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1.  Beneficial Impact of CCL2 and CCL12 Neutralization on Experimental Malignant Pleural Effusion 
PLoS ONE  2013;8(8):e71207.
Using genetic interventions, we previously determined that C-C motif chemokine ligand 2 (CCL2) promotes malignant pleural effusion (MPE) formation in mice. Here we conducted preclinical studies aimed at assessing the specific therapeutic potential of antibody-mediated CCL2 blockade against MPE. For this, murine MPEs or skin tumors were generated in C57BL/6 mice by intrapleural or subcutaneous delivery of lung (LLC) or colon (MC38) adenocarcinoma cells. Human lung adenocarcinoma cells (A549) were used to induce MPEs in severe combined immunodeficient mice. Intraperitoneal antibodies neutralizing mouse CCL2 and/or CCL12, a murine CCL2 ortholog, were administered at 10 or 50 mg/kg every three days. We found that high doses of CCL2/12 neutralizing antibody treatment (50 mg/kg) were required to limit MPE formation by LLC cells. CCL2 and CCL12 blockade were equally potent inhibitors of MPE development by LLC cells. Combined CCL2 and CCL12 neutralization was also effective against MC38-induced MPE and prolonged the survival of mice in both syngeneic models. Mouse-specific CCL2-blockade limited A549-caused xenogeneic MPE, indicating that host-derived CCL2 also contributes to MPE precipitation in mice. The impact of CCL2/12 antagonism was associated with inhibition of immune and vascular MPE-related phenomena, such as inflammation, new blood vessel assembly and plasma extravasation into the pleural space. We conclude that CCL2 and CCL12 blockade are effective against experimental MPE induced by murine and human adenocarcinoma in mice. These results suggest that CCL2-targeted therapies may hold promise for future use against human MPE.
PMCID: PMC3743892  PMID: 23967166
2.  Systemic Delivery of Neutralizing Antibody Targeting CCL2 for Glioma Therapy 
Journal of neuro-oncology  2010;104(1):83-92.
Tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) inhibit anti-tumor immune responses and facilitate tumor growth. Precursors for these immune cell populations migrate to the tumor site in response to tumor secretion of chemokines, such as monocyte chemoattractant protein-1 (MCP-1/CCL2), which was originally purified and identified from human gliomas. In syngeneic mouse GL261 glioma and human U87 glioma xenograft models, we evaluated the efficacy of systemic CCL2 blockade by monoclonal antibodies (mAb) targeting mouse and/or human CCL2. Intraperitoneal (i.p.) administration of anti-mouse CCL2 mAb as monotherapy (2 mg/kg/dose, twice a week) significantly, albeit modestly, prolonged the survival of C57BL/6 mice bearing intracranial GL261 glioma (p=0.0033), which was concomitant with a decrease in TAMs and MDSCs in the tumor microenvironment. Similarly, survival was modestly prolonged in severe combined immunodeficiency (SCID) mice bearing intracranial human U87 glioma xenografts treated with both anti-human CCL2 mAb and anti-mouse CCL2 antibodies (2 mg/kg/dose for each, twice a week) compared to mice treated with control IgG (p=0.0159). Furthermore, i.p. administration of anti-mouse CCL2 antibody in combination with temozolomide (TMZ) significantly prolonged the survival of C57BL/6 mice bearing GL261 glioma with 8 of 10 treated mice surviving longer than 70 days, while only 3 of 10 mice treated with TMZ and isotype IgG survived longer than 70 days (p=0.0359). These observations provide support for development of mAb-based CCL2 blockade strategies in combination with the current standard TMZ-based chemotherapy for treatment of malignant gliomas.
PMCID: PMC3068234  PMID: 21116835
glioma; chemokine; CCL2; monoclonal antibody; chemotherapy; temozolomide
3.  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
4.  CCL2 Blockade Augments Cancer Immunotherapy 
Cancer research  2009;70(1):109.
Since an immuno-inhibitory environment exists within tumors, successful vaccines will likely require additional approaches to alter the tumor microenvironment. Monocyte chemoattractant proteins (such as CCL2) are produced by many tumors and have both direct and indirect immuno-inhibitory effects. We hypothesized that CCL2 blockade would reduce immunosuppression and augment vaccine immunotherapy. Anti-murine-CCL2/CCL12 monoclonal antibodies were administered in three immunotherapy models: one aimed at the HPV-E7 antigen expressed by a non-small cell lung cancer line, one targeted to mesothelin expressed by a mesothelioma cell line, and one using an adenovirus expressing Interferon-α to treat a non-immunogenic, non-small cell lung cancer line. We evaluated the effect of the combination treatment on tumor growth and assessed the mechanism of these changes by evaluating cytotoxic T cells, immunosuppressive cells, and the tumor microenvironment. Administration of anti-CCL2/CCL12 antibodies along with the vaccines markedly augmented efficacy with enhanced reduction in tumor volume and cures of approximately half of the tumors. The combined treatment generated more total intra-tumoral CD8+ T-cells that were more activated and more anti-tumor antigen specific, as measured by tetramer evaluation. Another important potential mechanism was reduction in intratumoral T-regulatory (T-reg) cells. CCL2 appears to be a key proximal cytokine mediating immunosuppression in tumors. Its blockade augments CD8+ T cell immune response to tumors elicited by vaccines via multifactorial mechanisms. These observations suggest that combining CCL2 neutralization with vaccines should be considered in future immunotherapy trials.
PMCID: PMC2821565  PMID: 20028856
CCL2; Cancer immunotherapy; Lung Cancer; Mesothelioma; T-lymphocytes
5.  Novel CCL21-Vault Nanocapsule Intratumoral Delivery Inhibits Lung Cancer Growth 
PLoS ONE  2011;6(5):e18758.
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.
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.
PMCID: PMC3086906  PMID: 21559281
6.  A role for CCL2 in both tumor progression and immunosurveillance 
Oncoimmunology  2013;2(7):e25474.
The chemokine CCL2, which is best known for its chemotactic functions, is expressed not only by immune cells, but also by several types of malignant and stromal cells. CCL2 has been shown to exert both pro- and anti-tumor effects. However, recent results demonstrate a main role for CCL2 in tumor progression and metastasis, suggesting that this chemokine may constitute a therapeutic target for anticancer drugs. Mammary carcinoma models, including models of implantable, transgenic, and chemically-induced tumors, were employed in the setting of Ccl2 or Ccr2 knockout mice or CCL2 neutralization with a monoclonal antibody to further investigate the role of the CCL2/CCR2 signaling axis in tumor progression and metastatic spread. In our implantable tumor models, an anti-CCL2 monoclonal antibody inhibited the growth of primary malignant lesions in a biphasic manner and reduced the number of metastases. However, in Ccl2−/− or Ccr2−/− mice developing implanted or transgenic tumors, the number of pulmonary metastases was increased despite a reduction in the growth rate of primary neoplasms. Transgenic Mtag.Ccl2−/− or Mtag.Ccr2−/− mice also exhibited a significantly earlier of disease onset. In a chemical carcinogenesis model, anti-CCL2 monoclonal antibody inhibited the growth of established lesions but was ineffective in the tumor induction phase. In contrast to previous studies indicating a role for CCL2 in the establishment of metastases, we have demonstrated that the absence of CCL2/CCR2-signaling results in increased metastatic disease. Thus, the CCL2/CCR2 signaling axis appears to play a dual role in mediating early tumor immunosurveillance and sustaining the growth and progression of established neoplasms. Our findings support the use of anti-CCL2 therapies for the treatment of established breast carcinoma, although the complete abrogation of the CCL2 signaling cascade may also limit immunosurveillance and support metastatic spread.
PMCID: PMC3782157  PMID: 24073384
breast cancer; CCL2; CCR2; chemokine; immunosurveillance; metastases; monoclonal antibody therapy; tumor progression
7.  Inhibition of CCL2 Signaling in Combination with Docetaxel Treatment Has Profound Inhibitory Effects on Prostate Cancer Growth in Bone 
The C-C chemokine ligand 2 (CCL2) stimulates migration, proliferation, and invasion of prostate cancer (PCa) cells, and its signaling also plays a role in the activation of osteoclasts. Therefore targeting CCL2 signaling in regulation of tumor progression in bone metastases is an area of intense research. The objective of our study was to investigate the efficacy of CCL2 blockade by neutralizing antibodies to inhibit the growth of PCa in bone. We used a preclinical model of cancer growth in the bone in which PCa C4-2B cells were injected directly into murine tibiae. Animals were treated for ten weeks with neutralizing anti-CCL2 antibodies, docetaxel, or a combination of both, and then followed an additional nine weeks. CCL2 blockade inhibited the growth of PCa in bone, with even more pronounced inhibition in combination with docetaxel. CCL2 blockade also resulted in increases in bone mineral density. Furthermore, our results showed that the tumor inhibition lasted even after discontinuation of the treatment. Our data provide compelling evidence that CCL2 blockade slows PCa growth in bone, both alone and in combination with docetaxel. These results support the continued investigations of CCL2 blockade as a treatment for advanced metastatic PCa.
PMCID: PMC3676850  PMID: 23698775
prostate cancer; bone metastases; chemokine; CCL2; docetaxel; bone metastases
8.  A novel role of hematopoietic CCL5 in promoting triple-negative mammary tumor progression by regulating generation of myeloid-derived suppressor cells 
Cell Research  2012;23(3):394-408.
CCL5 is a member of the CC chemokine family expressed in a wide array of immune and non-immune cells in response to stress signals. CCL5 expression correlates with advanced human breast cancer. However, its functional significance and mode of action have not been established. Here, we show that CCL5-deficient mice are resistant to highly aggressive, triple-negative mammary tumor growth. Hematopoietic CCL5 is dominant in this phenotype. The absence of hematopoietic CCL5 causes aberrant generation of CD11b+/Gr-1+, myeloid-derived suppressor cells (MDSCs) in the bone marrow in response to tumor growth by accumulating Ly6Chi and Ly6G+ MDSCs with impaired capacity to suppress cytotoxicity of CD8+ T cells. These properties of CCL5 are observed in both orthotopic and spontaneous mammary tumors. Antibody-mediated systemic blockade of CCL5 inhibits tumor progression and enhances the efficacy of therapeutic vaccination against non-immunogenic tumors. CCL5 also helps maintain the immunosuppressive capacity of human MDSCs. Our study uncovers a novel, chemokine-independent activity of the hematopoietically derived CCL5 that promotes mammary tumor progression via generating MDSCs in the bone marrow in cooperation with tumor-derived colony-stimulating factors. The study sheds considerable light on the interplay between the hematopoietic compartment and tumor niche. Because of the apparent dispensable nature of this molecule in normal physiology, CCL5 may represent an excellent therapeutic target in immunotherapy for breast cancer as well as a broad range of solid tumors that have significant amounts of MDSC infiltration.
PMCID: PMC3587709  PMID: 23266888
CCL5; myeloid-derived suppressor cell; triple-negative breast cancer; immune response; mammary tumor; bone marrow; cytotoxic T lymphocyte
9.  Loss of Transforming Growth Factor-β Signaling in Mammary Fibroblasts Enhances CCL2 Secretion to Promote Mammary Tumor Progression through Macrophage-Dependent and -Independent Mechanisms1 
Neoplasia (New York, N.Y.)  2010;12(5):425-433.
Whereas the accumulation of fibroblasts and macrophages in breast cancer is a well-documented phenomenon and correlates with metastatic disease, the functional contributions of these stromal cells on breast cancer progression still remain largely unclear. Previous studies have uncovered a potentially important role for CCL2 inflammatory chemokine signaling in regulating metastatic disease through a macrophage-dependent mechanism. In these studies, we demonstrate a significant regulatory mechanism for CCL2 expression in fibroblasts in mediating mammary tumor progression and characterize multiple functions for CCL2 in regulating stromal-epithelial interactions. Targeted ablation of the transforming growth factor-β (TGF-β) type 2 receptor in fibroblasts (Tgfbr2FspKO) results in a high level of secretion of CCL2, and cografts of Tgfbr2FspKO fibroblasts with 4T1 mammary carcinoma cells enhanced tumor progression associated with recruitment of tumor-associated macrophages (TAMs). Antibody neutralization of CCL2 in tumor-bearing mice inhibits primary tumor growth and liver metastases as evidenced by reduced cell proliferation, survival, and TAM recruitment. Both high and low stable expressions of small interfering RNA to CCL2 in Tgfbr2FspKO fibroblasts significantly reduce liver metastases without significantly affecting primary tumor growth, cell proliferation, or TAM recruitment. High but not low knockdown of CCL2 enhances tumor cell apoptosis. These data indicate that CCL2 enhances primary tumor growth, survival, and metastases in a dose-dependent manner, through TAM-dependent and -independent mechanisms, with important implications on the potential effects of targeting CCL2 chemokine signaling in the metastatic disease.
PMCID: PMC2864480  PMID: 20454514
10.  Loss of p53 Attenuates the Contribution of IL-6 Deletion on Suppressed Tumor Progression and Extended Survival in Kras-Driven Murine Lung Cancer 
PLoS ONE  2013;8(11):e80885.
Interleukin-6 (IL-6) is involved in lung cancer tumorigenesis, tumor progression, metastasis, and drug resistance. Previous studies show that blockade of IL-6 signaling can inhibit tumor growth and increase drug sensitivity in mouse models. Clinical trials in non-small cell lung cancer (NSCLC) reveal that IL-6 targeted therapy relieves NSCLC-related anemia and cachexia, although other clinical effects require further study. We crossed IL-6-/- mice with KrasG12D mutant mice, which develop lung tumors after activation of mutant KrasG12D, to investigate whether IL-6 inhibition contributes to tumor progression and survival time in vivo. KrasG12D; IL-6-/- mice exhibited increased tumorigenesis, but slower tumor growth and longer survival, than KrasG12D mice. Further, in order to investigate whether IL-6 deletion contributes to suppression of lung cancer metastasis, we generated KrasG12D; p53flox/flox; IL-6-/- mice, which developed lung cancer with a trend for reduced metastases and longer survival than KrasG12D; p53flox/flox mice. Tumors from KrasG12D; IL-6-/- mice showed increased expression of TNFα and decreased expression of CCL-19, CCL-20 and phosphorylated STAT3 (pSTAT3) than KrasG12D mice; however, these changes were not present between tumors from KrasG12D; p53flox/flox; IL-6-/- and KrasG12D; p53flox/flox mice. Upregulation of pSTAT3 and phosphorylated AKT (pAKT) were observed in KrasG12D tumors with p53 deletion. Taken together, these results indicate that IL-6 deletion accelerates tumorigenesis but delays tumor progression and prolongs survival time in a Kras-driven mouse model of lung cancer. However, these effects can be attenuated by p53 deletion.
PMCID: PMC3829911  PMID: 24260500
11.  Role of CCL11/eotaxin-1 signaling in ovarian cancer 
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.
PMCID: PMC2669845  PMID: 19351767
12.  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.
PMCID: PMC1138266  PMID: 15689637
13.  Involvement of CCR6/CCL20/IL-17 Axis in NSCLC Disease Progression 
PLoS ONE  2011;6(9):e24856.
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.
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.
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.
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.
PMCID: PMC3174223  PMID: 21949768
14.  Inflammatory monocyte mobilization decreases patient survival in pancreatic cancer: a role for targeting the CCL2/CCR2 axis 
To determine the role of the CCL2/CCR2 axis and inflammatory monocytes (IM; CCR2+/CD14+) as immunotherapeutic targets in the treatment of pancreatic cancer (PC).
Experimental Design
Survival analysis was performed to determine if the prevalence of pre-operative blood monocytes correlates with survival in PC patients following tumor resection. IM prevalence in the blood and bone marrow of PC patients and controls was compared. The immunosuppressive properties of IM and macrophages in the blood and tumors, respectively, of PC patients were assessed. CCL2 expression by human PC tumors was compared to normal pancreas. A novel CCR2 inhibitor (PF-04136309) was tested in an orthotopic model of murine PC.
Monocyte prevalence in the peripheral blood correlates inversely with survival, and low monocyte prevalence is an independent predictor of increased survival in PC patients with resected tumors. IM are increased in the blood and decreased in the bone marrow of PC patients compared to controls. An increased ratio of IM in the blood versus the bone marrow is a novel predictor of decreased patient survival following tumor resection. Human PC produces CCL2, and immunosuppressive CCR2+ macrophages infiltrate these tumors. Patients with tumors that exhibit high CCL2 expression/low CD8 T cell infiltrate have significantly decreased survival. In mice, CCR2 blockade depletes IM and macrophages from the primary tumor and premetastatic liver resulting in enhanced anti-tumor immunity, decreased tumor growth, and reduced metastasis.
IM recruitment is critical to PC progression, and targeting CCR2 may be an effective immunotherapeutic strategy in this disease.
PMCID: PMC3700620  PMID: 23653148
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.
PMCID: PMC3208506  PMID: 21654748
16.  Chemokine Expression in Melanoma Metastases Associated with CD8+ T-Cell Recruitment 
Cancer research  2009;69(7):10.1158/0008-5472.CAN-08-2281.
Despite the frequent detection of circulating tumor antigen–specific T cells, either spontaneously or following active immunization or adoptive transfer, immune-mediated cancer regression occurs only in the minority of patients. One theoretical rate-limiting step is whether effector T cells successfully migrate into metastatic tumor sites. Affymetrix gene expression profiling done on a series of metastatic melanoma biopsies revealed a major segregation of samples based on the presence or absence of T-cell-associated transcripts. The presence of lymphocytes correlated with the expression of defined chemokine genes. A subset of six chemokines (CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10) was confirmed by protein array and/or quantitative reverse transcription-PCR to be preferentially expressed in tumors that contained T cells. Corresponding chemokine receptors were found to be up-regulated on human CD8+ effector T cells, and transwell migration assays confirmed the ability of each of these chemokines to promote migration of CD8+ effector cells in vitro. Screening by chemokine protein array identified a subset of melanoma cell lines that produced a similar broad array of chemokines. These melanoma cells more effectively recruited human CD8+ effector T cells when implanted as xenografts in nonobese diabetic/severe combined immunodeficient mice in vivo. Chemokine blockade with specific antibodies inhibited migration of CD8+ T cells. Our results suggest that lack of critical chemokines in a subset of melanoma metastases may limit the migration of activated T cells, which in turn could limit the effectiveness of antitumor immunity.
PMCID: PMC3886718  PMID: 19293190
17.  In vitro migration of cytotoxic T lymphocyte derived from a colon carcinoma patient is dependent on CCL2 and CCR2 
Infiltration of colorectal carcinomas (CRC) with T-cells has been associated with good prognosis. There are some indications that chemokines could be involved in T-cell infiltration of tumors. Selective modulation of chemokine activity at the tumor site could attract immune cells resulting in tumor growth inhibition. In mouse tumor model systems, gene therapy with chemokines or administration of antibody (Ab)-chemokine fusion proteins have provided potent immune mediated tumor rejection which was mediated by infiltrating T cells at the tumor site. To develop such immunotherapeutic strategies for cancer patients, one must identify chemokines and their receptors involved in T-cell migration toward tumor cells.
To identify chemokine and chemokine receptors involved in T-cell migration toward CRC cells, we have used our previously published three-dimensional organotypic CRC culture system. Organotypic culture was initiated with a layer of fetal fibroblast cells mixed with collagen matrix in a 24 well tissue culture plate. A layer of CRC cells was placed on top of the fibroblast-collagen layer which was followed by a separating layer of fibroblasts in collagen matrix. Anti-CRC specific cytotoxic T lymphocytes (CTLs) mixed with fibroblasts in collagen matrix were placed on top of the separating layer. Excess chemokine ligand (CCL) or Abs to chemokine or chemokine receptor (CCR) were used in migration inhibition assays to identify the chemokine and the receptor involved in CTL migration.
Inclusion of excess CCL2 in T-cell layer or Ab to CCL2 in separating layer of collagen fibroblasts blocked the migration of CTLs toward tumor cells and in turn significantly inhibited tumor cell apoptosis. Also, Ab to CCR2 in the separating layer of collagen and fibroblasts blocked the migration of CTLs toward tumor cells and subsequently inhibited tumor cell apoptosis. Expression of CCR2 in four additional CRC patients' lymphocytes isolated from infiltrating tumor tissues suggests their role in migration in other CRC patients.
Our data suggest that CCL2 secreted by tumor cells and CCR2 receptors on CTLs are involved in migration of CTLs towards tumor. Gene therapy of tumor cells with CCL2 or CCL2/anti-tumor Ab fusion proteins may attract CTLs that potentially could inhibit tumor growth.
PMCID: PMC3076246  PMID: 21450101
18.  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
19.  Proteinase-Activated Receptor-1, CCL2, and CCL7 Regulate Acute Neutrophilic Lung Inflammation 
PAR1 plays a central role in mediating the interplay between coagulation and inflammation, but its role in regulating acute neutrophilic inflammation is unknown. We report that antagonism of PAR1 was highly effective at reducing acute neutrophil accumulation in a mouse model of LPS-induced lung inflammation. PAR1 antagonism also reduced alveolar–capillary barrier disruption in these mice. This protection was associated with a reduction in the expression of the chemokines, CCL2 and CCL7, but not the proinflammatory cytokines, TNF and IL-6, or the classic neutrophil chemoattractants, CXCL1 and CXCL2. Antibody neutralization of CCL2 and CCL7 significantly reduced LPS-induced total leukocyte and neutrophil accumulation, recovered from the bronchoalveolar lavage fluid of challenged mice. Immunohistochemical analysis revealed that CCL2 predominantly localized to alveolar macrophages and pulmonary epithelial cells, whereas CCL7 was restricted to the pulmonary epithelium. In keeping with these observations, the intranasal administration of recombinant CCL2 (rCCL2) and rCCL7 led to the accumulation of neutrophils within the lung airspaces of naive mice in the absence of any underlying inflammation. Flow cytometry analysis further demonstrated an increase in Ly6Ghi neutrophils expressing the chemokine receptors, CCR1 and CCR2, isolated from mouse lungs compared with circulating neutrophils. Conversely, the expression of CXCR2 decreased on neutrophils isolated from the lung compared with circulating neutrophils. Furthermore, this switch in chemokine receptor expression was accentuated after acute LPS-induced lung inflammation. Collectively, these findings reveal a novel role for PAR1 and the chemokines, CCL2 and CCL7, during the early events of acute neutrophilic inflammation.
PMCID: PMC3930934  PMID: 23972264
inflammation; lung; neutrophil; chemokine; proteinase-activated receptor-1
20.  Exosome derived from epigallocatechin gallate treated breast cancer cells suppresses tumor growth by inhibiting tumor-associated macrophage infiltration and M2 polarization 
BMC Cancer  2013;13:421.
Tumor-associated macrophages (TAM) play an important role in tumor microenvironment. Particularly, M2 macrophages contribute to tumor progression, depending on the expression of NF-κB. Tumor-derived exosomes can modulate tumor microenvironment by transferring miRNAs to immune cells. Epigallocatechin gallate (EGCG) has well known anti-tumor effects; however, no data are available on the influence of EGCG on communication with cancer cells and TAM.
Murine breast cancer cell lines, 4T1, was used for in vivo and ex vivo studies. Exosome was extracted from EGCG-treated 4T1 cells, and the change of miRNAs was screened using microarray. Tumor cells or TAM isolated from murine tumor graft were incubated with exosomes derived from EGCG-treated and/or miR-16 inhibitor-transfected 4T1 cells. Chemokines for monocytes (CSF-1 and CCL-2), cytokines both with high (IL-6 and TGF-β) and low (TNF-α) expression in M2 macrophages, and molecules in NF-κB pathway (IKKα and Iκ-B) were evaluated by RT-qPCR or western blot.
EGCG suppressed tumor growth in murine breast cancer model, which was associated with decreased TAM and M2 macrophage infiltration. Expression of chemokine for monocytes (CSF-1 and CCL-2) were low in tumor cells from EGCG-treated mice, and cytokines of TAM was skewed from M2- into M1-like phenotype by EGCG as evidenced by decreased IL-6 and TGF-β and increased TNF-α. Ex vivo incubation of isolated tumor cells with EGCG inhibited the CSF-1 and CCL-2 expression. Ex vivo incubation of TAM with exosomes from EGCG-treated 4T1 cells led to IKKα suppression and concomitant I-κB accumulation; increase of IL-6 and TGF-β; and, decrease of TNF-α. EGCG up-regulated miR-16 in 4T1 cells and in the exosomes. Treatment of tumor cells or TAM with exosomes derived from EGCG-treated and miR-16-knock-downed 4T1 cells restored the above effects on chemokines, cytokines, and NF-κB pathway elicited by EGCG-treated exosomes.
Our data demonstrate that EGCG up-regulates miR-16 in tumor cells, which can be transferred to TAM via exosomes and inhibits TAM infiltration and M2 polarization. We suggest a novel mechanism by which EGCG exerts anti-tumor activity via regulation of TAM in tumor microenvironment.
PMCID: PMC3848851  PMID: 24044575
EGCG; Exosomes; miR-16; Tumor microenvironment; Tumor-associated macrophages (TAM)
21.  Myeloid Cell Receptor LRP1/CD91 Regulates Monocyte Recruitment and Angiogenesis in Tumors 
Cancer research  2013;73(13):3902-3912.
Recruitment of monocytes into sites of inflammation is essential in the immune response. In cancer, recruited monocytes promote invasion, metastasis, and possibly angiogenesis. LDL receptor-related protein (LRP1) is an endocytic and cell-signaling receptor that regulates cell migration. In this study, we isografted PanO2 pancreatic carcinoma cells into mice in which LRP1 is deleted in myeloid lineage cells. Recruitment of monocytes into orthotopic and subcutaneous tumors was significantly increased in these mice, compared with control mice. LRP1-deficient bone marrow-derived macrophages (BMDMs) expressed higher levels of multiple chemokines, including, most prominently, macrophage inflammatory protein-1α/CCL3, which is known to amplify inflammation. Increased levels of CCL3 were detected in LRP1-deficient tumor-associated macrophages (TAMs), isolated from PanO2 tumors, and in RAW 264.7 macrophage-like cells in which LRP1 was silenced. LRP1-deficient BMDMs migrated more rapidly than LRP1-expressing cells in vitro. The difference in migration was reversed by CCL3-neutralizing antibody, by CCR5-neutralizing antibody, and by inhibiting NFκB with JSH-23. Inhibiting NFκB reversed the increase in CCL3 expression associated with LRP1 gene-silencing in RAW 264.7 cells. Tumors formed in mice with LRP1-deficient myeloid cells demonstrated increased angiogenesis. Although VEGF mRNA expression was not increased in LRP1-deficient TAMs, at the single-cell level, the increase in TAM density in tumors with LRP1-deficient myeloid cells may have allowed these TAMs to contribute an increased amount of VEGF to the tumor microenvironment. Our results demonstrate that macrophage density in tumors is correlated with cancer angiogenesis in a novel model system. Myeloid cell LRP1 may be an important regulator of cancer progression.
PMCID: PMC3702673  PMID: 23633492
22.  Phenotypic characterisation of lung macrophages in asthma: over-expression of CCL17 
Studies using monocyte-derived macrophages (MDM) and animal models have suggested a role for alternatively-activated (M2) macrophages in asthmatic inflammation, but in vivo evidence for this phenotype in human asthma is lacking.
Phenotypically to characterize lung macrophages from asthmatic patients in relation to disease severity and treatment.
M2 biomarkers were first identified using MDM exposed to Th2 cytokines and then used to phenotype sputum and bronchoalveolar lavage (BAL) macrophages from 12 healthy controls, 12 mild and 14 moderate asthmatics and to assess the effects of corticosteroids and phosphatidylinositol-3-kinase (PI3K) inhibitors.
Sputum macrophages from asthmatics expressed significantly more CCL17 mRNA but less CD163 than macrophages from healthy individuals. However, none of the other M2 biomarkers were differentially expressed in asthma and ex vivo BAL cells spontaneously produced similar amounts of M2 cytokine/chemokines (IL-10, CCL17 and CCL22). CCL17 mRNA over-expression correlated weakly but significantly with sputum eosinophilia (p=0.0252) and was also observed in macrophages from moderate asthmatics treated with inhaled steroids, suggesting relative insensitivity to inhibition by corticosteroids. The PI3Kinase inhibitor LY294002 inhibited basal CCL17 release from BAL cells and IL-4-stimulated release from MDM.
This study does not support the existence in human asthma of the full M2 phenotype described to date, but points to upregulation of CCL17 in both mild and moderate asthma, providing a further source for this ligand of CCR4+ cells that contribute to airways inflammation. CCL17 expression is corticosteroid resistant but is suppressed by PI3Kinase enzyme inhibitors.
PMCID: PMC3805016  PMID: 22981793
23.  Macrophage Inflammatory Protein 1α/CCL3 Is Required for Clearance of an Acute Klebsiella pneumoniae Pulmonary Infection 
Infection and Immunity  2001;69(10):6364-6369.
The objective of these studies was to determine the role of macrophage inflammatory protein 1α/CCL3 in pulmonary host defense during Klebsiella pneumoniae infection. Following intratracheal inoculation, 7-day survival of CCL3−/− mice was less than 10%, compared to 60% for CCL3+/+ mice. Survival of CCR5−/− mice was equivalent to that of controls, indicating that the enhanced susceptibility of CCL3−/− mice to K. pneumoniae is mediated via another CCL3 receptor, presumably CCR1. At day 3, CFU burden in the lungs of CCL3−/− mice was 800-fold higher than in CCL3+/+ mice, demonstrating that CCL3 is critical for control of bacterial growth in the lung. Surprisingly, CCL3−/− mice had no differences in the recruitment of monocytes/macrophages and even showed enhanced neutrophil recruitment at days 1, 2, and 3 postinfection, compared to CCL3+/+ mice. Therefore, the defect in clearance was not due to insufficient recruitment of leukocytes. No significant differences in cytokine levels of monocyte chemoattractant protein 1 (MCP-1), interleukin 12, gamma interferon, or tumor necrosis factor alpha in lung lavages were found between CCL3+/+ and CCL3−/− mice. CCL3−/− alveolar macrophages were found to have significantly lower phagocytic activity toward K. pneumoniae than CCL3+/+ alveolar macrophages. These findings demonstrate that CCL3 production is critical for activation of alveolar macrophages to control the pulmonary growth of the gram-negative bacterium K. pneumoniae.
PMCID: PMC98771  PMID: 11553580
24.  Dynamic interaction networks in a hierarchically organized tissue 
We have integrated gene expression profiling with database and literature mining, mechanistic modeling, and cell culture experiments to identify intercellular and intracellular networks regulating blood stem cell self-renewal.Blood stem cell fate in vitro is regulated non-autonomously by a coupled positive–negative intercellular feedback circuit, composed of megakaryocyte-derived stimulatory growth factors (VEGF, PDGF, EGF, and serotonin) versus monocyte-derived inhibitory factors (CCL3, CCL4, CXCL10, TGFB2, and TNFSF9).The antagonistic signals converge in a core intracellular network focused around PI3K, Raf, PLC, and Akt.Model simulations enable functional classification of the novel endogenous ligands and signaling molecules.
Intercellular (between cell) communication networks are required to maintain homeostasis and coordinate regenerative and developmental cues in multicellular organisms. Despite the recognized importance of intercellular networks in regulating adult stem and progenitor cell fate, the specific cell populations involved, and the underlying molecular mechanisms are largely undefined. Although a limited number of studies have applied novel bioinformatic approaches to unravel intercellular signaling in other cell systems (Frankenstein et al, 2006), a comprehensive analysis of intercellular communication in a stem cell-derived, hierarchical tissue network has yet to be reported.
As a model system to explore intercellular communication networks in a hierarchically organized tissue, we cultured human umbilical cord blood (UCB)-derived stem and progenitor cells in defined, minimal cytokine-supplemented liquid culture (Madlambayan et al, 2006). To systematically explore the molecular and cellular dynamics underlying primitive progenitor growth and differentiation, gene expression profiles of primitive (lineage negative; Lin−) and mature (lineage positive; Lin+) populations were generated during phases of stem cell expansion versus depletion. Parallel phenotypic and subproteomic experiments validated that mRNA expression correlated with complex measures of proteome activity (protein secretion and cell surface expression). Using a curated list of secreted ligand–receptor interactions and published expression profiles of purified mature blood populations, we implemented a novel algorithm to reconstruct the intercellular signaling networks established between stem cells and multi-lineage progeny in vitro. By correlating differential expression patterns with stem cell growth, we predict cell populations, pathways, and secreted ligands associated with stem cell self-renewal and differentiation (Figure 3A).
We then tested the correlative predictions in a series of cell culture experiments. UCB progenitor cell cultures were supplemented with saturating amounts of 18 putative regulatory ligands, or cocultured with purified mature blood lineages (megakaryocytes, monocytes, and erythrocytes), and analyzed for effects on total cell, progenitor, and primitive progenitor growth. At the primitive progenitor level, 3/5 novel predicted stimulatory ligands (EGF, PDGFB, and VEGF) displayed significant positive effects, 5/7 predicted inhibitory factors (CCL3, CCL4, CXCL10, TNFSF9, and TGFB2) displayed negative effects, whereas only 1/5 non-correlated ligand (CXCL7) displayed an effect. Also consistent with predictions from gene expression data, megakaryocytes and monocytes were found to stimulate and inhibit primitive progenitor growth, respectively, and these effects were attributable to differential secretome profiles of stimulatory versus inhibitory ligands.
Cellular responses to external stimuli, particularly in heterogeneous and dynamic cell populations, represent complex functions of multiple cell fate decisions acting both directly and indirectly on the target (stem cell) populations. Experimentally distinguishing the mode of action of cytokines is thus a difficult task. To address this we used our previously published interactive model of hematopoiesis (Kirouac et al, 2009) to classify experimentally identified regulatory ligands into one of four distinct functional categories based on their differential effects on cell population growth. TGFB2 was classified as a proliferation inhibitor, CCL4, CXCL10, SPARC, and TNFSF9 as self-renewal inhibitors, CCL3 a proliferation stimulator, and EGF, VEGF, and PDGFB as self-renewal stimulators.
Stem and progenitor cells exposed to combinatorial extracellular signals must propagate this information through intracellular molecular networks, and respond appropriately by modifying cell fate decisions. To explore how our experimentally identified positive and negative regulatory signals are integrated at the intracellular level, we constructed a blood stem cell self-renewal signaling network through extensive literature curation and protein–protein interaction (PPI) network mapping. We find that signal transduction pathways activated by the various stimulatory and inhibitory ligands converge on a limited set of molecular control nodes, forming a core subnetwork enriched for known regulators of self-renewal (Figure 6A). To experimentally test the intracellular signaling molecules computationally predicted as regulators of stem cell self-renewal, we obtained five small molecule antagonists against the kinases Phosphatidylinositol 3-kinase (PI3K), Raf, Akt, Phospholipase C (PLC), and MEK1. Liquid cultures were supplemented with the five molecules individually, and resultant cell population outputs compared against model simulations to deconvolute the functional effects on proliferation (and survival) versus self-renewal. This analysis classifies inhibition of PI3K and Raf activity as selectively targeting self-renewal, PLC as selectively targeting survival, and Akt as selectively targeting proliferation; MEK inhibition appears non-specific for these processes.
This represents the first systematic characterization of how cell fate decisions are regulated non-autonomously through lineage-specific interactions with differentiated progeny. The complex intercellular communication networks can be approximated as an antagonistic positive–negative feedback circuit, wherein progenitor expansion is modulated by a balance of megakaryocyte-derived stimulatory factors (EGF, PDGF, VEGF, and possibly serotonin) versus monocyte-derived inhibitory factors (CCL3, CCL4, CXCL10, TGFB2, and TNFSF9). This complex milieu of endogenous regulatory signals is integrated and processed within a core intracellular signaling network, resulting in modulation of cell-level kinetic parameters (proliferation, survival, and self-renewal). We reconstruct a stem cell associated intracellular network, and identify PI3K, Raf, Akt, and PLC as functionally distinct signal integration nodes, linking extracellular and intracellular signaling. These findings lay the groundwork for novel strategies to control blood stem cell self-renewal in vitro and in vivo.
Intercellular (between cell) communication networks maintain homeostasis and coordinate regenerative and developmental cues in multicellular organisms. Despite the importance of intercellular networks in stem cell biology, their rules, structure and molecular components are poorly understood. Herein, we describe the structure and dynamics of intercellular and intracellular networks in a stem cell derived, hierarchically organized tissue using experimental and theoretical analyses of cultured human umbilical cord blood progenitors. By integrating high-throughput molecular profiling, database and literature mining, mechanistic modeling, and cell culture experiments, we show that secreted factor-mediated intercellular communication networks regulate blood stem cell fate decisions. In particular, self-renewal is modulated by a coupled positive–negative intercellular feedback circuit composed of megakaryocyte-derived stimulatory growth factors (VEGF, PDGF, EGF, and serotonin) versus monocyte-derived inhibitory factors (CCL3, CCL4, CXCL10, TGFB2, and TNFSF9). We reconstruct a stem cell intracellular network, and identify PI3K, Raf, Akt, and PLC as functionally distinct signal integration nodes, linking extracellular, and intracellular signaling. This represents the first systematic characterization of how stem cell fate decisions are regulated non-autonomously through lineage-specific interactions with differentiated progeny.
PMCID: PMC2990637  PMID: 20924352
cellular networks; hematopoiesis; intercellular signaling; self-renewal; stem cells
25.  A Destructive Cascade Mediated by CCL2 Facilitates Prostate Cancer Growth in Bone 
Cancer research  2009;69(4):1685-1692.
Monocyte chemoattractant protein 1 (MCP-1; CCL2) is a recently identified prominent regulator of prostate cancer growth and metastasis. The purpose of this study was to investigate the mechanistic role of CCL2 in prostate cancer growth in bone. The present study found that CCL2 was upregulated in osteoblasts (3-fold by PC3 and 2-fold by VCaP conditioned media) and endothelial cell (2-fold by PC3 and VCaP conditioned media). PTHrP treatment of osteoblastic cells upregulated CCL2 and was blocked by a PTHrP antagonist suggesting prostate cancer-derived PTHrP plays an important role in elevation of osteoblast derived CCL2. CCL2 indirectly increased blood vessel formation in endothelial cells through VEGF-A, which was up-regulated 2-fold with administration of CCL2 in prostate cancer cells. In vivo, anti-CCL2 treatment suppressed tumor growth in bone. The decreased tumor burden was associated with decreased bone resorption (serum TRAP5b levels were decreased by 50~60% in anti-CCL2 treated animals from VCaP or PC-3 cell osseous lesions) and micro-vessel density was decreased by 70% in anti-CCL2 treated animals with bone lesions from VCaP cells. These data suggest that a destructive cascade is driven by tumor cell-derived PTHrP mediated induction of CCL2, which facilitates tumor growth via enhanced osteoclastic and endothelial cell activity in bone marrow. Taken together, CCL2 mediates the interaction between tumor-derived factors and host-derived chemokines acting in cooperation to promote skeletal metastasis.
PMCID: PMC2698812  PMID: 19176388
CCL2; prostate cancer; parathyroid hormone related protein

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