Ovarian cancer is typically accompanied by the occurrence of malignant ascites containing large number of macrophages. It has been suggested that these tumor-associated macrophages (TAMs) are skewed to alternative polarization (M2) and thereby play an essential role in therapy resistance and metastatic spread. In our study, we have investigated the nature, regulation and clinical correlations of TAM polarization in serous ovarian cancer. Macrophage polarization markers on TAMs and ascites cytokine levels were analyzed for 30 patients and associated with relapse-free survival (RFS) in a prospective study with 20 evaluable patients. Surface expression of the M2 marker CD163 on TAMs was inversely associated with RFS (p < 0.01). However, global gene expression profiles determined for 17 of these patients revealed a mixed-polarization phenotype unrelated to the M1/M2 classification. CD163 surface expression also correlated with the ascites levels of IL-6 and IL-10 (p < 0.05), both cytokines induced CD163 expression, and their ascites levels showed a clear inverse association with RFS (p < 0.01). These findings define a subgroup of patients with high CD163 expression, high IL-6 and/or IL-10 levels and poor clinical outcome.

The reciprocal interplay of cancer cells and host cells is an indispensable prerequisite for tumor growth and progression. Cells of both the innate and adaptive immune system, in particular tumor-associated macrophages (TAMs) and T cells, as well as cancer-associated fibroblasts enter into a malicious liaison with tumor cells to create a tumor-promoting and immunosuppressive tumor microenvironment (TME). Ovarian cancer, the most lethal of all gynecological malignancies, is characterized by a unique TME that enables specific and efficient metastatic routes, impairs immune surveillance, and mediates therapy resistance. A characteristic feature of the ovarian cancer TME is the role of resident host cells, in particular activated mesothelial cells, which line the peritoneal cavity in huge numbers, as well as adipocytes of the omentum, the preferred site of metastatic lesions. Another crucial factor is the peritoneal fluid, which enables the transcoelomic spread of tumor cells to other pelvic and peritoneal organs, and occurs at more advanced stages as a malignancy-associated effusion. This ascites is rich in tumor-promoting soluble factors, extracellular vesicles and detached cancer cells as well as large numbers of T cells, TAMs, and other host cells, which cooperate with resident host cells to support tumor progression and immune evasion. In this review, we summarize and discuss our current knowledge of the cellular and molecular interactions that govern this interplay with a focus on signaling networks formed by cytokines, lipids, and extracellular vesicles; the pathophysiologial roles of TAMs and T cells; the mechanism of transcoelomic metastasis; and the cell type selective processing of signals from the TME.

BackgroundSoluble protein and lipid mediators play essential roles in the tumor environment, but their cellular origins, targets, and clinical relevance are only partially known. We have addressed this question for the most abundant cell types in human ovarian carcinoma ascites, namely tumor cells and tumor-associated macrophages.ResultsTranscriptome-derived datasets were adjusted for errors caused by contaminating cell types by an algorithm using expression data derived from pure cell types as references. These data were utilized to construct a network of autocrine and paracrine signaling pathways comprising 358 common and 58 patient-specific signaling mediators and their receptors. RNA sequencing based predictions were confirmed for several proteins and lipid mediators. Published expression microarray results for 1018 patients were used to establish clinical correlations for a number of components with distinct cellular origins and target cells. Clear associations with early relapse were found for STAT3-inducing cytokines, specific components of WNT and fibroblast growth factor signaling, ephrin and semaphorin axon guidance molecules, and TGFβ/BMP-triggered pathways. An association with early relapse was also observed for secretory macrophage-derived phospholipase PLA2G7, its product arachidonic acid (AA) and signaling pathways controlled by the AA metabolites PGE2, PGI2, and LTB4. By contrast, the genes encoding norrin and its receptor frizzled 4, both selectively expressed by cancer cells and previously not linked to tumor suppression, show a striking association with a favorable clinical course.ConclusionsWe have established a signaling network operating in the ovarian cancer microenvironment with previously unidentified pathways and have defined clinically relevant components within this network.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0956-6) contains supplementary material, which is available to authorized users.

The nuclear receptor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is a lipid ligand-inducible transcription factor associated with macrophage polarization. However, its function in tumor-associated macrophages (TAMs) has not been investigated to date. Here, we report the PPARβ/δ-regulated transcriptome and cistrome for TAMs from ovarian carcinoma patients. Comparison with monocyte-derived macrophages shows that the vast majority of direct PPARβ/δ target genes are upregulated in TAMs and largely refractory to synthetic agonists, but repressible by inverse agonists. Besides genes with metabolic functions, these include cell type-selective genes associated with immune regulation and tumor progression, e.g., LRP5, CD300A, MAP3K8 and ANGPTL4. This deregulation is not due to increased expression of PPARβ/δ or its enhanced recruitment to target genes. Instead, lipidomic analysis of malignancy-associated ascites revealed high concentrations of polyunsaturated fatty acids, in particular linoleic acid, acting as potent PPARβ/δ agonists in macrophages. These fatty acid ligands accumulate in lipid droplets in TAMs, thereby providing a reservoir of PPARβ/δ ligands. These observations suggest that the deregulation of PPARβ/δ target genes by ligands of the tumor microenvironment contributes to the pro-tumorigenic polarization of ovarian carcinoma TAMs. This conclusion is supported by the association of high ANGPTL4 expression with a shorter relapse-free survival in serous ovarian carcinoma.

Besides its established functions in intermediary metabolism and developmental processes, the nuclear receptor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) has a less defined role in tumorigenesis. In the present study, we have identified a function for PPARβ/δ in cancer cell invasion. We show that two structurally divergent inhibitory ligands for PPARβ/δ, the inverse agonists ST247 and DG172, strongly inhibit the serum- and transforming growth factor β (TGFβ)-induced invasion of MDA-MB-231 human breast cancer cells into a three-dimensional matrigel matrix. To elucidate the molecular basis of this finding, we performed chromatin immunoprecipitation sequencing (ChIP-Seq) and microarray analyses, which identified the gene encoding angiopoietin-like 4 (ANGPTL4) as the major transcriptional PPARβ/δ target in MDA-MB-231 cells, previously implicated in TGFβ-mediated tumor progression and metastatic dissemination. We show that the induction of ANGPTL4 by TGFβ and other oncogenic signals is strongly repressed by ST247 and DG172 in a PPARβ/δ-dependent fashion, resulting in the inhibition of ANGPTL4 secretion. This effect is attributable to these ligands' ability to induce a dominant transcriptional repressor complex at the site of transcription initiation that blocks preinitiation complex formation through an histone deacetylase-independent, non-canonical mechanism. Repression of ANGPTL4 transcription by inverse PPARβ/δ agonists is functionally linked to the inhibition of cancer cell invasion into a three-dimensional matrix, as (i) invasion of MDA-MB-231 cells is critically dependent on ANGPTL4 expression, (ii) recombinant ANGPTL4 stimulates invasion, and (iii) reverses the inhibitory effect of ST247 and DG172. These findings indicate that a PPARβ/δ–ANGPTL4 pathway is involved in the regulation of tumor cell invasion and that its pharmacological manipulation by inverse PPARβ/δ agonists is feasible.

Ovarian cancer is characterized by early transcoelomic metastatic spread via the peritoneal fluid, where tumor cell spheroids (TU), tumor-associated T cells (TAT), and macrophages (TAM) create a unique microenvironment promoting cancer progression, chemoresistance, and immunosuppression. However, the underlying signaling mechanisms remain largely obscure. To chart these signaling networks, we performed comprehensive proteomic and transcriptomic analyses of TU, TAT, and TAM from ascites of ovarian cancer patients. We identify multiple intercellular signaling pathways driven by protein or lipid mediators that are associated with clinical outcome. Beyond cytokines, chemokines and growth factors, these include proteins of the extracellular matrix, immune checkpoint regulators, complement factors, and a prominent network of axon guidance molecules of the ephrin, semaphorin, and slit families. Intriguingly, both TU and TAM from patients with a predicted short survival selectively produce mediators supporting prometastatic events, including matrix remodeling, stemness, invasion, angiogenesis, and immunosuppression, whereas TAM associated with a longer survival express cytokines linked to effector T-cell chemoattraction and activation. In summary, our study uncovers previously unrecognized signaling networks in the ovarian cancer microenvironment that are of potential clinical relevance.

Macrophages occur as resident cells of fetal origin or as infiltrating blood monocyte-derived cells. Despite the critical role of tumor-associated macrophages (TAMs) in tumor progression, the contribution of these developmentally and functionally distinct macrophage subsets and their alteration by the tumor microenvironment are poorly understood. We have addressed this question by comparing TAMs from human ovarian carcinoma ascites, resident peritoneal macrophages (pMPHs) and monocyte-derived macrophages (MDMs). Our study revealed striking a similarity between TAMs and pMPHs, which was considerably greater that the resemblance of TAMs and MDMs, including their transcriptomes, their inflammation-related activation state, the presence of receptors mediating immune functions and the expression of tumor-promoting mediators. Consistent with these results, TAMs phagocytized bacteria, presented peptide antigens and activated cytotoxic T cells within their pathophysiological environment. These observations support the notion that tumor-promoting properties of TAMs may reflect, at least to some extent, normal features of resident macrophages rather than functions induced by the tumor microenvironment. In spite of these surprising similarities between TAMs and pMPHs, bioinformatic analyses identified a TAM-selective signature of 30 genes that are upregulated relative to both pMPHs and MDMs. The majority of these genes is linked to extracellular matrix (ECM) remodeling, supporting a role for TAMs in cancer cell invasion and ovarian cancer progression.

Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is a lipid ligand-inducible transcription factor with established metabolic functions, whereas its anti-inflammatory function is poorly understood. To address this issue, we determined the global PPARβ/δ-regulated signaling network in human monocyte-derived macrophages. Besides cell type-independent, canonical target genes with metabolic and immune regulatory functions we identified a large number of inflammation-associated NFκB and STAT1 target genes that are repressed by agonists. Accordingly, PPARβ/δ agonists inhibited the expression of multiple pro-inflammatory mediators and induced an anti-inflammatory, IL-4-like morphological phenotype. Surprisingly, bioinformatic analyses also identified immune stimulatory effects. Consistent with this prediction, PPARβ/δ agonists enhanced macrophage survival under hypoxic stress and stimulated CD8+ T cell activation, concomitantly with the repression of immune suppressive target genes and their encoded products CD274 (PD-1 ligand), CD32B (inhibitory Fcγ receptor IIB) and indoleamine 2,3-dioxygenase 1 (IDO-1), as well as a diminished release of the immune suppressive IDO-1 metabolite kynurenine. Comparison with published data revealed a significant overlap of the PPARβ/δ transcriptome with coexpression modules characteristic of both anti-inflammatory and pro-inflammatory cytokines. Our findings indicate that PPARβ/δ agonists induce a unique macrophage activation state with strong anti-inflammatory but also specific immune stimulatory components, pointing to a context-dependent function of PPARβ/δ in immune regulation.