Cancer-associated inflammation mobilizes a variety of leukocyte populations that can inhibit or enhance tumor cell growth in situ. These subsets include ?? T cells, which can infiltrate tumors and typically provide large amounts of antitumor cytokines, such as IFN-?. By contrast, we report here that in a well-established transplantable (ID8 cell line) model of peritoneal/ovarian cancer, ?? T cells promote tumor cell growth. ?? 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 ?? T cells revealed preferential production of interleukin-17A (IL-17), rather than IFN-?. Consistent with this finding, both T cell receptor (TCR)?-deficient and IL-17-deficient mice displayed reduced ID8 tumor growth compared with wild-type animals. IL-17 production by ?? T cells in the tumor environment was essentially restricted to a highly proliferative CD27((-)) subset that expressed V?6 instead of the more common V?1 and V?4 TCR chains. The preferential expansion of IL-17-secreting CD27((-)) V?6((+)) ?? 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 ?? T cells and SPMs that promotes tumor cell growth and thus counteracts cancer immunosurveillance.
Junctional adhesion molecule C (JAM-C) is a transmembrane protein with significant roles in regulation of endothelial cell (EC) functions, including immune cell recruitment and angiogenesis. As these responses are important in promoting tumor growth, the role of EC JAM-C in tumor development was investigated using the ID8 syngeneic model of ovarian cancer. Within 10-15 wk, intraperitoneally injected ID8 cells form multiple tumor deposits and ascites that resemble human high-grade serous ovarian cancer. Compared to wild-type mice, survival in this model was increased in EC JAM-C knockouts (KOs; 88 vs. 96 d, P=0.04) and reduced in EC JAM-C transgenics (88 vs. 78.5 d, P=0.03), mice deficient in or overexpressing EC JAM-C, respectively. While tumor growth was significantly reduced in EC JAM-C KOs (87% inhibition at 10 wk, P<0.0005), this was not associated with alterations in tumor vessel density or immune cell infiltration. However, tumor microvessels from EC JAM-C-deficient mice exhibited reduced pericyte coverage and increased vascular leakage, suggesting a role for EC JAM-C in the development of functional tumor vessels. These findings provide evidence for a role for EC JAM-C in tumor growth and aggressiveness as well as recruitment of pericytes to newly formed blood vessels in a model of ovarian cancer.
The mechanisms by which oncolytic vaccinia virus induces tumor cell death are poorly understood. We have evaluated cell death pathways following infection of ovarian cancer cells with both wild-type and thymidine kinase-deleted (dTK) Lister strain vaccinia. We show that death does not rely upon classical apoptosis despite the appearances of some limited apoptotic features, including phosphatidylserine externalization and appearance of sub-G1 DNA populations. Vaccinia infection induces marked lipidation of LC3 proteins, but there is no general activation of the autophagic process and cell death does not rely upon autophagy induction. We show that vaccinia induces necrotic morphology on transmission electron microscopy, accompanied by marked by reductions in intracellular adenosine triphosphate, altered mitochondrial metabolism, and release of high mobility group box 1 (HMGB1) protein. This necrotic cell death appears regulated, as infection induces formation of a receptor interacting protein (RIP1)/caspase-8 complex. In addition, pharmacological inhibition of both RIP1 and substrates downstream of RIP1, including MLKL, significantly attenuate cell death. Blockade of TNF-?, however, does not alter virus efficacy, suggesting that necrosis does not result from autocrine cytokine release. Overall, these results show that, in ovarian cancer cells, vaccinia virus causes necrotic cell death that is mediated through a programmed series of events.
The CC-chemokine ligand 2 (CCL2) is highly expressed in various malignancies and promotes carcinogenesis. Blocking CCL2 has preclinical antitumor activity. A phase 1 trial of carlumab (CNTO 888), a human anti-CCL2 IgG1? mAb, was conducted to evaluate the safety, tolerability, pharmacokinetic-pharmacodynamic profile, and antitumor activity.
Constitutive production of inflammatory cytokines is a characteristic of many human malignant cell lines; however, the in vitro and in vivo interdependence of these cytokines, and their significance to the human cancer microenvironment, are both poorly understood. Here, we describe for the first time how three key cytokine/chemokine mediators of cancer-related inflammation, TNF, CXCL12, and interleukin 6, are involved in an autocrine cytokine network, the "TNF network," in human ovarian cancer. We show that this network has paracrine actions on angiogenesis, infiltration of myeloid cells, and NOTCH signaling in both murine xenografts and human ovarian tumor biopsies. Neutralizing antibodies or siRNA to individual members of this TNF network reduced angiogenesis, myeloid cell infiltration, and experimental peritoneal ovarian tumor growth. The dependency of network genes on TNF was shown by their downregulation in tumor cells from patients with advanced ovarian cancer following the infusion of anti-TNF antibodies. Together, the findings define a network of inflammatory cytokine interactions that are crucial to tumor growth and validate this network as a key therapeutic target in ovarian cancer.
There have been major advances in our understanding of the cellular and molecular biology of the human malignancies that are collectively referred to as ovarian cancer. At a recent Helene Harris Memorial Trust meeting, an international group of researchers considered actions that should be taken to improve the outcome for women with ovarian cancer. Nine major recommendations are outlined in this Opinion article.
Chemokines (chemo-attractant cytokines) are a group of small proteins that act together with their cell surface receptors, in development, normal physiology and immune responses, to direct cells to specific locations throughout the body. Cancer cells acquire the ability to subvert the chemokine system, such that these molecules and their receptors become important regulators of cell movement into and out of the tumour microenvironment and major players in cancer biology.
Inflammatory cells and mediators are an essential component of the tumor microenvironment. Inflammatory circuits can differ considerably in different tumors in terms of cellular and cytokine networks and molecular drivers. However, macrophages are a common and fundamental component of cancer promoting inflammation. Drivers of macrophage functional orientation include tumor cells, cancer-associated fibroblasts, T cells and B cells. Dissection of the diversity of cancer-related inflammation is instrumental to the design of therapeutic approaches that target cancer-related inflammation.
The majority of human pancreatic cancers have activating mutations in the KRAS proto-oncogene. These mutations result in increased activity of the NF-?B pathway and the subsequent constitutive production of proinflammatory cytokines. Here, we show that inhibitor of ?B kinase 2 (Ikk2), a component of the canonical NF-?B signaling pathway, synergizes with basal Notch signaling to upregulate transcription of primary Notch target genes, resulting in suppression of antiinflammatory protein expression and promotion of pancreatic carcinogenesis in mice. We found that in the Kras(G12D)Pdx1-cre mouse model of pancreatic cancer, genetic deletion of Ikk2 in initiated pre-malignant epithelial cells substantially delayed pancreatic oncogenesis and resulted in downregulation of the classical Notch target genes Hes1 and Hey1. Tnf-? stimulated canonical NF-?B signaling and, in collaboration with basal Notch signals, induced optimal expression of Notch targets. Mechanistically, Tnf-? stimulation resulted in phosphorylation of histone H3 at the Hes1 promoter, and this signal was lost with Ikk2 deletion. Hes1 suppresses expression of Pparg, which encodes the antiinflammatory nuclear receptor Ppar?. Thus, crosstalk between Tnf-?/Ikk2 and Notch sustains the intrinsic inflammatory profile of transformed cells. These findings reveal what we believe to be a novel interaction between oncogenic inflammation and a major cell fate pathway and show how these pathways can cooperate to promote cancer progression.
The inflammatory cytokine TNF-? 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-?-mediated promotion of cancer development. Using an adoptive transfer strategy and the 7,12-dimethylbenz[?]anthracene/terephthalic acid (DMBA/TPA) two-stage model of skin carcinogenesis, we found that both B cells and TNF-? 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-? was eliminated selectively in B cells. Resistance to papilloma development in Tnf(-/-) mice was associated with increased IFN-? and CD8(+) T cells in skin and a significant reduction in IL-10-producing B regulatory cells alongside an increase in IFN-?-producing CD8(+) T cells in the spleen. These data indicate that during DMBA/TPA-induced squamous carcinogenesis TNF-? mediates tumor-promoting activity via regulatory B cells that repress antitumor immunity.
Ovarian clear cell adenocarcinoma (OCCA) is an uncommon histotype that is generally refractory to platinum-based chemotherapy. We analyze here the most comprehensive gene expression and copy number data sets, to date, to identify potential therapeutic targets of OCCA.
Oncolytic adenoviruses show promise as a cancer treatment. However, they generate acute inflammatory responses with production of cytokines, including tumor necrosis factor-? (TNF-?). We investigated whether inhibition of TNF-? augments efficacy of the E1A CR2-deleted adenovirus dl922-947 in ovarian cancer. dl922-947 induced transcription of TNF-? and its downstream signaling targets interleukin-6 and -8 (IL-6 and IL-8) in ovarian cancer cells. In vitro, RNAi-mediated knockdown of TNF-? reduced production of multiple inflammatory cytokines after infection and increased ovarian cancer cell sensitivity to virus cytotoxicity, as did treatment with the anti-TNF-? antibody infliximab. In vivo, stable knockdown of TNF-? in IGROV-1 xenografts increased the anticancer activity of dl922-947. In addition, inhibition of TNF-? using monoclonal antibodies also improved dl922-947 efficacy. This increased efficacy resulted from suppression of cellular inhibitor of apoptosis-1 and -2 (cIAP1 and cIAP2) transcription in malignant cells and a consequent increase in caspase-mediated apoptosis. These findings suggest that TNF-? acts as a survival factor in adenovirus-infected cells. Combining TNF-? inhibition with oncolytic adenoviruses could improve antitumor activity in clinical trials.
The oncolytic adenovirus dl922-947 replicates selectively within and lyses cells with a dysregulated Rb pathway, a finding seen in > 90% human cancers. dl922-947 is more potent than wild type adenovirus and the E1B-deletion mutant dl1520 (Onyx-015). We wished to determine which host cell factors influence cytotoxicity. SV40 large T-transformed MRC5-VA cells are 3-logs more sensitive to dl922-947 than isogenic parental MRC5 cells, confirming that an abnormal G1/S checkpoint increases viral efficacy. The sensitivity of ovarian cancer cells to dl922-947 varied widely: IC50 values ranged from 51 (SKOV3ip1) to 0.03 pfu/cell (TOV21G). Cells sensitive to dl922-947 had higher S phase populations and supported earlier E1A expression. Cytotoxicity correlated poorly with both infectivity and replication, but well with expression of p21 by microarray and western blot analyses. Matched p21+/+ and -/- Hct116 cells confirmed that p21 influences dl922-947 activity in vitro and in vivo. siRNA-mediated p21 knockdown in sensitive TOV21G cells decreases E1A expression and viral cytotoxicity, whilst expression of p21 in resistant A2780CP cells increases virus activity in vitro and in intraperitoneal xenografts. These results highlight that host cell factors beyond simple infectivity can influence the efficacy of oncolytic adenoviruses. p21 expression may be an important biomarker of response in clinical trials.
We recently reported that human T-lymphotropic virus type 1 (HTLV-1) infection is accompanied by a high frequency of CD4(+)FoxP3(+) cells in the circulation. In asymptomatic carriers of HTLV-1 and in patients with HTLV-1-associated inflammatory and malignant diseases, a high FoxP3(+) cell frequency correlated with inefficient cytotoxic T cell-mediated killing of HTLV-1-infected cells. In adult T cell leukemia/lymphoma (ATLL), the FoxP3(+) population was distinct from the leukemic T cell clones. However, the cause of the increase in FoxP3(+) cell frequency in HTLV-1 infection was unknown. In this study, we report that the plasma concentration of the chemokine CCL22 is abnormally high in HTLV-1-infected subjects and that the concentration is strongly correlated with the frequency of FoxP3(+) cells, which express the CCL22 receptor CCR4. Further, we show that CCL22 is produced by cells that express the HTLV-1 transactivator protein Tax, and that the increased CCL22 enhances the migration and survival of FoxP3(+) cells in vitro. Finally, we show that FoxP3(+) cells inhibit the proliferation of ex vivo, autologous leukemic clones from patients with ATLL. We conclude that HTLV-1-induced CCL22 causes the high frequency of FoxP3(+) cells observed in HTLV-1 infection; these FoxP3(+) cells may both retard the progression of ATLL and HTLV-1-associated inflammatory diseases and contribute to the immune suppression seen in HTLV-1 infection, especially in ATLL.
The stromal compartment is increasingly recognized to play a role in cancer. However, its role in the transition from preinvasive to invasive disease is unknown. Most gastrointestinal tumors have clearly defined premalignant stages, and Barretts esophagus (BE) is an ideal research model. Supervised clustering of gene expression profiles from microdissected stroma identified a gene signature that could distinguish between BE metaplasia, dysplasia, and esophageal adenocarcinoma (EAC). EAC patients overexpressing any of the five genes (TMEPAI, JMY, TSP1, FAPalpha, and BCL6) identified from this stromal signature had a significantly poorer outcome. Gene ontology analysis identified a strong inflammatory component in BE disease progression, and key pathways included cytokine-cytokine receptor interactions and TGF-beta. Increased protein levels of inflammatory-related genes significantly up-regulated in EAC compared with preinvasive stages were confirmed in the stroma of independent samples, and in vitro assays confirmed functional relevance of these genes. Gene set enrichment analysis of external datasets demonstrated that the stromal signature was also relevant in the preinvasive to invasive transition of the stomach, colon, and pancreas. These data implicate inflammatory pathways in the genesis of gastrointestinal tract cancers, which can affect prognosis.
Tumour necrosis factor (TNF) is a major inflammatory cytokine that was first identified for its ability to induce rapid haemorrhagic necrosis of experimental cancers. When efforts to harness this anti-tumour activity in cancer treatments were underway, a paradoxical tumour-promoting role of TNF became apparent. Now that links between inflammation and cancer are appreciated, is TNF a target or a therapeutic in malignant disease -- or both?
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.
In this study, we established an in vitro organoid model of normal human ovarian surface epithelial (HOSE) cells. The spheroids of these normal HOSE cells resembled epithelial inclusion cysts in human ovarian cortex, which are the cells of origin of ovarian epithelial tumor. Because there are strong correlations between chronic inflammation and the incidence of ovarian cancer, we used the organoid model to test whether protumor inflammatory cytokine tumor necrosis factor alpha would induce malignant phenotype in normal HOSE cells. Prolonged treatment of tumor necrosis factor alpha induced phenotypic changes of the HOSE spheroids, which exhibited the characteristics of precancerous lesions of ovarian epithelial tumors, including reinitiation of cell proliferation, structural disorganization, epithelial stratification, loss of epithelial polarity, degradation of basement membrane, cell invasion, and overexpression of ovarian cancer markers. The result of this study provides not only an evidence supporting the link between chronic inflammation and ovarian cancer formation but also a relevant and novel in vitro model for studying of early events of ovarian cancer.
B regulatory cells are a newly described subpopulation of B cells that appear to play important roles in autoimmunity and more recently, in cancer. In this review we summarize our current knowledge of B regulatory cells, as well as the body of evidence pointing towards a role for B cells in general, and B regulatory cells in particular, in promoting tumor growth.
High-grade serous ovarian cancer (HGSC) disseminates early and extensively throughout the peritoneal space, causing multiple lesions that are a major clinical problem. The aim of this study was to investigate the cellular composition of peritoneal tumour deposits in patient biopsies and their evolution in mouse models using immunohistochemistry, intravital microscopy, confocal microscopy, and 3D modelling. Tumour deposits from the omentum of HGSC patients contained a prominent leukocyte infiltrate of CD3(+) T cells and CD68(+) macrophages, with occasional neutrophils. Alpha-smooth muscle actin(+) (?-SMA(+) ) pericytes and/or fibroblasts surrounded these well-vascularized tumour deposits. Using the murine bowel mesentery as an accessible mouse peritoneal tissue that could be easily imaged, and two different transplantable models, we found multiple microscopic tumour deposits after i.p. injection of malignant cells. Attachment to the peritoneal surface was rapid (6-48 h) with an extensive CD45(+) leukocyte infiltrate visible by 48 h. This infiltrate persisted until end point and in the syngeneic murine ID8 model, it primarily consisted of CD3(+) T lymphocytes and CD68(+) macrophages with ?-SMA(+) cells also involved from the earliest stages. A majority of tumour deposits developed above existing mesenteric blood vessels, but in avascular spaces new blood vessels tracked towards the tumour deposits by 2-3 weeks in the IGROV-1 xenografts and 6 weeks in the ID8 syngeneic model; a vigorous convoluted blood supply was established by end point. Inhibition of tumour cell cytokine production by stable expression of shRNA to CXCR4 in IGROV-1 cells did not influence the attachment of cells to the mesentery but delayed neovascularization and reduced tumour deposit size. We conclude that the multiple peritoneal tumour deposits found in HGSC patients can be modelled in the mouse. The techniques described here may be useful for assessing treatments that target the disseminated stage of this disease.
We have investigated the role of cytokine lymphotoxin in tumor-stromal interactions in human ovarian cancer. We found that lymphotoxin over-expression is commonly shared by the cancer cells of various ovarian cancer subtypes, and lymphotoxin-beta receptor (LTBR) is expressed ubiquitously in both the cancer cells and cancer-associated fibroblasts (CAFs). In monoculture, we showed that ovarian cancer cells are not the major lymphotoxin-responsive cells. On the other hand, our co-culture studies demonstrated that the cancer cell-derived lymphotoxin induces chemokine expression in stromal fibroblasts through LTBR-NF?B signalling. Amongst the chemokines being produced, we found that fibroblast-secreted CXCL11 promotes proliferation and migration of ovarian cancer cells via the chemokine receptor CXCR3. CXCL11 is highly-expressed in CAFs in ovarian cancer biopsies, while CXCR3 is found on malignant cells in primary ovarian tumours. Additionally, the over-expression of CXCR3 is significantly associated with the tumour grade and lymph node metastasis of ovarian cancer, further supporting the role of CXCR3, which interacts with CXCL11, in promoting growth and metastasis of human ovarian cancer. Taken together, these results demonstrated that cancer-cell derived lymphotoxin mediates reciprocal tumour-stromal interactions in human ovarian cancer by inducing CXCL11 in fibroblasts. Our findings suggest that lymphotoxin-LTBR and CXCL11-CXCR3 signalling represent therapeutic targets in ovarian cancer.
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