A Dynamic Inflammatory Cytokine Network in the Human Ovarian Cancer Microenvironment

A majority of human malignant cell lines constitutively secrete cytokines and chemokines as a consequence of oncogenic mutations and disregulated signaling pathways (1). However, it is not clear whether this cytokine/chemokine expression has relevance to the regulation of complex human tumor microenvironments.

We previously reported that the cytokines TNF and interleukin (IL)-6, the chemokine CXCL12 and its CXCR4 receptor were constitutively expressed and coregulated in ovarian cancer cell lines in tissue culture (2, 3). Stable knockdown of TNF mRNA in one of these cell lines provided evidence for an autocrine cytokine network with paracrine actions on blood vessel development in a peritoneal xenograft model (3).

The aim of this study was to investigate whether this autocrine cytokine network was relevant to the human tumor microenvironment of ovarian cancer. We present evidence, for the first time, that the cytokine network exists in human cancer biopsies. TNF, CXCL12, and IL-6 are coexpressed and coregulated in human ovarian cancer biopsies in what we describe as the “TNF network.” We show that high TNF network pathway gene expression in the tumor microenvironment associates with genes involved in angiogenesis, inflammation, leukocyte infiltrates, and NOTCH signaling. This was confirmed by inhibition of the TNF network in experimental ovarian cancer models and in tumor cells from ovarian cancer patients after infusion of an anti-TNF antibody.

IGROV-1 high-grade serous ovarian cancer cells and TOV21G clear-cell cancer cells were cultured as described (3). All cell lines have undergone 16 loci STR authentication (LGC Standards) and were most recently authenticated in September 2011. The cells were cultured in RPMI-1640 10% fetal calf serum.

Paraffin-embedded biopsies, tissue arrays, and xenografts were stained with antibodies for CXCR4 (MAB173; R&D Systems), CXCL12 (MAB350; R&D Systems), TNF (MAB 610; R&D systems), IL-6 (SC-7920; Santa Cruz Biotechnology), F4/80 (MCA497; Serotec) CD68 (Dako), and HES1 (Sc-25392; Santa Cruz Biotechnology Inc.). Negative controls were isotype matched.

Cell extract (15 μg) was run on an SDS 10% acrylamide gel and transferred to a nylon membrane. The membrane was blocked overnight (4°C in PBS with 0.1% Tween and 10% milk powder) and probed using the anti-JAG1 antibody (AF1272; R&D Systems). A horseradish peroxidase–conjugated secondary antibody was used for detection (1:5,000) dilution at room temperature for 1 hour. Protein concentration equivalence was confirmed by anti–β-actin antibody

Cells were counterstained with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Sigma) and analyzed on a FACScan flow cytometer using CellQuest software (BD Pharmingen).

Cells were plated at 3 × 10⁵ cells per well, cell culture supernatants removed after 48 hours of culture and cytokine concentrations measured using Quantikine ELISA Kits (R&D Systems). Frozen tissue was ground in liquid nitrogen and lysed in Tris-HCl, pH 8/50 mmol/L/NaCl 150 mmol/L/1% Triton X-100/DTT 1 mmol/L/1× inhibitor mixture mix (Calbiochem)/100 μg/mL phenylmethylsulfonylfluoride. Protein concentrations were determined by Bradford assay (Rad Laboratories).

IGROV-1 cells were transfected with SUPER RNAi plasmids containing 2 different short hairpin RNA (shRNA) sequences targeting CXCR4, or a control plasmid containing scrambled RNA (IGROV-Mock) and isolated according to Brummelkamp and colleagues (4). Lipofectamine was used for transfection as described previously (3). For transient knockdown, the ON-TARGET plus SMART pool of oligos targeting CXCR4 gene expression was transfected using Dharmafect1 transfection reagent (Dharmacon). SiCONTROL nontargeting siRNA pool served as control. Lentiviral vectors containing the luciferase reporter construct were as described (3).

RNA was extracted using Tri Reagent (Sigma) and treated with 10 U DNase (Pharmacia). DNase-treated RNA (2 μg) was reverse transcribed with M-MLV reverse transcriptase (Promega). Multiplex real-time reverse transcriptase (RT-PCR) analysis was done using premade TaqMan probes (FAM) and 18s rRNA [individually ventilated cages (VIC)] specific primers and probes with the ABI PRISM 7700 Sequence Detection System instrument and software (PE Applied Biosystems). Expression values were normalized (ΔC_t) to 18s rRNA by subtracting the cycle threshold (C_t) value of 18s rRNA from the C_t value of the experimental value.

SPF female nude mice (Cancer Research UK) 6 to 8 weeks of age, housed in sterile IVC were injected intraperitoneally with 5 × 10⁶ cells. Twice weekly treatment (5 mg/kg i.p.) was with monoclonal antibody infliximab or nonspecific, Gamimune N polyclonal human IgG control. Bioluminescence imaging was carried out and blood vessels quantified as before (3).

Total RNA was isolated using the standard TRIzol protocol and purified further with the RNeasy Kit (Qiagen). RNA was quantified using a 2100 Bioanalyzer (Agilent Technologies). Expression profiles of all specimens were compared with a commercial universal reference RNA (Clontech). The Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays were used. Probe synthesis and microarray hybridization were carried out according to standard Affymetrix protocols.

Three Affymetrix datasets were obtained in triplicate. The NCBI GEO accession number for the arrays is GSE 13763. Data were analyzed using Bioconductor 1.9 running on R 2.6.0. Probeset expression measures (5, 6) were calculated using the Affymetrix package's Robust Multichip Average (RMA) default method (7). Differential gene expression was assessed between control (IGROV-1 and IGROV-Mock) and shRNAi (I) and (II) replicate groups, using an empirical Bayes t test (limma package; ref. 6); P values were adjusted for multiple testing using the Benjamini–Hochberg method (8). Any probe sets that exhibited an adjusted P value of 0.05 were called differentially expressed. In addition, any probe sets that exhibited an absolute fold change of greater than 2 were used to generate a heatmap. Two-dimensional hierarchical clustering of expression data using differentially expressed genes across control and shCXCR4 (I) and (II) samples was done. Samples were clustered using a 1–Pearson correlation distance matrix and average linkage clustering. Genes were clustered using a Euclidean distance matrix and average linkage clustering. The NCBI GEO accession number for the cell lines is GSE 13763. The NCBI accession number for datasets from the ascites cells is GSE18681.

Differentially expressed probes were selected on the basis of meeting criteria of false discovery rate (FDR) < 0.05. Probes were divided into positive and negative fold change lists and used to determine enrichment using GeneGO processes within MetaCore pathway tool (GeneGo, Inc.). The analysis employs a hypergeometric distribution to determine the most enriched gene set

The microarray datasets GSE6008, GSE3149, and GSE9899 were downloaded from the GEO website. Datasets GSE6008 and GSE3149 were merged using Bioconductor 1.9 running on R 2.6.0. Probeset expression measures were calculated using the Affymetrix package's RMA default method. The microarray dataset from The Cancer Genome Atlas Network (TCGA) comprising 590 biopsies of high-grade serous ovarian cancer was downloaded from the TCGA data portal site (9).

The function GeneSetTest from the limma package was used to assess whether each sample had a tendency to be associated with an up- or downregulation of members of the, for example, TNF pathway. The individual genes in the TNF, CXCL12, and IL-6 pathways, as defined by MetaCore, are listed in Supplementary Table S1. Fourteen percent of the genes are shared among the 3 pathways. The function employs a Wilcoxon t test to generate P values. All samples were ranked on this enrichment, from the most significant to the least significant. The top and bottom 50 samples were extracted from the dataset as “high-TNF” and “low-TNF.” The same process was used to identify samples that were enriched with members of the CXCL12 and IL-6 pathway. We also used immune cell-specific signatures (10).

The phase I/II trial was a single center, open-label, study of infliximab at 2 dose levels (5 mg/kg and 10 mg/kg) in patients with advanced epithelial ovarian cancer; full details in Charles and colleagues (11).

Fifty-three cases of stage III/IV high-grade serous tumors were used for tissue microarray (TMA) construction as described in Brennan and colleagues (12), 4 cores per patient. The Aperio ScanScope XT Slide Scanner (Aperio Technologies) system was used to capture whole slide digital images with a 20× objective. Slides were dearrayed to visualize individual cores, using Spectrum (Aperio). Genie histology pattern recognition software (Aperio) was used to identify tumor from stroma in individual cores, and a color deconvolution algorithm (Aperio) was used to quantify TNF, CXCL12, and IL-6. For assay of CD68⁺ cells in the Australian Ovarian Cancer Study (AOCS) ovarian cancer biopsies, microscope fields that contained tumor and adjacent stroma were counted blind at ×40 magnification.

The clinical study was approved by North East London and the City Health Authority Research Ethics Committee (LREC P/02/150) and Lothian Research Ethics Committee (LREC2000/4/60, LREC/2002/8/31) and conducted according to the declaration of Helsinki. All patients gave voluntary, written informed consent. The TMA study was approved by the Research Ethics Committee at the National Maternity Hospital, Dublin, Ireland. Access to human biopsy samples satisfied requirements of the East London and City Health Authority Research Ethics Subcommittee (LREC number 07/Q0604/25).

Statistical analysis of in vitro and animal experiments used 1-way ANOVA, χ² test, or unpaired t test with Welch correction (GraphPad Prism version 4 Software).

Using gene expression microarray datasets from ovarian cancer biopsies in which more than 90% of samples were stage III/IV high-grade serous cancer, we first looked for associations between gene expression levels of the TNF, CXCL12, and IL-6 signaling pathways. Biopsies from the AOCS were ranked by expression levels of genes in the TNF, CXCL12, and IL-6 signaling pathways in each sample relative to the mean levels of gene expression in all samples (Method, Supplementary Fig. S1; individual genes in TNF, CXCL12, and IL-6 pathways, Supplementary Table S1). The top 50 samples with the highest levels of gene expression were compared with the bottom 50 samples with the lowest levels of gene expression in the TNF, CXCL12, and IL-6 pathways, so that each sample was associated with 3 P values. Using a binomial distribution, we found that samples that were highly enriched in one pathway, that is, ranked in the top 50, were highly likely to have high expression levels of genes in the other 2 pathways (Table 1). We then merged 2 additional ovarian cancer gene expression microarray datasets to give another 245 ovarian cancer biopsy samples and also conducted the same analysis on the 590 biopsies of high-grade serous cancer from the TCGA dataset, this time analyzing those samples that were in the highest 25% and lowest 25% of samples. The associations between high CXCL12, TNF, and IL-6 signaling pathway gene expression were fully validated (Table 1). The same significant associations were found if expression levels of individual receptor/ligand pairs, rather than pathways, were tested (data not shown). Hence, there was a 3-way interdependency of the cytokines in the human tumor biopsy samples, with the expression of each ligand and its signaling pathway related to the other.

We used automated immunohistochemistry (IHC) to localize TNF, CXCL12, and IL-6 and determine whether the proteins were coexpressed at cellular level using a TMA of 53 cases of stage III/IV high-grade serous ovarian cancer (12). Using an automated algorithm, staining was expressed as a score that combined both the intensity and density of positive pixels and partitioned into epithelial and stromal fractions using image analysis software. In the malignant cell compartment, we found a significant association between expression of TNF and CXCL12 (P < 0.004) as well as between TNF and IL-6 (P < 0.05). There was also significant correlation between TNF levels in the stromal compartment and CXCL12 (P < 0.004) as well as between TNF and IL-6 (<0.001). We named the coexpression of these 3 mediators the TNF network.

We previously reported that stable expression of shRNA to TNF inhibited CXCL12 and IL-6 production and CXCR4 expression in IGROV-1 cells (3). To look for further in vitro evidence of the TNF network, we stably expressed shRNA to CXCR4 in these cells (Fig. 1A). This also reduced constitutive production of TNF, CXCL12, and IL-6 (Fig. 1B) but did not affect production of TGF-β1 or basic fibroblast growth factor. Transient transfection of RNAi to CXCR4 in IL-6 and TNF producing TOV21G clear-cell carcinoma cells also attenuated TNF and IL-6 expression (Supplementary Fig. S2).

We then compared gene expression patterns in high and low TNF network biopsies and the IGROV-1 cells to obtain information on the actions of the TNF network. We prepared in triplicate microarrays from the mock transfected and shCXCR4 IGROV-1 cell lines described in Fig. 1A and B in which the TNF network was inhibited. An eBayes t test was used to determine a list of differentially expressed probes between IGROV1 cells treated with shCXCR4 versus IGROV-1 controls. The heatmap in Fig. 1C shows a hierarchical clustering using probes differentially expressed in shCXCR4 IGROV-1 cells compared with control/mock transfected IGROV-1 cells. We next selected gene expression data from biopsies that were in the highest or lowest 50 for gene expression levels for all 3 signaling pathways (n = 28 high TNF network biopsies, n = 25 low TNF network biopsies) from the AOCS dataset.

We then identified, using gene set enrichment analysis (GSEA; ref. 13), pathways and processes significantly increased in the high TNF network biopsies and cell lines compared with low TNF biopsies and cell lines, pathways and processes that were also significantly reduced in the cell lines when the network was inhibited by shRNA to CXCR4. We validated the results using the TCGA dataset, this time comparing 52 high TNF network samples versus 45 low TNF network samples defined as above for AOCS samples.

GSEA revealed a strong association between high TNF network expression and angiogenesis, cell adhesion, cell cycle, and inflammation signaling (Table 2). Strikingly, there was also a significant association with NOTCH signaling. As the biopsies would also contain infiltrating leukocytes, we looked for associations with immune cell signatures (10). High TNF network biopsy samples had significant enhancement of gene expression profiles for T cells, neutrophils, myeloid cells, monocytes, dendritic, and B cells (P < 0.0001) but not NK cells (P = 0.053) compared with the low TNF network group (Table 2 column 1). The complete gene list associated with high TNF network in the IGROV-1 cell lines and biopsies is available in Supplementary Tables S2 and S3, respectively.

Stable knockdown of CXCR4 in the IGROV-1 cells was maintained when they were grown as intraperitoneal xenografts (Supplementary Fig. S3A). Levels of CXCL12 and TNF protein were significantly decreased in tumors derived from shCXCR4-transfected cells (Supplementary Fig. S3B), and IL-6 levels were also significantly reduced in the tumors (16 pg ± 13 pg/100 μg protein compared with mean 846 pg ± 70 pg/100 μg protein in mock transfected tumors).

Stable knockdown of CXCR4 inhibited growth of peritoneal ovarian cancer xenografts measured by bioluminescence imaging (Fig. 2A), although it had no effect on cell growth in vitro (data not shown). Median survival of mice injected with IGROV-Mock cells was 46 days in contrast to 92 and 79 days in mice bearing tumors derived from 2 different clones of IGROV-shCXCR4 cells (P < 0.0001; Fig. 2B). The number of tumor deposits and the extent of organ involvement were significantly reduced in mice injected with IGROV-shCXCR4 cells (P <0.0001 for both parameters) after 42 days (data not shown). F4/80+ macrophages were significantly reduced comparing size-matched shCXCR4 and mock transfected tumors (Fig. 2C). The vascular area of size-matched tumor deposits was significantly reduced in IGROV-shCXCR4 compared with IGROV-Mock tumors (P < 0.0001; Fig. 2D).

The anti-human TNF antibody infliximab had similar actions to CXCR4 knockdown. After 4 weeks intraperitoneal growth and treatment in vivo, there was a reduction in tumor growth as evidenced by reduced luciferase expression in the anti-TNF–treated mice [Mean relative light units (RLU) of 1.1 × 10⁷ and 1.1 × 10⁷ for PBS and control IgG groups, respectively, compared with mean RLU 0.3 × 10⁷ for anti-TNF–treated mice]. The vascular area and the F4/80+ infiltrate were significantly reduced following anti-TNF treatment (Fig. 3A and B). There was also a reduction in IL-6 protein as assessed by IHC in the tumors treated with anti-TNF antibodies (Fig. 3C).

As the TNF network associated with the myeloid cell infiltrate, we asked whether TNF network gene expression correlated with myeloid cells in human tumor biopsies. We obtained 21 high-grade serous ovarian cancer biopsies from the AOCS series that matched those used in the analyses of Tables 1 and 2; 7 were from high and 14 from low TNF network biopsies. There was a striking association between CD68⁺ macrophages in the stromal areas and high TNF network expression (P = 0.01; Fig. 3D). In 10 of 14 of the low TNF network biopsies, no CD68⁺ cells could be detected in either tumor or stromal areas at all, whereas 7 of 7 high TNF network biopsies had visible CD68⁺ cells.

To further investigate the association between the TNF network and NOTCH signaling (Table 2), we focused on NOTCH3 and JAG1 as both have been implicated in ovarian cancer (14, 15).

In the AOCS dataset, high TNF, CXCL12, and IL-6 signaling pathway expression significantly associated with JAG1 (P = 0.03; P = 7.7 × 10⁻⁷; P = 0.00053, respectively) and high CXCL12 and IL-6 with NOTCH3 (P = 0.00016; P = 0.0044, respectively). In IGROV-1 cells, NOTCH3 and JAG1 mRNA expression was significantly diminished when the TNF network was inhibited by shRNA to CXCR4 (Fig. 4A). Anti–IL-6 antibodies reduced constitutive JAG1 expression in IGROV-1 as well as in the TOV21G cells. Exogenous IL-6 stimulated JAG1 mRNA expression in a STAT3-dependent manner, and the effects of TNF could be inhibited by anti–IL-6 (Fig. 4B). NOTCH3 and JAG1 mRNA levels were also reduced in the tumor lysates from IGROV-1 shCXCR4 tumors. The percent of cell nuclei that stained positive for the HES1 transcription factor, downstream of NOTCH signaling, was also significantly inhibited in sections from the shCXCR4 tumors compared with controls (Fig. 4C, Supplementary Fig. S3C). Treatment of tumors with the anti-TNF antibody also significantly reduced nuclear HES1 protein staining (Supplementary Fig. S4). We concluded that key members of the TNF network regulate NOTCH3, JAG1, and HES1.

We then sought evidence that the TNF network could be inhibited in ovarian cancer patients. Serial samples of ovarian cancer ascites were obtained from 9 patients with advanced disease, who had been treated with the anti-human TNF antibody infliximab (11). We compared ascites cell TNF network gene expression levels pretreatment, 24 and 48 hours after infliximab infusion. The results are shown as a heatmap (Fig. 5A), with green indicating lower and red indicating higher expression relative to pretreatment. Four patients showed a significant downregulation of TNF network gene expression 24 and/or 48 hours after anti-TNF antibody infusion. We compared the global gene expression patterns in pretreatment tumors from patients who showed robust downregulation of the TNF network versus those with little attenuation (patients 1–4 vs. patients 5–9; Fig. 5A). An empirical Bayes t test (6) identified 280 probes that were significantly different between the groups (with an FDR of P < 0.001). Probes and samples were clustered with the 280 genes, using average linkage clustering to generate a hierarchical clustering heatmap (Fig. 5B). We tested whether the gene sets representing the TNF, CXCL12, and IL-6 pathways were enriched in either group. TNF (P = 2.68 × 10⁻⁹), CXCL12 (P = 3.83 × 10⁻⁹), and IL-6 (P = 5.37 × 10⁻⁷) pathways were statistically enriched in patients 1 to 4 compared with patients 5 to 9, as was NOTCH signaling (P = 1.64 × 10⁻³) and enhanced gene expression profiles of T cells, neutrophils, myeloid, monocytes, dendritic, and B cells (P < 0.0001). Patients who had the highest level of TNF network expression showed the most intense downregulation of the network following infliximab anti-TNF antibody.

We have used a combination of molecular biology, bioinformatics, and cancer biology techniques to show that key pathways in cancer-related inflammation and Notch signaling are part of an autocrine malignant cell network in human ovarian cancer, a network with paracrine actions on angiogenesis and myeloid cell infiltration into tumors. The starting point of the current work was an observation in ovarian cancer cell lines (3). Here, we have found that information obtained from these cell lines is relevant to biopsies advanced human high-grade serous ovarian cancer. Our data also suggest that malignant cells regulate the inflammatory cytokine network in the human ovarian cancer microenvironment.

The factors coregulated in the TNF network have been individually considered as targets for cancer treatment. In 2 clinical studies of TNF antagonists in women with advanced ovarian cancer (11, 16), there was some evidence of transient disease stabilization and biological effects consistent with our knowledge of the actions of TNF. In a phase II trial of the anti–IL-6 antibody siltuximab in 18 patients with relapsed progressing ovarian cancer, there was one partial response, and 7 patients achieved periods of disease stabilization (17). In the context of the TNF network data described here, patients receiving siltuximab for 6 months had a significant decline in plasma levels of CXCL12.

The most common and lethal form of ovarian cancer is high-grade serous ovarian cancer (18). More than 95% of all the biopsies investigated (mRNA samples and biopsy sections) in our work were of this subtype. The IGROV-1 cell line is derived from a high-grade serous case. Clear-cell carcinoma of the ovary is characterized by overexpression of the IL-6–STAT3–HIF pathway (19) and has distinct genetic drivers compared with high-grade serous cancers (20, 21). TOV21G cells produced the highest levels of IL-6 but also produced other TNF network members. We recently found that 1 other high-grade serous and 6 clear-cell carcinoma cell lines constitutively coproduce TNF, CXCL12, and IL-6 (H. Kulbe, unpublished data). There are many examples in the literature of human tumor cell lines that produce TNF, IL-6, or CXCL12 but to our knowledge, few studies have assessed all 3 of these cytokines together. We believe it is likely that the TNF network is active in tumors with distinct genetic lesions.

Our data show that targeting cytokines, such as TNF and IL-6, is more likely to influence the tumor microenvironment than to kill malignant cells directly. Therefore anticytokine treatments, which are generally well tolerated in patients with inflammatory or malignant disease, are likely to be most useful in combination with conventional chemotherapy or treatments that target the malignant cell directly.

The TNF network defined here has features of a robust network as described by Yarden and colleagues for EGF–ERBB (22, 23). Further characterization of the TNF network using a systems biology approach may suggest new ways of treating the high-grade serous and clear-cell carcinoma. This approach may also help determine the best treatments to combine with anticytokine/chemokine agents. Because other malignant cell types produce TNF, IL-6, and CXCL12, these therapeutic strategies may be widely applicable in other cancers.

No potential conflicts of interest were disclosed.

H. Kulbe, T. Schioppa, P. Chakravarty were funded by Cancer Research United Kingdom; J. Kwong was funded by AICR; D.A. Leinster and J. Kwong were funded by MRC; S.C. Robinson was funded by Barts and the London Charity, M.A. Salako was funded by Ovarian Cancer Action; T. Hagemann was funded by the MRC; R.G. Thompson and F.R. Balkwill were funded by HEFCE; and D.D. Bowtell is funded by the National Health and Medical Research Council of Australia, Cancer Councils of Australia, and the US Department of Defense Ovarian Cancer Research Program. The UCD Conway Institute is funded by the PRTLI administered by the Higher Education Authority of Ireland.

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