in distinct human cancers. However, the mechanism by which aggressive tumors avoid infiltration with NKT and other T cells remains poorly understood. Here, we report that the v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN), the hallmark of aggressive neuroblastoma, repressed expression of monocyte chemoattractant protein–1/CC chemokine ligand 2 (MCP-1/CCL2), a chemokine required for NKT cell chemoattraction. MYCN knockdown in MYCN-amplified neuroblastoma cell lines restored CCL2 production and NKT cell chemoattraction. Unlike other oncogenes, MYCN repressed chemokine expression in a STAT3-independent manner, requiring an E-box element in the CCL2 promoter to mediate transcriptional repression. MYCN overexpression in neuroblastoma xenografts in NOD/SCID mice severely inhibited their ability to attract human NKT cells, T cells, and monocytes. Patients with MYCN-amplified neuroblastoma metastatic to bone marrow had 4-fold fewer NKT cells in their bone marrow than did their nonamplified counterparts, indicating that the MYCN-mediated immune escape mechanism, which we believe to be novel, is operative in metastatic cancer and should be considered in tumor immunobiology and for the development of new therapeutic strategies.
doi:10.1172/JCI30751
PMCID: PMC1940236  PMID: 17710228
18.  Cross talk between follicular helper T cells and tumor cells in human follicular lymphoma promotes immune evasion in the tumor microenvironment 
The microenvironment of human follicular lymphoma (FL), an incurable B-cell non-Hodgkin lymphoma, is thought to play a major role in its pathogenesis and course. Microenvironmental cells of likely importance include follicular helper T cells (TFH) and regulatory T cells (Tregs), and understanding their interactions with FL tumor cells is necessary to develop novel therapeutic strategies. We found that IL-4 and CD40L are expressed by intratumoral TFH and induce production of CCL17 and CCL22 by FL tumor cells. IL-4 alone induces only CCL17, but enhances stimulation by CD40L of both CCL17 and CCL22. Consistent with our in vitro results, mRNA transcripts of IL-4 correlated with CCL17 but not CCL22 in gene expression profiling studies of FL biopsies, whereas CD40L correlated with both CCL17 and CCL22. Tumor supernatants induced preferential migration of Tregs and IL-4–producing T cells rather than IFN-γ–producing T cells, and antibodies to CCR4 significantly abrogated the migration of Tregs. Our results suggest that through two distinct mechanisms, intratumoral TFH induce production of CCL17 and CCL22 by FL tumor cells and facilitate active recruitment of Tregs and IL-4–producing T cells, which in turn may stimulate more chemokine production in a feed-forward cycle. Thus, TFH appear to play a major role in generating an immunosuppressive tumor microenvironment that promotes immune escape and tumor survival and growth. Our results provide novel insights into the cross talk between TFH, tumor cells, and Tregs in FL and offer potential targets for development of therapeutic strategies to overcome immune evasion.
doi:10.4049/jimmunol.1201363
PMCID: PMC3680117  PMID: 23686488
19.  Serum Level of CC-Chemokine Ligand 18 Is Increased in Patients with Non-Small-Cell Lung Cancer and Correlates with Survival Time in Adenocarcinomas 
PLoS ONE  2012;7(7):e41746.
CC-chemokine ligand 18 (CCL18) is mainly expressed by alternatively activated macrophages and DCs and plays an important role in lung fibrosis, arthritis and other diseases. Here CCL18 was measured in sera of 31 healthy volunteers and 170 patients with lung cancer and correlated these data with histology, tumor stage and clinical parameters. Mean CCL18 serum level of the patients with non-small-cell lung cancer was 150(857) ng/ml vs. 32(61) ng/ml in the healthy control group. Patient groups differ significantly according their histology (adenocarcinoma 143(528) ng/ml vs squamous cell carcinoma 187(857) ng/ml, p<0.02). In addition, we found a significant difference between patients with lower versus higher T-stage (p<0.003). Receiver operating characteristic (ROC) analyses revealed a cutoff point of 83 ng/ml (area under the curve (AUC): 0.968; p<0.0001) to discriminate between healthy controls and non-small-cell lung cancer patients. ROC analyses to discriminate between patients, who died because of cancer related death and those who died for other reasons did not lead to a valid AUC. To stratify the tumor patients, a criterion value plot was performed leading to a point of equal sensitivity and specificity (54%) of 162 ng/ml. Patients with a CCL18 serum level higher than 160 ng/ml had a mean survival time of 623 days. In contrast, those in patients with a baseline level between 83 ng/ml and 160 ng/ml the mean survival time was 984 days (p<0.005). Survival-analysis revealed in adenocarcinoma a mean survival of 1152 days in the group below 83 ng/ml. In the median group the mean survival time was 788 days and in the group with the highest levels the mean survival time was 388 days (p<0.001). In contrast, we found no correlation between the FEV1 and the CCL18 baseline level. In conclusion, in patients suffering from adenocarcinoma increased serum CCL18 levels predict a diminished survival time.
doi:10.1371/journal.pone.0041746
PMCID: PMC3404958  PMID: 22848587
20.  Preclinical evaluation of MORAb-009, a chimeric antibody targeting tumor-associated mesothelin 
Novel therapeutic agents that are safe and effective are needed for the treatment of pancreatic, ovarian, lung adenocarcinomas and mesotheliomas. Mesothelin is a glycosyl-phosphatidyl inositol (GPI)-linked membrane protein of 40 kDa over-expressed in all pancreatic adenocarcinoma and mesothelioma, in >70% of ovarian adenocarcinoma, and in non-small cell lung and colorectal cancers. The biological functions of mesothelin are not known, although it appears to be involved in cell adhesion via its interaction with MUC16. We have recently developed MORAb-009, a mouse-human chimeric IgG1κ monoclonal antibody with an affinity of 1.5 nM for human mesothelin. Here we provide evidence that MORAb-009 prevents adhesion of mesothelin-bearing tumor cells to MUC16 positive cells and can elicit cell-mediated cytotoxicity on mesothelin-bearing tumor cells. Treatment that included MORAb-009 in combination with chemotherapy led to a marked reduction in tumor growth of mesothelin-expressing tumors in nude mice compared to chemotherapy or MORAb-009 treatment alone. No adverse effects of MORAb-009 were noted during toxicology studies conducted in non-human primates. The preclinical data obtained from our studies warrants pursuing clinical testing of MORAb-009. We have in fact initiated a Phase I clinical study enrolling patients with mesothelin-positive pancreatic, mesothelioma, non-small cell lung and ovarian cancers.
PMCID: PMC2935758  PMID: 18088084
preclinical drug evaluation; monoclonal antibody; mesothelin; antibody-dependent cellular cytotoxicity
21.  Quantification of Mesenchymal Stem Cells (MSCs) at Sites of Human Prostate Cancer 
Oncotarget  2012;4(1):106-117.
Circulating bone marrow-derived Mesenchymal Stem Cells (BM-MSCs) have an innate tropism for tumor tissue in response to the inflammatory microenvironment present in malignant lesions. The prostate is bombarded by numerous infectious & inflammatory insults over a lifetime. Chronic inflammation is associated with CXCL12, CCL5, and CCL2, which are highly overexpressed in prostate cancer. Among other cell types, these chemoattractant stimuli recruit BM-MSCs to the tumor. MSCs are minimally defined as plastic-adhering cells characterized by the expression of CD90, CD73, and CD105 in the absence of hematopoietic markers, which can differentiate into osteoblasts, chondrocytes, and adipocytes. MSCs are immunoprivileged and have been implicated in tumorigenesis through multiple mechanisms, including promoting proliferation, angiogenesis, and metastasis, in addition to the generation of an immunosuppressive microenvironment. We have demonstrated that MSCs represent 0.01-1.1% of the total cells present in core biopsies from primary human prostatectomies. Importantly, these analyses were performed on samples prior to expansion in tissue culture. MSCs in these prostatectomy samples are FAP-, CD90-, CD73-, and CD105-positive, and CD14-, CD20-, CD34-, CD45-, and HLA-DR-negative. Additionally, like BM-MSCs, these prostate cancer-derived stromal cells (PrCSCs) were shown to differentiate into osteoblasts, adipocytes, & chondrocytes. In contrast to primary prostate cancer-derived epithelial cells, fluorescently-labeled PrCSCs & BM-MSCs were both shown to home to CWR22RH prostate cancer xenografts following IV injection. These studies demonstrate that not only are MSCs present in sites of prostate cancer where they may contribute to carcinogenesis, but these cells may also potentially be used to deliver cytotoxic or imaging agents for therapeutic and/or diagnostic purposes.
PMCID: PMC3702211  PMID: 23362217
Mesenchymal Stem Cells; MSC; Inflammation; Prostate Cancer; Drug Delivery; CAF
22.  Dendritic Cells (DC) Facilitate Detachment of Squamous Carcinoma Cells (SCC), While SCC Promote an Immature CD16+ DC Phenotype and Control DC Migration 
Cancer Microenvironment  2011;6(1):41-55.
In the inflammatory mucosal microenvironment of head and neck SCC (HNSCC), DC express CD16 and are usually in direct contact with tumor cells. Mucosal and inflammation-associated DC develop from monocytes, and monocyte-derived DC are used in HNSCC immunotherapy. However, beyond apoptotic tumor cell uptake and presentation of tumor antigens by DC, HNSCC cell interactions with DC are poorly understood. Using co-cultures of monocyte-derived DC and two established HNSCC cell lines that represent well- and poorly-differentiated SCC, respectively, we found that carcinoma cells induced significant increases in CD16 expression on DC while promoting a CD1a+CD86dim immature phenotype, similar to that observed in HNSCC specimens. Moreover, HNSCC cells affected steady-state and CCL21-induced migration of DC, and these effects were donor-dependent. The CCL21-induced migration directly correlated with HNSCC-mediated effects on CCR7 and CD38 expression on DC-SIGN-high DC. The dominant pattern seen in six out of nine donors was the increase in steady-state and CCL21-induced DC migration in co-cultures with HNSCC, while the reverse pattern, i.e., decreased DC migration in co-cultures with SCC, was identified in two donors. A split in migratory DC behavior, i.e. increase with one HNSCC cell line and a decrease with the second cell line, was observed in one donor. Remarkably, the numbers of live detached HNSCC cells were orders of magnitude higher in DC-HNSCC co-cultures than in parallel HNSCC cell cultures without DC. This study provides novel insights into the effects of DC-HNSCC interactions relevant to the tumor microenvironment.
Electronic supplementary material
The online version of this article (doi:10.1007/s12307-011-0077-4) contains supplementary material, which is available to authorized users.
doi:10.1007/s12307-011-0077-4
PMCID: PMC3601220  PMID: 21809059
CD16; Dendritic cells; Head and neck squamous cell carcinoma; Tumor microenvironment
23.  A Novel Human Monoclonal Antibody that Binds with High Affinity to Mesothelin-Expressing Cells and Kills them by ADCC 
Molecular cancer therapeutics  2009;8(5):1113-1118.
Mesothelin is a potential new target for cancer immunotherapy because it is present at relatively low levels only in mesothelial cells of pleura, peritoneum and pericardium of healthy people but is significantly elevated in a number of tumors, including mesothelioma, ovarian, pancreatic and lung cancers. However, all currently available antibodies against mesothelin are either murine or chimeric which could limit their use because of increased likelihood of immunogenicity compared to fully human antibodies. Here we report the identification and characterization of a novel fully human monoclonal antibody, m912, which was isolated from a human Fab library by panning against recombinant mesothelin. This antibody in scFv, Fab and IgG1 formats bound specifically and with high affinity (equilibrium dissociation constant in the nM range) to cell surface-associated human mesothelin and to recombinant mesothelin. It specifically lysed cancer cells engineered to express mesothelin in the presence of peripheral blood mononuclear cells isolated from healthy donors most likely by antibody-dependent cellular cytotoxicity (ADCC). M912 is the first reported fully human monoclonal antibody to mesothelin, which has potential for cancer treatment and diagnosis
doi:10.1158/1535-7163.MCT-08-0945
PMCID: PMC2891957  PMID: 19417159
mesothelin; mesothelioma; ovarian cancer; lung cancer; therapeutic antibody
24.  CC chemokine ligand 2 (CCL2) promotes prostate cancer tumorigenesis and metastasis 
CCL2 is a chemokine known to recruit monocytes and macrophages to sites of inflammation. A growing body of research suggests CCL2 is progressively overexpressed in tumor beds and may play a role in the clinical progression of solid tumors. Cancer cells derived from several solid tumor types demonstrate functional receptors for CCL2, suggesting this chemokine may achieve tumorigenicity through direct effects on malignant cells; however, a variety of normal host cells that co-exist with cancer in the tumor microenvironment also respond to CCL2. These cells include macrophages, osteoclasts, endothelial cells, T-lymphocytes, and myeloid-derived immune suppressor cells (MDSCs). CCL2 mediated interactions between normal and malignant cells in the tumor microenvironment and plays a multi-faceted role in tumor progression.
doi:10.1016/j.cytogfr.2009.11.009
PMCID: PMC2857769  PMID: 20005149
CCL2; CCR2; tumorigenesis; metastasis; prostate cancer
25.  Role of CCL11/eotaxin-1 signaling in ovarian cancer 
Purpose
Tumor cell growth and migration can be directly regulated by chemokines. In the present study the association of CCL11 with ovarian cancer has been investigated.
Experimental design and results
Circulating levels of CCL11 in sera of patients with ovarian cancer were significantly lower than those in healthy women or women with breast, lung, liver, pancreatic or colon cancers. Cultured ovarian carcinoma cells absorbed soluble CCL11 indicating that absorption by tumor cells could be responsible for the observed reduction of serum level of CCL11 in ovarian cancer. Postoperative CCL11 levels in women with ovarian cancer negatively correlated with relapse-free survival. Ovarian tumors overexpressed three known cognate receptors of CCL11, CCR2, CCR3, and CCR5. Strong positive correlation was observed between expression of individual receptors and tumor grade. CCL11 potently stimulated proliferation and migration/invasion of ovarian carcinoma cell lines, and these effects were inhibited by neutralizing antibodies against CCR2,3, and 5. The growth stimulatory effects of CCL11 were likely associated with activation of ERK1/2, MEK-1, and STAT3 phosphoproteins and with increased production of multiple cytokines, growth and angiogenic factors. Inhibition of CCL11 signaling by the combination of neutralizing antibodies against the ligand and its receptors significantly increased sensitivity to cisplatin in ovarian carcinoma cells. We conclude that CCL11 signaling plays an important role in proliferation and invasion of ovarian carcinoma cells and CCL11 pathway could be targeted for therapy in ovarian cancer. Furthermore, CCL11 could be used as a biomarker and a prognostic factor of relapse-free survival in ovarian cancer.
doi:10.1158/1078-0432.CCR-08-2024
PMCID: PMC2669845  PMID: 19351767

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