The combined action of IL-15 and IL-12 gene transfer can induce tumor cell rejection without T and NK cell involvement

The cooperative antitumor effects of IL-12 and IL-15 gene transfer were studied in the N592 MHC class I-negative small cell lung cancer cell line xenotransplanted in nude mice. N592 cells engineered to secrete IL-15 displayed a significantly reduced tumor growth kinetics, and a slightly reduced tumor take rate, while N592 engineered with IL-12 displayed only minor changes in their growth in nude mice. However, N592 cells producing both cytokines were completely rejected, and produced a potent local bystander effect, inducing rejection of coinjected wild-type tumor cells. N592/IL-12/IL-15 cells were completely and promptly rejected also in NK-depleted nude mice, while in granulocyte-depleted animals a slight delay in the rejection process was observed. Immunohistochemical analyses of the N592/IL-12/IL-15 tumor area in intact nude mice revealed the presence of infiltrating macrophages, granulocytes, and NK cells, and expression of inducible NO synthase and of secondary cytokines such as IL-1beta, TNF-alpha, and IFN-gamma, and at higher levels GM-CSF, macrophage-inflammatory protein-2, and monocyte chemoattractant protein-1. In NK cell-depleted nude mice, numerous macrophages and granulocytes infiltrated the tumor, and a strong expression of macrophage-inflammatory protein-2 and inducible NO synthase was also observed. Finally, macrophages cocultured with N592/IL-12/IL-15 produced NO in vitro, and inhibited tumor cell growth, further suggesting their role as effector cells in this model.     

Irradiated tumor cells adenovirally engineered to secrete granulocyte/macrophage-colony-stimulating factor establish antitumor immunity and eliminate pre-existing tumors in syngeneic mice

The specific aim of this study was to examine the prophylactic as well as the therapeutic efficacies of irradiated mouse CT26 colon cancer cells, infected with recombinant adenoviruses harboring cDNAs specific for granulocyte macrophage-colony-stimulating factor (GM-CSF), interferon (IFN-γ) and monocyte chemotactic protein1 (MCP-1). Results showed that tumor cells secrete the respective cytokines for several days after infection and subsequent irradiation. Vaccination with irradiated GM-CSF-secreting CT26 cells protected 90% of syngeneic mice challenged with live parental cells. On the other hand, vaccination with irradiated IFNγ or MCP-1-secreting CT26 cells totally failed to protect mice from tumor development after challenge with parental cells. None of the tumor-free mice initially vaccinated with irradiated GM-CSF-producing CT26 cells developed tumor upon repeated challenge with parental cells during the entire observation period. The establishment of specific and long-lasting antitumor immunity following vaccination with GM-CSF-producing tumor cells requires the simultaneous presence of GM-CSF and tumor antigen at the vaccine site. Depletion of CD8⁺ cells, but not CD4⁺ cells, blocked the vaccine efficacy of GM-CSF-producing tumor cells. Subcutaneous injection of irradiated GM-CSF-producing CT26 cells also effectively prevented the growth of a small load of parental tumor that was implanted 3 days earlier or the development of metastatic foci in the lung from intravenously injected parental cells either 7 days before or 3 days after vaccination. Our data thus show that, in these experimental tumor models, subcutaneous injection of irradiated tumor cells adenovirally, transduced with the GM-CSF gene leads not only to prevention of growth of subsequently implanted tumor but also to elimination of pre-existing and metastatic tumors.     

Chimeric antigen receptor T cells shape myeloid cell function within the tumor microenvironment through IFN-γ and GM-CSF

The infiltration of suppressive myeloid cells into the tumor microenvironment restrains anti-tumor immunity. However, cytokines may alter the function of myeloid lineage cells to support tumor rejection, regulating the balance between pro- and anti-tumor immunity. In this study, it is shown that effector cytokines secreted by adoptively transferred T cells expressing a chimeric Ag receptor (CAR) shape the function of myeloid cells to promote endogenous immunity and tumor destruction. Mice bearing the ovarian ID8 tumor were treated with T cells transduced with a chimeric NKG2D receptor. GM-CSF secreted by the adoptively transferred T cells recruited peripheral F4/80(lo)Ly-6C(+) myeloid cells to the tumor microenvironment in a CCR2-dependent fashion. T cell IFN-γ and GM-CSF activated local, tumor-associated macrophages, decreased expression of regulatory factors, increased IL-12p40 production, and augmented Ag processing and presentation by host macrophages to Ag-specific T cells. In addition, T cell-derived IFN-γ, but not GM-CSF, induced the production of NO by F4/80(hi) macrophages and enhanced their lysis of tumor cells. The ability of CAR T cell therapy to eliminate tumor was moderately impaired when inducible NO synthase was inhibited and greatly impaired in the absence of peritoneal macrophages after depletion with clodronate encapsulated liposomes. This study demonstrates that the activation of host macrophages by CAR T cell-derived cytokines transformed the tumor microenvironment from immunosuppressive to immunostimulatory and contributed to inhibition of ovarian tumor growth.     

Low-level monocyte chemoattractant protein-1 stimulation of monocytes leads to tumor formation in nontumorigenic melanoma cells

Tumors commonly produce chemokines for recruitment of host cells, but the biological significance of tumor-infiltrating inflammatory cells, such as monocytes/macrophages, for disease outcome is not clear. Here, we show that all of 30 melanoma cell lines secreted monocyte chemoattractant protein-1 (MCP-1), whereas normal melanocytes did not. When low MCP-1-producing melanoma cells from a biologically early, nontumorigenic stage were transduced to overexpress the MCP-1 gene, tumor formation depended on the level of chemokine secretion and monocyte infiltration; low-level MCP-1 secretion with modest monocyte infiltration resulted in tumor formation, whereas high secretion was associated with massive monocyte/macrophage infiltration into the tumor mass, leading to its destruction within a few days after injection into mice. Tumor growth stimulated by monocytes/macrophages was due to increased angiogenesis. Vessel formation in vitro was inhibited with mAbs against TNF-alpha, which, when secreted by cocultures of melanoma cells with human monocytes, induced endothelial cells under collagen gels to form branching, tubular structures. These studies demonstrate that the biological effects of tumor-derived MCP-1 are biphasic, depending on the level of secretion. This correlates with the degree of monocytic cell infiltration, which results in increased tumor vascularization and TNF-alpha production.     

CD40 ligand-CD40 interaction induces chemokines in cervical carcinoma cells in synergism with IFN-gamma

Cellular immunity plays a major role in controlling human papilloma virus infection and development of cervical carcinoma. Mononuclear cell infiltration possibly due to the action of chemokines becomes prominent in the tumor tissue. In fact, the macrophage chemoattractant protein-1, MCP-1, was detected in cervical squamous cell carcinoma in situ, whereas absent in cultured cells. From this, unknown environmental factors were postulated regulating chemokine expression in vivo. In this study, we show high CD40 expression on cervical carcinoma cells and CD40 ligand (CD40L) staining on attracted T cells in tumor tissue, suggesting a paracrine stimulation mechanism via CD40L-CD40 interactions. We therefore investigated chemokine synthesis in nonmalignant and malignant human papilloma virus-positive cell lines after CD40L exposure. Constitutive expression of MCP-1, MCP-3, RANTES, and IFN-gamma-inducible protein-10 was almost undetectable in all cell lines tested. CD40L was able to induce MCP-1 production; however, despite much higher CD40 expression in malignant cells, MCP-1 induction was significantly lower compared with nontumorigenic cells. After sensitization with IFN-gamma, another T cell-derived cytokine showing minimal effects on CD40 expression levels, CD40 ligation led to a more than 20-fold MCP-1 induction in carcinoma cell lines. An even stronger effect was observed for IFN-gamma-inducible protein-10. Our study highlights the synergism of T cell-derived mediators such as CD40L and IFN-gamma for chemokine responses in cervical carcinoma cells, helping to understand the chemokine expression patterns observed in vivo.     

Role of chemokines and formyl peptides in pneumococcal pneumonia-induced monocyte/macrophage recruitment

Host-derived chemoattractant factors are suggested to play crucial roles in leukocyte recruitment elicited by inflammatory stimuli in vitro and in vivo. However, in the case of acute bacterial infections, pathogen-derived chemoattractant factors are also present, and it has not yet been clarified how cross-talk between chemoattractant receptors orchestrates diapedesis of leukocytes in this context of complex chemoattractant arrays. To investigate the role of chemokine (host-derived) and formyl peptide (pathogen-derived) chemoattractants in leukocyte extravasation in life-threatening infectious diseases, we used a mouse model of pneumococcal pneumonia. We found an increase in mRNA expression of eight chemokines (RANTES, macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, IP-10, monocyte chemoattractant protein (MCP)-1, T cell activation 3, and KC) within the lungs during the course of infection. KC and MIP-2 protein expression closely preceded pulmonary neutrophil recruitment, whereas MCP-1 protein production coincided more closely than MIP-1alpha with the kinetics of macrophage infiltration. In situ hybridization of MCP-1 mRNA suggested that MCP-1 expression started at peribronchovascular regions and expanded to alveoli-facing epithelial cells and infiltrated macrophages. Interestingly, administration of a neutralizing Ab against MCP-1, RANTES, or MIP-1alpha alone did not prevent macrophage infiltration into infected alveoli, whereas combination of the three Abs significantly reduced macrophage infiltration without affecting neutrophil recruitment. The use of an antagonist to N-formyl peptides, N-t-Boc-Phe-D-Leu-Phe-D-Leu-Phe, reduced both macrophages and neutrophils significantly. These data demonstrate that a complex chemokine network is activated in response to pulmonary pneumococcal infection, and also suggest an important role for fMLP receptor in monocyte/macrophage recruitment in that model.     

Stat3 activity in melanoma cells affects migration of immune effector cells and nitric oxide-mediated antitumor effects

Infiltration of immune effector cells in tumors is critical for antitumor immune responses. However, what regulates immune cell infiltration of tumors remains to be identified. Stat3 is constitutively activated with high frequency in diverse cancers, promoting tumor cell growth and survival. Blocking Stat3 signaling in tumors in vivo results in tumor growth inhibition that involves killing of nontransfected tumor cells and infiltration of immune effector cells, suggesting that Stat3 activity in tumor cells might affect immune cell recruitment. However, dying tumor cells can also attract immune cells. In this study, we show in isogenic murine melanomas that natural Stat3 activity is associated with tumor growth and reduction of T cell infiltration. Blocking Stat3 signaling in the melanoma cells containing high Stat3 activity results in expression of multiple chemoattractants, leading to increased migration of lymphocytes, NK cells, neutrophils, and macrophages. In addition, blocking Stat3 triggers tumor cells to produce soluble factors capable of activating macrophage production of NO in vitro and in vivo. TNF-alpha and IFN-beta, which are secreted by Stat3-inhibited tumor cells, are able to activate macrophage NO production, whereas neutralizing TNF-alpha in the tumor supernatant from Stat3-blocked tumor cells abrogates nitrite production. Moreover, interrupting Stat3 signaling in tumor cells leads to macrophage-mediated, nitrite-dependent cytostatic activity against nontransduced tumor cells. These results suggest that tumor Stat3 activity affects recruitment of diverse immune effectors and it can be manipulated to activate the effector phase of innate immune responses.     

Poly I:C activated macrophages are tumoricidal for TNF-alpha-resistant 3LL tumor cells

Successive coculture of Lewis lung carcinoma (3LL) cells with T cell-derived lymphokines and LPS-activated macrophages has led to the acquisition of 3LL tumor variants (macrophage-resistant 3LL tumor variants (3LL-R)), manifesting a highly reduced sensitivity to the cytotoxic potential of T cell-derived lymphokines and LPS-activated macrophages and TNF-alpha. However, when 3LL-R cells are cocultured with Poly I:C-activated macrophages or with conditioned medium derived from these effector cells a significant lysis is observed. TNF-alpha participates in the cytolytic process of Poly I:C-activated macrophages as anti-TNF-alpha antibodies abolish the cytotoxic effect of these effector cells. In addition, class I IFN is involved because IFN-alpha and IFN-beta act synergistically on TNF-alpha mediated lysis of 3LL-R cells within 18 h. Moreover, anticlass I IFN antibodies abolish the cytolytic capacity of Poly I:C-activated macrophages. Hence, Poly I:C-induced macrophage-mediated cytolysis of 3LL-R cells may result from 1) the induction of macrophages by Poly I:C to secrete high amounts of TNF-alpha and class I IFN and 2) a synergism between IFN-alpha/IFN-beta and TNF-alpha on lysis of 3LL-R cells. This synergism does not result from a class I IFN-mediated enhancement of TNF-alpha receptor expression on 3LL-R cells. Therefore, the sensitivity of 3LL-R cells to TNF-alpha-mediated lysis in the presence of class I IFN is most probably regulated at the post-TNF-alpha receptor level. Furthermore, treatment of mice with Poly I:C strongly reduces the metastatic capacity of 3LL-R tumor cells, suggesting the participation of macrophages in the eradication of the established metastasis. Hence, TNF-alpha-resistant 3LL-R tumor cells may serve as a useful tool for the detection of alternative macrophage-related cytotoxins leading to the destruction of neoplastic cells both in vitro and in vivo.     

Modulation of the invasive phenotype of human colon carcinoma cells by organ specific fibroblasts of nude mice

We examined whether fibroblasts from subcutaneous, colon or lung tissues of nude mice influence the invasive potential of highly metastatic human colon carcinoma KM12SM cells. Primary cultures of nude mouse fibroblasts from skin, lung and colon were established. Invasive and metastatic KM12SM cells were cultured alone or with fibroblasts. Growth and invasive properties of the KM12SM cells were evaluated as well as their production of gelatinase activity. KM12SM cells were able to grow on monolayers of all three fibroblast cultures but did not invade through skin fibroblasts. The conditioned media of KM12SM cells cocultured with skin, colon or lung fibroblasts were examined for the presence of type IV collagenase (gelatinase). KM12SM growing on plastic and on colon or lung fibroblasts produced significant levels of latent and active forms of 64 kDa type IV collagenase, whereas KM12SM cells cocultivated with nude mouse skin fibroblasts did not. In contrast, human squamous cell carcinoma A431 cells produced significant levels of collagenase type IV when cocultured with nude mouse skin fibroblasts, a tissue they invaded and completely penetrated. Incubation of KM12SM cells in serum-free medium containing recombinant human interferon-beta (fibroblast interferon) was associated with significant reduction in gelatinase activity. Since the production of type IV collagenase by human colon cancer cells is specifically inhibited by mouse skin fibroblasts but not by colon or lung fibroblasts the data suggest that organ-specific fibroblasts can influence the invasive and metastatic properties of KM12SM cells.     

GM-CSF production by glioblastoma cells has a functional role in eosinophil survival, activation, and growth factor production for enhanced tumor cell proliferation

Medicinal interventions of limited efficacy are currently available for the treatment of glioblastoma multiforme (GBM), the most common and lethal primary brain tumor in adults. The eosinophil is a pivotal immune cell in the pathobiology of atopic disease that is also found to accumulate in certain tumor tissues. Inverse associations between atopy and GBM risk suggest that the eosinophil may play a functional role in certain tumor immune responses. To assess the potential interactions between eosinophils and GBM, we cultured human primary blood eosinophils with two separate human GBM-derived cell lines (A172, U87-MG) or conditioned media generated in the presence or absence of TNF-α. Results demonstrated differential eosinophil adhesion and increased survival in response to coculture with GBM cell lines. Eosinophil responses to GBM cell line-conditioned media included increased survival, activation, CD11b expression, and S100A9 release. Addition of GM-CSF neutralizing Abs to GBM cell cultures or conditioned media reduced eosinophil adhesion, survival, and activation, linking tumor cell-derived GM-CSF to the functions of eosinophils in the tumor microenvironment. Dexamethasone, which has been reported to inhibit eosinophil recruitment and shrink GBM lesions on contrast-enhanced scans, reduced the production of tumor cell-derived GM-CSF. Furthermore, culture of GBM cells in eosinophil-conditioned media increased tumor cell viability, and generation of eosinophil-conditioned media in the presence of GM-CSF enhanced the effect. These data support the idea of a paracrine loop between GM-CSF-producing tumors and eosinophil-derived growth factors in tumor promotion/progression.     

Hsp110 over-expression increases the immunogenicity of the murine CT26 colon tumor

Several studies have suggested a positive correlation between heat shock protein (hsp) expression and tumor immunogenicity. Independently, many studies have shown that hsp purified from tumors can be used as a tumor-specific vaccine. In this study, we have explored the connection between hsp expression and anti-tumor immunity by transducing murine CT26 colon carcinoma cells with the cDNA of a major hsp, i.e. hsp110. We have shown that over-expression of hsp110 has no effect on CT26 tumor cell growth in vitro, and does not inhibit their anchorage-independent growth capacity. However, in situ, hsp110 over-expressing CT26 tumor (CT26-hsp110) grew at a significantly reduced rate as compared to the wild-type CT26 tumor in immunocompetent mice. Moreover, immunization of mice with inactivated CT26-hsp110 cells significantly inhibited the growth of wild-type CT26 tumor. This immunity was associated with an increased frequency of tumor-specific T cells after vaccination. An in vivo antibody depletion assay demonstrated that inactivated CT26-hsp110 cells elicited anti-tumor responses involving CD8(+) T cells and natural killer (NK) cells, but not CD4(+) T cells. Lastly, the effect of the addition of granulocyte-macrophage colony stimulating factor (GM-CSF) to these vaccine formulations was determined. Mice immunized with irradiated CT26-hsp110 cells combined with GM-CSF-producing bystander cells revealed a complete inhibition of CT26 tumor growth, indicating a synergy between inactivated CT26-hsp110 vaccine activity and GM-CSF. These observations demonstrate that manipulation of hsp110 expression in tumors, specifically when combined with GM-CSF, represents a potentially powerful approach to cancer vaccine formulation.     

Alveolar macrophage-derived cytokines induce monocyte chemoattractant protein-1 expression from human pulmonary type II-like epithelial cells

Many acute and chronic lung diseases are characterized by the presence of increased numbers of activated macrophages. These macrophages are derived predominantly from newly recruited peripheral blood monocytes and may play a role in the amplification and perpetuation of an initial lung insult. The process of inflammatory cell recruitment is poorly understood, although the expression of inflammatory cell-specific chemoattractants and subsequent generation of chemotactic gradients is likely involved. Although immune cells such as macrophages and lymphocytes are known to generate several inflammatory cell chemoattractants, parenchymal cells can also synthesize and secrete a number of bioactive factors. We now demonstrate the generation of significant monocyte chemotactic activity from tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta-treated pulmonary type II-like epithelial cells (A549). The predominant inducible monocyte chemotaxin had an estimated molecular mass of approximately 14-15 kDa and was neutralized by specific antibody to human monocyte chemotactic protein-1 (MCP-1). Induction of activity was accompanied by increases in steady-state mRNA level for MCP-1. These data are consistent with the induction of MCP-1 expression from A549 cells by TNF and IL-1. MCP-1 production from A549 cells could be induced by lipopolysaccharide (LPS)-stimulated alveolar macrophage (AM)-conditioned media, but not by LPS alone. The inducing activity in AM-conditioned media was neutralized with specific antibodies to IL-1 beta, but not TNF-alpha. Our findings suggest that the alveolar epithelium can participate in inflammatory cell recruitment via the production of MCP-1 and that cytokine networking between contiguous alveolar macrophages and the pulmonary epithelium may be essential for parenchymal cell MCP-1 expression.     

Expression of monocyte chemoattractant protein-1 and distribution of immune cell populations in the bovine corpus luteum throughout the estrous cycle.

This study characterizes the expression of monocyte chemoattractant protein-1 (MCP-1) and the relative distribution of immune cell populations in the bovine corpus luteum throughout the estrous cycle. Immunodetectable MCP-1 was evident in corpora lutea of cows at Days 6, 12, and 18 postovulation (Day 0 = ovulation, n = 4 cows/stage). Day 6 corpora lutea contained minimal MCP-1 that was confined primarily to blood vessels. In contrast, relatively intense staining for MCP-1 was observed in corpora lutea from Days 12 and 18 postovulation. MCP-1 was again most evident in the cells of the vasculature, but it was also observed surrounding individual luteal cells, particularly by Day 18. An increase in immunohistochemical expression of MCP-1 on Days 12 and 18 postovulation corresponded with increases in MCP-1 mRNA and protein in corpora lutea as determined by Northern blot analysis and ELISA. Monocytes and macrophages were the most abundant immune cells detected in the bovine corpus luteum, followed by CD8+ and CD4+ T lymphocytes. In all instances, Day 6 corpora lutea contained fewer immune cells than corpora lutea from Days 12 and 18. In conclusion, increased expression of MCP-1 was accompanied by the accumulation of immune cells in the corpora lutea of cows during the latter half of the estrous cycle (Days 12-18 postovulation). These results support the hypothesis that MCP-1 promotes immune cell recruitment into the corpus luteum to facilitate luteal regression. These results also raise a provocative issue, however, concerning the recruitment of immune cells several days in advance of the onset of luteal regression.     

Amphiregulin and hepatocyte-derived extracellular matrix regulate proliferation and autocrine growth factor expression in colon cancer cell lines of varying liver-colonizing capability

We studied the effect of two members of the epidermal growth factor (EGF) family--amphiregulin and heparin-binding EGF-like growth factor (HB-EGF)-on cell proliferation, growth factor and growth factor receptor expression, and cell differentiation in two human colon cell lines of varying liver-colonizing potential. The effect of amphiregulin and HB-EGF was assessed both in cells grown on plastic, as well as on cells grown on hepatocyte-derived extracellular matrix (ECM). We found that both colon cell lines were sensitive to HB-EGF stimulation of cell proliferation. Amphiregulin inhibited cell proliferation in KM12 cells and stimulated the strongly metastatic cell line KM12SM to a slight extent. When the cells were cultured on hepatocyte-derived ECM, amphiregulin inhibited the weakly metastatic KM12 and stimulated the growth of KM12SM. HB-EGF synergistically acted with hepatocyte-derived ECM to enhance cell proliferation in both colon cell lines. Expression of ligands of the EGF family, such as transforming growth factor-alpha (TGF-alpha) and amphiregulin, was decreased in both cell lines when cultured on ECM. Hepatocyte-derived ECM decreased expression of cripto in KM12 and increased it in KM12SM cells. Neither cripto nor TGF-alpha mRNA levels was affected by growing the cells in the presence of amphiregulin. However, amphiregulin increased expression of its own mRNA in the weakly metastatic KM12 and decreased it in the strongly metastatic KM12SM when the cells were cultured on plastic. Amphiregulin and HB-EGF stimulated expression of erb-B2 in both cell lines cultured on plastic. Surprisingly, when the cells were grown on hepatocyte-derived ECM, amphiregulin inhibited erb-B2 expression in both cell lines. We observed no effect of amphiregulin on cell differentiation as assessed by alkaline phosphatase expression. Our studies demonstrate one mechanism that could play a role in site-specific metastasis. We found an inhibitory response to an autocrine growth factor in the context of hepatocyte-derived ECM in a weakly metastatic cell and a stimulatory effect of the same growth factor when strongly metastatic cells were cultured on the same ECM.     

Expression of theJE/MCP-1 gene suppresses metastatic potential in murine colon carcinoma cells

The purpose of this study was to determine whether the expression of theJE/MCP-1 gene encoding for the monocyte chemottractant protein, MCP-1 (also known as monocyte chemotactic and activating factor MCAF, TDCF, and SMC-CF) can influence the metastatic properties of tumor cells. The highly metastatic murine colon carcinoma CT-26 cells, syngeneic to BALB/c mice that do not produce endogenous JE/MCP-1 protein, were transfected with a BCMGS-Neo expression vector (control) or a vector containing full-lengthJE cDNA. CT-26 parental cells, CT-26 Neo, and CT-26 JE/MCP-1-positive cells were injected into syngeneic or nude mice. The CT-26 JE/MCP-1-positive cells produced significantly fewer lung metastases. The decrease in incidence of metastasis was not due to the inability of the transfected cells to arrest in the lung vasculature or to differences in cell cycle time. CT-26 cells producing JE/MCP-1 were highly susceptible to lysis by syngeneic macrophages treated with subthreshold concentrations of lipopolysaccharide. In addition, culture supernatants of JE/MCP-1-expressing cells plus lipopolysaccharide synergistically activated tumoricidal properties in syngeneic macrophages. This activity was blocked by anti-JE/MCP-1 antibodies, indicating the involvement of the JE/MCP-1 molecule in this process. Moreover, purified JE/MCP-1 added to lipopolysaccharide-containing medium resulted in significant activation of macrophages against parental CT-26 cells. These data suggest that, in addition to its chemotactic properties, JE/MCP-1 can synergize with bacterial endotoxins to activate macrophages to become tumoricidal and, hence, could suppress metastasis.     

GCP-2/CXCL6 synergizes with other endothelial cell-derived chemokines in neutrophil mobilization and is associated with angiogenesis in gastrointestinal tumors

The precise role of chemokines in neovascularization during inflammation or tumor growth is not yet fully understood. We show here that the chemokines granulocyte chemotactic protein-2 (GCP-2/CXCL6), interleukin-8 (IL-8/CXCL8), and monocyte chemotactic protein-1 (MCP-1/CCL2) are co-induced in microvascular endothelial cells after stimulation with pro-inflammatory stimuli. In contrast with its weak proliferative effect on endothelial cells, GCP-2 synergized with MCP-1 in neutrophil chemotaxis. This synergy may represent a mechanism for tumor development and metastasis by providing efficient leukocyte infiltration in the absence of exogenous immune modulators. To mimic endothelial cell-derived GCP-2 in vivo, GCP-2 was intravenously injected and shown to provoke a dose-dependent systemic response, composed of an immediate granulopenia, followed by a profound granulocytosis. By immunohistochemistry, GCP-2 was further shown to be expressed by endothelial cells from human patients with gastrointestinal (GI) malignancies. GCP-2 staining correlated with leukocyte infiltration into the tumor and with the expression of the matrix metalloproteinase-9 (MMP-9/gelatinase B). Together with previous findings, these data suggest that the production of GCP-2 by endothelial cells within the tumor can contribute to tumor development through neovascularization due to endothelial cell chemotaxis and to tumor cell invasion and metastasis by attracting and activating neutrophils loaded with proteases that promote matrix degradation.