Richard G. Thompson

“Re-educating” tumor-associated macrophages by targeting NF-κB

The nuclear factor κB (NF-κB) signaling pathway is important in cancer-related inflammation and malignant progression. Here, we describe a new role for NF-κB in cancer in maintaining the immunosuppressive phenotype of tumor-associated macrophages (TAMs). We show that macrophages are polarized via interleukin (IL)-1R and MyD88 to an immunosuppressive “alternative” phenotype that requires IκB kinase β–mediated NF-κB activation. When NF-κB signaling is inhibited specifically in TAMs, they become cytotoxic to tumor cells and switch to a “classically” activated phenotype; IL-12high, major histocompatibility complex IIhigh, but IL-10low and arginase-1low. Targeting NF-κB signaling in TAMs also promotes regression of advanced tumors in vivo by induction of macrophage tumoricidal activity and activation of antitumor activity through IL-12–dependent NK cell recruitment. We provide a rationale for manipulating the phenotype of the abundant macrophage population already located within the tumor microenvironment; the potential to “re-educate” the tumor-promoting macrophage population may prove an effective and novel therapeutic approach for cancer that complements existing therapies.

Interleukin-6 as a Therapeutic Target in Human Ovarian Cancer

Purpose: We investigated whether inhibition of interleukin 6 (IL-6) has therapeutic activity in ovarian cancer via abrogation of a tumor-promoting cytokine network. Experimental Design: We combined preclinical and in silico experiments with a phase 2 clinical trial of the anti-IL-6 antibody siltuximab in patients with platinum-resistant ovarian cancer. Results: Automated immunohistochemistry on tissue microarrays from 221 ovarian cancer cases showed that intensity of IL-6 staining in malignant cells significantly associated with poor prognosis. Treatment of ovarian cancer cells with siltuximab reduced constitutive cytokine and chemokine production and also inhibited IL-6 signaling, tumor growth, the tumor-associated macrophage infiltrate and angiogenesis in IL-6–producing intraperitoneal ovarian cancer xenografts. In the clinical trial, the primary endpoint was response rate as assessed by combined RECIST and CA125 criteria. One patient of eighteen evaluable had a partial response, while seven others had periods of disease stabilization. In patients treated for 6 months, there was a significant decline in plasma levels of IL-6–regulated CCL2, CXCL12, and VEGF. Gene expression levels of factors that were reduced by siltuximab treatment in the patients significantly correlated with high IL-6 pathway gene expression and macrophage markers in microarray analyses of ovarian cancer biopsies. Conclusion: IL-6 stimulates inflammatory cytokine production, tumor angiogenesis, and the tumor macrophage infiltrate in ovarian cancer and these actions can be inhibited by a neutralizing anti-IL-6 antibody in preclinical and clinical studies. Clin Cancer Res; 17(18); 6083–96. ©2011 AACR.

The tumor-promoting actions of TNF-α involve TNFR1 and IL-17 in ovarian cancer in mice and humans

Cytokines orchestrate the tumor-promoting interplay between malignant cells and the immune system. In many experimental and human cancers, the cytokine TNF-alpha is an important component of this interplay, but its effects are pleiotropic and therefore remain to be completely defined. Using a mouse model of ovarian cancer in which either TNF receptor 1 (TNFR1) signaling was manipulated in different leukocyte populations or TNF-alpha was neutralized by antibody treatment, we found that this inflammatory cytokine maintained TNFR1-dependent IL-17 production by CD4+ cells and that this led to myeloid cell recruitment into the tumor microenvironment and enhanced tumor growth. Consistent with this, in patients with advanced cancer, treatment with the TNF-alpha-specific antibody infliximab substantially reduced plasma IL-17 levels. Furthermore, expression of IL-1R and IL-23R was downregulated in CD4+CD25- cells isolated from ascites of ovarian cancer patients treated with infliximab. We have also shown that genes ascribed to the Th17 pathway map closely with the TNF-alpha signaling pathway in ovarian cancer biopsy samples, showing particularly high levels of expression of genes encoding IL-23, components of the NF-kappaB system, TGF-beta1, and proteins involved in neutrophil activation. We conclude that chronic production of TNF-alpha in the tumor microenvironment increases myeloid cell recruitment in an IL-17-dependent manner that contributes to the tumor-promoting action of this proinflammatory cytokine.

Expression of both TNF-α receptor subtypes is essential for optimal skin tumour development

Keratinocyte-derived TNF-α acts as an endogenous tumour promoter and can also regulate AP-1 activity in mouse epidermis. To gain further insight into TNF-α signalling during skin tumour formation, mice deficient in TNFR1 (TNFR1−/− mice) or TNFR2 (TNFR2−/− mice) were subjected to chemical carcinogenesis. Tumour multiplicity was significantly reduced in TNFR1−/− and TNFR2−/− mice compared to wild-type (wt) mice, suggesting that both receptors have protumour activity. However, TNFR1−/− mice were markedly more resistant to tumour development than TNFR2−/− mice indicating that TNFR1 is the major mediator of TNF-α-induced tumour formation. TNFR1 and TNFR2 were both expressed in wt epidermis during tumour promotion and by primary keratinocytes in vitro. TPA-induced c-Jun expression was transient in TNFR1−/− and TNFR2−/− compared to wt epidermis and this was reflected by reduced induction of the AP-1-responsive genes granulocyte/macrophage-colony stimulating factor, matrix metalloproteinase-9 and matrix metalloproteinase-3. These genes were differentially regulated in TNFR1−/− compared to TNFR2−/− epidermis, suggesting that the TNF-α receptors act independently via different AP-1 complexes to transduce TNF-α signals during tumour promotion. In addition, TNFR2 cooperated with TNFR1 to optimise TNFR1-mediated TNF-α bioactivity on keratinocytes in vitro. Our data provide further insight into TNF-α signalling in malignancy and provide some rationale for the use of TNF-α antagonists in the treatment of cancer.

B regulatory cells and the tumor-promoting actions of TNF-α during squamous carcinogenesis

The inflammatory cytokine TNF-α has been recognized as a critical tumor promoter, but the effector cells that mediate its action have not been fully characterized. Because B cells regulate squamous and prostate carcinogenesis, and Tnf−/− mice harbor B-cell defects, we investigated the hypothesis that B cells are important effector cells for TNF-α–mediated promotion of cancer development. Using an adoptive transfer strategy and the 7,12-dimethylbenz[α]anthracene/terephthalic acid (DMBA/TPA) two-stage model of skin carcinogenesis, we found that both B cells and TNF-α are critical for the development of DMBA/TPA-induced papilloma. Transfer of B cells from DMBA/TPA-treated wild-type mice to Tnf−/− mice rescued papilloma development to a wild-type level, a result not observed when B cells from Tnf−/− mice were transferred to Rag2−/− mice or when TNF-α was eliminated selectively in B cells. Resistance to papilloma development in Tnf−/− mice was associated with increased IFN-γ and CD8+ T cells in skin and a significant reduction in IL-10–producing B regulatory cells alongside an increase in IFN-γ–producing CD8+ T cells in the spleen. These data indicate that during DMBA/TPA-induced squamous carcinogenesis TNF-α mediates tumor-promoting activity via regulatory B cells that repress antitumor immunity.

Ovarian cancer cell–derived migration inhibitory factor enhances tumor growth, progression, and angiogenesis

In view of our previous findings that tumor cell–derived macrophage migration inhibitory factor (MIF) increased macrophage-mediated ovarian cancer cell invasiveness in vitro, we investigated the wider significance of ovarian cancer cell–derived MIF for tumor growth, metastasis, and angiogenesis. We found that MIF is expressed in borderline and malignant ovarian tumors, and active MIF is found in malignant ascitic fluid. We next investigated the expression and function of MIF in a syngeneic ovarian cancer model. Stable knockdown of MIF in the murine ovarian cancer cell line ID8 decreased in vivo tumor burden and overall survival. Tumors arising from MIF knockdown cells had decreased proliferation and significantly increased apoptosis. This was associated with an increased phosphorylation of p53 and reduced Akt phosphorylation. MIF knockdown led to a changed cytokine profile in the ascitic microenvironment; tumor necrosis factor-α, interleukin-6 (IL-6), and IL-10 expression were all significantly decreased. Accompanying this decrease in cytokine expression was a significant decrease in macrophage infiltration into ascites. Additionally, MIF knockdown reduced the expression of proangiogenic cytokines vascular endothelial growth factor and keratinocyte chemoattractant (KC) and reduced the amount of endothelial cells in the malignant ascites. We conclude that autocrine production of MIF by ovarian cancer cells stimulates other cytokines, chemokines, and angiogenic factors that may promote colonization of the peritoneum and neovascularization of tumor deposits. [Mol Cancer Ther 2007;6(7):1993–2002]

Activity of human monocytes in IgE antibody-dependent surveillance and killing of ovarian tumor cells

We have previously shown that a chimeric IgE antibody against the folic acid receptor (MOv18 IgE) inhibits tumor growth in a SCID mouse model of ovarian carcinoma. MOv18 IgE gave greater protection than the corresponding chimeric MOv18 IgG1. We have now confirmed these effects in a nude‐mouse model of ovarian carcinoma and have demonstrated for the first time that human monocytes are active in IgE antibody‐dependent cell‐mediated cytotoxicity. Injection of tumor‐bearing mice with PBMC and MOv18 IgE led to infiltration of monocytes into the tumors and prolonged survival of the mice.Incubation of PBMC or purified monocytes and MOv18 IgE with ovarian tumor cells in vitro resulted in tumor cell killing proportional to the expression of unoccupied FcϵRI on monocytes.We observed phagocytosis of tumor cells by the monocytes in vitro. Our results suggest that tumor‐specific IgE antibodies may be exploited for immunotherapy of cancer.

TNF-α regulates epithelial expression of MMP-9 and integrin αvβ6 during tumour promotion. A role for TNF-α in keratinocyte migration?

Mice deficient in TNF-α (TNF-α−/− mice) are resistant to skin carcinogenesis and expression of MMP-9 is inhibited in TNF-α−/− mice during skin tumour development. In the early stages of tumour promotion, MMP-9 protein initially localized to the follicular epidermis but subsequently began to accumulate in the interfollicular epidermis of wild-type but not TNF-α−/− mice. Inhibition of TNF-α or MMP-9 function reduced keratinocyte migration in vitro. In addition, a deficiency of TNF-α delayed re-epithelialization in vivo and this correlated with reduced MMP-9 expression. Collectively, these data suggest that MMP-9 regulates keratinocyte migration in a TNF-α-dependent manner. Expression profiling of genes that control cell adhesion and migration revealed markedly lower levels of the integrin subunits αv and β6 in TNF-α−/− compared with wild-type keratinocytes in vitro. αvβ6 expression was upregulated by keratinocytes in vitro and during tumour promotion in vivo in a TNF-α-dependent manner. Furthermore, αvβ6 blockade significantly inhibited keratinocyte migration and TNF-α-stimulated MMP-9 expression in vitro. These data illustrate a novel TNF-α-dependent mechanism for the control of αvβ6 expression and suggest one pathway for TNF-α regulation of MMP-9. Increased MMP-9 and αvβ6 expression may stimulate epithelial cell migration during tumour formation and may be one mechanism whereby TNF-α acts as an endogenous tumour promoter.

Murine CD27(-) Vγ6(+) γδ T cells producing IL-17A promote ovarian cancer growth via mobilization of protumor small peritoneal macrophages.

Significance Tumor development is impacted by a set of diverse infiltrating leukocyte populations that can either inhibit or, paradoxically, enhance tumor cell growth. This study characterizes a cellular cross-talk between γδ T lymphocytes and small peritoneal macrophages (SPMs) that is mediated by the proinflammatory cytokine, IL-17, and promotes ovarian cancer growth. IL-17 is preferentially produced by a population of γδ T cells, displaying a distinctive CD27(−) Vγ6(+) phenotype, that strongly proliferate in response to tumor challenge. This associates with the mobilization of SPMs that express protumor and proangiogenic molecular mediators upregulated by IL-17. Critically, these SPMs can directly enhance ovarian cancer cell growth. Our work identifies an IL-17–dependent γδ T cell/SPM axis that promotes tumor development and thus counteracts cancer immunosurveillance. Cancer-associated inflammation mobilizes a variety of leukocyte populations that can inhibit or enhance tumor cell growth in situ. These subsets include γδ T cells, which can infiltrate tumors and typically provide large amounts of antitumor cytokines, such as IFN-γ. By contrast, we report here that in a well-established transplantable (ID8 cell line) model of peritoneal/ovarian cancer, γδ T cells promote tumor cell growth. γδ T cells accumulated in the peritoneal cavity in response to tumor challenge and could be visualized within solid tumor foci. Functional characterization of tumor-associated γδ T cells revealed preferential production of interleukin-17A (IL-17), rather than IFN-γ. Consistent with this finding, both T cell receptor (TCR)δ-deficient and IL-17–deficient mice displayed reduced ID8 tumor growth compared with wild-type animals. IL-17 production by γδ T cells in the tumor environment was essentially restricted to a highly proliferative CD27(−) subset that expressed Vγ6 instead of the more common Vγ1 and Vγ4 TCR chains. The preferential expansion of IL-17–secreting CD27(−) Vγ6(+) γδ T cells associated with the selective mobilization of unconventional small peritoneal macrophages (SPMs) that, in comparison with large peritoneal macrophages, were enriched for IL-17 receptor A, and for protumor and proangiogenic molecular mediators, which were up-regulated by IL-17. Importantly, SPMs were uniquely and directly capable of promoting ovarian cancer cell proliferation. Collectively, this work identifies an IL-17–dependent lymphoid/myeloid cross-talk involving γδ T cells and SPMs that promotes tumor cell growth and thus counteracts cancer immunosurveillance.

Interleukin-6 stimulates defective angiogenesis

The cytokine IL6 has a number of tumor-promoting activities in human and experimental cancers, but its potential as an angiogenic agent has not been fully investigated. Here, we show that IL6 can directly induce vessel sprouting in the ex vivo aortic ring model, as well as endothelial cell proliferation and migration, with similar potency to VEGF. However, IL6-stimulated aortic ring vessel sprouts had defective pericyte coverage compared with VEGF-stimulated vessels. The mechanism of IL6 action on pericytes involved stimulation of the Notch ligand Jagged1 as well as angiopoietin2 (Ang2). When peritoneal xenografts of ovarian cancer were treated with an anti-IL6 antibody, pericyte coverage of vessels was restored. In addition, in human ovarian cancer biopsies, there was an association between levels of IL6 mRNA, Jagged1, and Ang2. Our findings have implications for the use of cancer therapies that target VEGF or IL6 and for understanding abnormal angiogenesis in cancers, chronic inflammatory disease, and stroke.