BACKGROUND
ovarian tumors ranks fifth as the cause of cancer-related death in women and represents the deadliest of all gynecological tumors  <cit> .
more than 90% of ovarian cancers are carcinomas that originate from the ovarian surface or fallopian tube epithelium.
high grade serous adenocarcinoma is the most common ovarian carcinoma subtype, with most patients presenting with advanced stage disease and disseminated tumor masses at the time of diagnosis.
although most ovarian cancers are highly sensitive to first-line adjuvant chemotherapy, the disease has a dire prognosis with an overall 5-year survival rate of less than 40%  <cit> .
several characteristic features contribute to the fatal nature of high grade serous ovarian adenocarcinoma, including the shedding of tumor cells at a very early stage of the disease, their spreading via the peritoneal fluid to form transcoelomic metastases and the tumor-promoting and immune suppressive effect of the peritoneal tumor environment, frequently formed by the malignancy-associated effusion within the peritoneal cavity, commonly referred to as malignant ascites.
this tumor microenvironment, consisting to a large extent of host-derived cells, is crucial for the growth, progression, therapy resistance and immune escape of malignant tumors, including ovarian cancer  <cit> .
the most common cell types in ovarian hgsc-associated ascites are macrophages and t lymphocytes  <cit> .
tumor-associated macrophages  can be derived from both blood monocytes  or resident tissue macrophages , with the latter most likely representing the major origin of tams in ovarian cancer  <cit> .
tam activation is skewed by factors of the tumor microenvironment to adopt a spectrum of phenotypes that represent mixed forms of alternatively activated  and pro-inflammatory macrophages  <cit> , which has also been clearly demonstrated for tams in ovarian cancer ascites  <cit> .
tams do not possess tumoricidal activity, but are rather thought to promote immune suppression and various aspects of cancer growth and progression, including tumor cell invasion, angiogenesis and metastasis  <cit> .
consistent with these tumor-promoting functions of tams, expression of the alternative activation marker cd <dig> in tams from malignancy-associated ascites showed a strong correlation with early relapse of serous ovarian carcinoma after first-line therapy  <cit> .
among the soluble factors contributing to tam polarization, tumor progression and a poor clinical outcome, interleukin  <dig> , il- <dig>  transforming growth factor β  and arachidonic acid play a prominent role .
to date, transcriptional signatures of human tams that distinguish subgroups of patients have not been described.
in the present study, we address this issue by determining the transcriptome of tams from different patients in conjunction with principal component analysis  and coexpression analysis to define distinct subgroups.
these analyses lead to the identification of two subgroups differing in the expression of genes associated with cytokine signaling, immune regulation, extracellular matrix reorganization and overall survival.
of note, an interferon  related signature showed a striking association with a favorable clinical outcome.
furthermore, ifnγ counteracted repression by ovarian cancer ascites of il- <dig>  a key mediator of an anti-tumor response mediated by natural killer cells  and t lymphocytes, consistent with the observed association of an ifn signaling-associated signature with ovarian cancer survival.
methods
patient samples
ascites was collected from patients undergoing primary surgery at the university hospital in marburg.
patient characteristics are presented in additional file 1: table s <dig>
isolation and immunophenotyping of cells from ovarian cancer ascites
mononuclear cells were isolated from ascites by lymphocyte separation medium  <dig>  density gradient centrifugation and further purified by magnetic cell sorting  using cd <dig> microbeads   <cit> .
tams were analyzed by flow cytometry for surface expression of cd <dig>  cd <dig>  cd <dig>  cd <dig>  cd <dig> and cd <dig> as described  <cit> .
tumor cell spheroids and t cells were analyzed as previously published  <cit> .
isolation and culture of monocyte-derived macrophages
monocyte-derived macrophages  were generated from monocytes  from healthy donors as described  <cit>  and cultured in rpmi medium with 5% human male ab serum  or cell-free ascites from ovarian cancer patients, as indicated.
t cell from peripheral blood were isolated as described for ascites  <cit> .
buffy coats were obtained from the blood bank at ukgm giessen, germany.
rt-qpcr
isolation of rna and rt-qpcr were carried out as described  <cit> .
the following primers were used:rpl27_fw: 5′aaagctgtcatcgtgaagaac
rpl27_rv: 5′gctgtcactttgcgggggtag
il12b_fw: 5′gcgaggttctaagccattcg
il12b_rev: 5′actccttgttgtcccctctg
cxcl10_fw: 5′aagcagttagcaaggaaaggtc
cxcl10_rv: 5′gacatatactccatgtagggaagtga
gbp4_fw: 5′ttcaaaggcattagagattcttga
gbp4_rv: 5′gtggagcccagagggaag
gpnmb_fw: 5′ctatgagaagaactgcagaaatg
gpnmb_rv: 5′gttatgatggctttggccgg
lgals2_fw: 5′ccacgagttgagccctgag
lgals2_rv: 5′cggcttcatgtccatgttc
kitlg_fw: 5′gccaagtcttacaagggcag
kitlg_rv: 5′gaaactctctctctttctcttgc
mrc1_fw: 5′cct cgg acc tgg tta ggg
mrc1_rv: 5′ggatgtgtggtcctccttgg
elisa
concentrations of p <dig>  in cell culture supernatants were determined using an elisa kit from biolegend/biozol  according to the instructions of the manufacturer.
ifnγ, il- <dig> and il- <dig> levels in ascites were quantified by elisa kits purchased from ebioscience .
rna sequencing
rna isolation and rna-seq was carried out on an illumina hiseq  <dig> as described  <cit> .
genome assembly and gene model data were retrieved from ensembl revision  <dig>  sequencing data were deposited at ebi arrayexpress .
statistical analysis of experimental data
paired and unpaired t tests were carried out with the python functions scipy.stats.ttest_rel () and scipy.stats.ttest_ind (), respectively.
results were expressed as follows: *p <  <dig> ; **p <  <dig> ; ***p <  <dig> ; ****p <  <dig> .
confidence intervals were calculated using the bootstrap method.
further statistical analyses were performed using the python functions numpy.percentile () and pandas.dataframe.boxplot ().
analysis of rna-seq data
rna-seq data was aligned to ensembl v <dig> using star  and processed as reported  <cit> .
the number of mapped reads was in the range of  <dig> – <dig>  million .
tpm  values were calculated based on the total gene read counts and length of merged exons and corrected for contamination by tumor cells as described  <cit> .
the source code for implementing the algorithm for tpm correction has also been deposited at github .
genes were considered expressed if they had a minimum tpm value of  <dig>  tam samples with tpm > <dig> for epcam or msln were excluded due to presumed tumor cell contamination.
all genomic sequence and gene annotation data were retrieved from ensembl release  <dig>  genome assembly hg <dig>
pca and delineation of differentially expressed gene clusters
pca was carried out on using the sklearnpca and sklearn_pca.fit_transform () functions  on rna-seq data.
pearson correlation coefficients  were determined with scipy.stats.pearsonr ().
the bioconductor package edger  <cit>  was used for the delineation of high variance gene clusters differentially regulated in subgroups of tams identified by pca .
coexpression analysis
genes with the greatest expression variance were identified by pandas.dataframe.var ().
pearson correlation coefficients  were determined for the  <dig> top genes using scipy.stats.pearsonr ().
sets of genes with r >  <dig>  and n >  <dig> were combined  and analyzed by hierarchical clustering using the scipy.cluster.hierarchy functions linkage  and dendrogram .
the resulting  <dig> clusters  were analyzed for intersections the signatures identified by pca , which revealed close relationships of cluster i with signature b and cluster iii with signature a.
pathway analyses
gene sets were analyzed for upstream regulators using the ingenuity pathway analysis  database  as described  <cit> .
functional annotations were performed by gene ontology  enrichment analysis .
survival analyses
overall survival  data were retrieved from precog   <cit> .
associations between gene expression and relapse-free survival  were analyzed by the web-based tool “km plotter”  <cit>   using the following settings: ‘auto select best cutoff’, probe set option: ‘jetset best probe’, histology: serous, datasets: all; other settings: default.
RESULTS
clustering of ovarian carcinoma tam samples
the transcriptomes determined for  <dig> independent samples of tam isolated from the ascites of ovarian cancer patients  were analyzed for potential similarity patterns by different approaches, as schematically summarized in fig.
<dig>  these samples were selected for very low contamination with tumor cells, as indicated by low tpm values  for the epithelial marker genes epcam and msln.
furthermore, we excluded all genes  highly expressed in tumor cells or t cells versus tams  to minimize interference by contaminating cells.fig.
<dig> schematic representation of data analysis and summary of results.
nomenclature for designation of clusters: tam samples clustered by pca: letters ; genes clustered by coexpression analysis: roman numbers .
genes identified by edger and upregulated in tam subgroup a or b were defined as signature a and b, respectively
genewise normalized tam transcriptomes were used for the bioinformatic delineation of similarity patterns.
principal component analysis  did not yield a clear partitioning of tam samples into subgroups, although eigenvalues suggested that the first two components can explain most of the data.
we therefore additionally grouped samples according to the expression of cd <dig>  previously established as a tam marker associated with a poor clinical outcome of ovarian cancer  <cit> .
combining pca with the the expression pattern of this surrogate marker revealed two discernible subgroups , which was confirmed by pearson correlation  and distance-based multidimensional scaling analysis .
two other markers associated with pro-tumorigenic functions, i.e., il <dig>   and pcolce <dig> , showed a very similar pattern of expression, except for il <dig> expression in tam <dig> .
based on this data we defined subgroup a as tam <dig>  tam <dig>  tam <dig>  tam <dig>  tam <dig> and tam <dig> expressing cd <dig>  pcolce <dig> and il <dig> at high levels relative to the subgroup b samples tam <dig>  tam <dig>  tam <dig>  tam <dig>  tam <dig> and tam <dig>  these subgroups were confirmed by flow cytometry showing a significantly higher fraction of cd163+ and cd163+cd206+ cells in subgroup a versus subgroup b tams, which was not observed for other macrophage markers .fig.
<dig> clustering of ovarian carcinoma tam samples based on rna-seq data.
a principal component analysis  of tam transcriptomes.
samples with high expression of cd <dig>  are shown in red , samples with low expression of cd <dig> in blue .
b heatmap based on pearson correlation coefficients  calculated for the tam transcriptomes identified by pca .
c-e expression of il <dig>  pcolce <dig> and cd <dig> in tam samples of clusters a  and b .
dotted lines show the quantiles used in panel .
f flow cytometry analysis of cluster a and b samples.
the plot shows the fraction of cd16+, cd32+, cd64+, cd163+, cd206+ and cd163+cd206+ cells .
g concentrations of il- <dig> and il <dig> in the ascites of cluster a and b patients determined by elisa.
boxes show the upper and lower quartiles, whiskers the 95% confidence intervals , and horizontal lines the median.
asterisks indicate the statistical significance determined by unpaired t test .
n/a, not applicable since all values >97%; ns: not significant
taken together, these data indicate that cluster a tams are skewed towards alternative activation , extracellular matrix  remodeling  and promotion of tumor growth .
as these markers are associated with a short relapse-free survival, it is likely that cluster b is linked to a favorable clinical outcome.
this conclusion is supported by the observation that the ascites concentrations of il- <dig>  highly predictive of a poor survival of ovarian cancer  <cit>  was consistently and dramatically increased in subgroup a versus subgroup b patients .
a similar pattern was observed with il- <dig> , also associated with a short time to relapse  <cit> .
cluster-specific gene expression
to gain more insight into the specific phenotypes of subgroup a and b tams, we analyzed the rna-seq data sets with edger, a bioconductor package for reliable gene-specific dispersion estimation in small datasets  <cit> .
the edger tool identified a group of  <dig> genes differentially expressed in the two subgroups of tams with an fdr of < <dig>  .
of these,  <dig> genes were upregulated in subgroup a versus b, and  <dig> genes showed the opposite pattern .
these gene sets were termed signature a and signature b. in contrast to these protein coding rnas, edger did not identify any differentially expressed lncrnas  in our rna-seq data set .fig.
<dig> identification of differentially expressed genes in subgroup a versus b tams by edger.
a
scatter plot showing the expression of genes identified by the edger tool  in tam subgroups a or b identified by pca in fig.
<dig>  data represent the ratio  of median tpm values for subgroup a versus subgroup b. b functional annotation of genes upregulated in subgroup a  or subgroup b  by gene ontology  enrichment analysis.
p values are plotted against fold enrichment.
only specific non-redundant terms with p values < <dig>  and enrichment > <dig> are shown.
c upstream regulator analysis  of upregulated genes with p < 10− <dig>  d expression of the ifn signaling-associated genes of signature b identified by go enrichment analysis .
boxes show the upper and lower quartiles, whiskers the 95% ci, and horizontal lines the median.
e validation of rna-seq data.
analysis by rt-qpcr of signature a and b genes  in tam samples from subgroup a and b .
error bars show the standard deviation and horizontal lines the mean.
red: cluster a samples; blue: cluster b samples.
asterisks indicate the statistical significance determined by unpaired t test ; ns: not significant
gene ontology  enrichment analysis identified significant associations of signature a with diverse biological processes, including ecm remodeling, locomotion, endocytosis as well as lipid and carbohydrate catabolism .
in contrast, innate immune defense mechanisms, t cell activation and ifn signaling were strongly associated with signature b .
consistent with these findings, ingenuity upstream regulator analysis indicated that signature b genes are major targets of proinflammatory pathways triggered by lipopolysaccharide , tumor necrosis factor α  and infγ , with all three target gene sets showing strong overlaps .
the differential expression patterns of signature a and b genes are shown in fig.
3d and additional file 3: figure s <dig> for the ifn and ecm gene sets identified by go enrichment analysis as representative examples.
as expected, these gene sets showed opposite patterns of regulation and the subgroup-selective expression was clearly significant for the vast majority of genes .
the rna-seq data for several genes identified by edger were confirmed by rt-qpcr .
taken together, these observations point to a relatively high complexity of biological functions affected by signature a genes, while the role of signature b genes appears to be specifically associated with ifn-stimulated immune defense mechanisms.
confirmation of clustering by coexpression analysis
to obtain independent evidence for the robustness of the clusters defined by pca and edger we performed coexpression analysis the genes showing the highest variance across all tam samples.
pearson correlation and hierarchical clustering yielded three large clusters of coregulated genes .
hierarchical clustering of tam samples using a combined set of these genes  yielded the identical partitioning into subgroups a and b as the pca-based approach in fig.
2a.
consistent with this observation, two of these gene clusters showed substantial and specific overlaps with signatures a and b, respectively, i.e., cluster i with signature a ; cluster iii with signature b , as shown in fig.
4c as well as additional file 2: datasets s <dig> and s <dig> fig.
<dig> coexpression analysis of all tam samples.
a correlation based heatmap of gene clusters  defined by coexpression analysis of genes with the highest variance across all tam samples, followed by hierarchical clustering .
b hierarchical clustering of patients based on the genes identified in panel .
c overlap of genes in clusters i - iv with signatures a and b identified by pca.
d functional annotation of cluster i , cluster ii  and cluster iii  genes by gene ontology  enrichment analysis.
p values are plotted against fold enrichment.
only specific non-redundant terms with p values < <dig>  and enrichment > <dig> are shown.
e expression of cluster i genes  in subgroup a  and b  samples.
f as panel , but for cluster ii genes .
g as panel , but for cluster iii genes .
boxes show the upper and lower quartiles, whiskers the 95% confidence intervals , and horizontal lines the median
functional annotations revealed endocytosis and chemotaxis for cluster i, chromatin modification and splicing for cluster ii, and immune defense and interferon signaling for cluster iii .
consistent with the overlaps of described in the preceding paragraph, the go terms for cluster i were also found for signature b, and the terms for cluster iii correspond to those for signature a .
finally, expression of cluster i genes was higher in subgroup b versus subgroup a , and vice versa, expression of cluster iii genes was higher in subgroup a relative to subgroup b .
in contrast, no differential expression was observed for cluster ii genes .
taken together, these findings clearly suggest that cluster iii corresponds to signature a, while cluster i corresponds to signature b. thus, both strategies, pca of patient samples followed by edger and coexpression analysis of high variance genes yielded very similar results, and identified ifn signaling as a hallmark of signature b upregulated in subtype b tams.
association of signature b, interferon signaling and survival
in order to identify potential associations between the expression of this genes in ovarian cancer with clinical outcome we made use of the precog database, which contains the results of a meta-analysis of  <dig> patients from  <dig> studies  <cit> .
these studies used solid ovarian tumor tissue containing substantial amounts of myeloid cells  <cit>  for transcriptome analysis, suggesting that the precog data are also suitable for testing survival associations for genes expressed in abundant tumor-associated host cells, such as tams.
signature a, cluster iii and their overlap comprised similar fractions of genes associated with poor or favorable os .
only when genes with distinct functional annotations were analyzed separately a clear os association was detectable.
thus, signature a genes linked to ecm remodeling were strongly associated with a short os .
in contrast, signature b, cluster i and their overlap were clearly linked to a favorable clinical outcome .
this applied in particular to the ifn signaling-associated genes of signature b, as shown in detail in fig.
5b.
a similar inverse association with relapse-free survival was also found based on data from the km plotter database, as exemplified for irf <dig> and tap <dig> in fig.
5c and d. all precog z-scores for signature a and b genes are also listed in additional file 2: dataset s <dig> fig.
<dig> association of cluster-specific gene expression with ovarian cancer survival.
a mean z-scores  for signature a and b genes; the ecm-related and ifn signaling-associated genes of signature a and b, respectively; cluster iii and i genes; and genes representing the intersection of signature a with cluster iii or of signature b with cluster i. survival data were obtained from the precog database with  <dig> ovarian cancer patients  <cit> .
b os z-scores for signature b genes that are associated with ifn signaling.
significant associations with a favorable clinical outcome are shown in blue .
the corresponding data for the complete signatures a and b are shown in additional file 3: figure s <dig>  c-f kaplan-meier plots analyzing the association of irf <dig>  tap <dig>  cd <dig> and cd <dig> with rfs of high-grade serous ovarian cancer determined by km plotter  <cit> .
g expression of the signature b genes hla-dpb <dig>  hla-dra and kynu in tams , tumor cells  and cd3+ tats  isolated from ovarian cancer ascites
these associations with a favorable clinical course are not simply consequence of the extent of myeloid cell infiltration.
first, activation state-independent myeloid marker genes, such as cd <dig> or cd <dig> did not show any significant association with ovarian cancer os   <cit>  or rfs  <cit>  .
second, tumor infiltration by myeloid cells inferred from rna expression data   <cit>  showed no significant association with os, with a trend towards a worse clinical outcome for monocytes .
third, a number of the ifn signaling-associated genes of signature b are expressed at high levels  selectively in tams compared to tumor cells or tumor-associated cd3+ lymphocytes  isolated from ovarian cancer ascites, as exemplified by hla-dra, hla-dpb <dig> and kynu in fig.
5g).
expression of ifn-encoding genes is linked to ovarian cancer survival
analysis of the precog database also revealed a significant association of ifng expression with a longer survival .
similar associations were also found for several genes coding for type i ifns, i.e., ifna <dig>  ifna <dig>  ifna <dig> and ifnb <dig>  evaluation of rna-seq data for different cell types  <cit>   showed that tumor-associated t cells  express ifng at relatively high levels , while tams and tumor cells do not .
interestingly, all tat samples expressed ifng at higher levels than normal cd3+ t cells isolated from the blood of healthy donors, with >10-fold higher levels observed with three out of the five patients analyzed.
it is therefore likely, that partially activated tats are a major source of ifnγ within the tumor microenvironment and the malignancy-associated ascites.
this is consistent with the presence of readily detectable ifnγ levels in the ascites of a subgroup of patients .
by contrast, all type i ifn genes associated with a favorable os were expressed a very low levels by all three cell types, if at all .fig.
<dig> associations of ifn gene expression with survival and abrogation by ifnγ of the ascites-induced il12b activation block.
a z-scores for the association of ifn genes with os .
blue bars: significant associations with a favorable clinical outcome .
b expression of ifng in tams , tumor cell  and tats  samples from ovarian carcinoma ascites, and in cd3+ t cells from healthy donors .
each dot represents an individual sample .
c ifnγ concentrations in the ascites from n =  <dig> ovarian cancer patients determined by elisa.
d
il12b expression in mdms differentiated for  <dig> d either in rpmi plus 5% human a/b serum  or in ovarian cancer ascites in the absence or presence of ifnγ .
cultures were stimulated with lps  plus ifnγ  or solvent only  for 24 h and rna was analyzed by rt-qpcr.
the experiment was performed with  <dig> independent samples .
e p <dig>  protein concentrations in the culture medium of the experiments in panel .
each dot represents an independent sample.
horizontal lines: median.
significance was determined by t-test between unstimulated and ifnγ + lps-stimulated cells
ifnγ prevents the ascites-induced il12b activation block in macrophages
interleukin- <dig>  is a particularly interesting cytokine in the context of ovarian cancer due to its immune stimulatory anti-tumor effects and its inverse associations with disease progression patients .
a hallmark of tams in ovarian cancer ascites is their defect to release il- <dig> in response to inflammatory stimuli, which results from a transcriptional block of the il12b gene encoding the p <dig> subunit  <cit> .
another cytokine with beneficial immune stimulatory and anti-tumor effects in ovarian cancer patients is ifnγ , consistent with the observation that ifnγ can prevent the skewing of monocyte differentiation by ovarian cancer ascites from immunostimulatory il-10lowil-12high macrophages to tam-like il-10highil-12low cells  <cit> .
to assess the relevance of the ifn signaling-associated signature b we explored the relationship between il- <dig> and ifnγ in further detail.
toward this end, we measured the inducibility of il12b mrna by lipopolysaccharide  plus ifnγ in monocyte-derived macrophages that were differentiated either in the presence of regular cell culture medium  or in ascites, both in the absence or presence of ifnγ during macrophage differentiation.
as expected, all ascites samples tested blocked il12b induction, which, however, was almost completely prevented by ifnγ .
in full agreement with these findings, secretion of p <dig> was strongly induced by lps plus fnγ in r <dig>  which was blocked by ascites only in the absence of ifnγ during differentiation .
these results clearly point to a clinically beneficial ifnγ – p40/il12b axis in differentiating myeloid cells in the ovarian cancer microenvironment, thus providing a potential explanation for the association of subgroup b tams with clinically favorable parameters.
discussion
delineation of subgroups of tams
transcriptional signatures of tams distinguishing subgroups of patients in a biologically or clinically meaningful way have not been reported to date.
we have used the ovarian cancer-associated ascites as an experimental system to address this issue by an unbiased approach .
by applying pca to data derived by next generation sequencing we were able to split tam samples from different patients into subgroups characterized by distinct gene expression patterns .
remarkably, cluster b is basically congruent with the subgroup of patients with low expression of the il <dig>  pcolce <dig> and cd <dig> genes, a low fraction of anti-inflammatory cd163+/cd206+ tams and low ascites concentrations of il- <dig> and il- <dig> .
these features are known as negative prognostic factors for different tumor entities, including ovarian cancer .
the same subgroups were also identified by an another unbiased approach, i.e., coexpression analysis of all tam samples .
therefore, these findings clearly suggest that subgroups a and b represent patients with a poor and favorable clinical outcome, respectively.
ifn signaling is a hallmark of signature b tams
intriguingly, a hallmark of subgroup b is the upregulation of target genes of the ifn signaling network .
consistent with this finding, our analysis of the precog database revealed a strong association of these genes with a longer os .
this is in agreement with previous studies which associated a high protein expression of several ifn signaling components with a favorable clinical outcome, including irf <dig>  <cit> .
furthermore, ifnγ inclusion in the first-line therapy of ovarian cancer resulted in an effector immune cell response  <cit>  and a prolongation of progression-free survival  <cit> , while type i ifns had no benefit  <cit> .
in keeping with these observations, elevated ifng expression in ovarian cancer tumor tissue correlated with an improved clinical outcome in patients  <cit> .
our upstream regulator and functional annotations  yielded ifnγ and type i ifn as activated signaling pathways.
we attribute this apparent ambiguity to the fact that type i and ii target genes show substantial overlaps.
type i ifns signal through their common receptor complex via jak1/tyk <dig> to the heterotrimer isgf <dig>  and, to a lesser extent, through stat <dig> homodimers, whereas ifnγ, the single type ii ifn, uses only stat <dig> homodimers phosphorylated by ifnγ receptor-associated jak1/jak <dig>  <cit> .
furthermore, stat-independent pathways could be activated differentially by the different receptor complexes.
taken together, this explains the overlapping, but not identical, effects of the two types of ifns.
interestingly, jak <dig> is among the signature b genes identified in this study  and might thus contribute to ifnγ-mediated effects.
taken together with our observation that, in contrast to ifng , none of the type i ifn genes associated with a longer os is expressed at significant levels by tams, tats or tumor cells  it is conceivable that upregulation of the ifn target genes of signature b is due to activated ifnγ signaling.
collectively, these findings suggest that the increased expression of ifn target genes in cluster b tams results, at least in part, from an elevated level of ifnγ in the tumor microenvironment.
of note, a substantial fraction of the ifn signature genes upregulated in cluster b tams are also target genes of pro-inflammatory pathways .
this suggests that other cytokines present in ascites, notably tnfα  <cit> , might contribute to the induction of these genes.
this would be consistent with the prevailing opinion that pro-inflammatory macrophages inhibit tumor progression.
since rna-seq measures mean transcript levels within a cell population, the fraction of tams expressing a given signature cannot be determined.
it is therefore possible that elevated expression of ifn genes reflects the higher relative occurrence of a subpopulation of cells.
these might be a newly recruited, cd163-negative, monocyte-like tam subset similar to the macrophage fraction that is able to elicit inflammatory responses in immune privileged reproductive organs  <cit> .
this hypothesis is in agreement with the flow cytometry data in fig.
2f, indicating that cd163+ and cd163− cells occur in both subgroup a and b, even though their ratio is clearly different in both subpopulations.
likewise, it is possible that a few tam samples could not be fitted into either subgroup  due to the presence of similar fractions of functionally different subpopulations.
role of t cells
ascites contains substantial numbers of different types of t cells, in particular cd4+ and cd8+ cells  <cit> , known as important ifnγ producers under physiological conditions.
a functional role of t cells in ovarian cancer is supported by many published observations strongly associating infiltrating t cells with a longer survival, with a high ratio of cd8+ versus regulatory t cells having a strong impact .
consistent with these findings, transcriptome analyses defined distinct high-grade serous ovarian carcinoma subtypes, of which the immune reactive subtype was associated with the best prognosis  <cit> .
transcriptome profiling also identified several genes contributing to cytotoxic t lymphocyte recruitment as being differentially expressed in tumors with high versus low cd <dig> t cell infiltration, including irf <dig> , providing another link between t cells, ifnγ and a favorable clinical outcome.
furthermore, the analysis in fig.
6a revealed a positive impact of ifng expression on ovarian cancer os.
it is thus possible that ifnγ in the tumor microenvironment and the expression of ifn target genes in tams are indicators of the presence of activated, ifnγ secreting t and/or nk cells mediating anti-tumor immune responses.
our rna-seq data support the conclusion that tats are a major source of ifnγ in ascites .
since tats showed a considerably higher level of ifng expression compared to normal t cells, it is likely that the former are partially activated, at least in a subset of patients.
this would be consistent with the observed clonal expansion of t cell subpopulations of unknown biological relevance in ovarian cancer ascites  <cit> .
however, tats apparently are not functional with respect to an anti-tumor response, as suggested by progression of the disease, presumably due the inhibitory effect of ascites on t cell activation  <cit> .
in line with this conclusion other makers of an activated th <dig>  th <dig>  th <dig> or th <dig> response were only weakly upregulated in a subset of tats relative to normal t cells , expressed at similar levels in both  or not expressed at all  .
inflammatory signaling and il- <dig> induction
there is a large body of evidence to suggest that ifnγ and il- <dig> are key determinants of a beneficial immune response in many cancers  <cit> .
physiologically, il- <dig> is released by macrophages and other antigen-presenting cells in response to proinflammatory signals, including toll-like receptor ligands and ifnγ from t or nk cells.
il- <dig> in turn stimulates a cytotoxic response by inducing multiple immune stimulatory processes, including the differentiation of naive t cells into th <dig> cells, the activation of cd8 + t cells and the maturation or activation of nk cells  <cit> .
multiple observations strongly support the hypothesis that il- <dig>  and probably its induction by ifnγ, are crucial determinants of ovarian cancer outcome.
for example, il- <dig> locally produced significantly delayed peritoneal disease development in a mouse model  <cit> , engineered tumor-targeted t cells ectopically expressing a fused il12a/il12b cdna have been reported to eradicate ovarian tumors in vivo  <cit>  and a highly significant association was found between high ifng and il12b expression and a more favorable clinical outcome of advanced stage ovarian carcinoma  <cit> .
in tams, the acquisition of a proinflammatory phenotype, including the secretion of il- <dig>  in response to proinflammatory signals is non-functional.
in lps-stimulated murine macrophages, il12b induction is selectively dependent on the nfκb family member rel  <cit>  and on tlr signaling-induced chromatin remodeling which is independent of rel  <cit> .
furthermore, ifnγ has been shown to enhance the synthesis of il- <dig> by priming macrophages for lps-mediated induction of the il12b gene  <cit> .
finally, the nuclear accumulation of a nfκb p <dig> homodimer with presumed inhibitory function has been suggested for the acquisition of a tam phenotype characterized by the defective production of il- <dig>  <cit> .
intriguingly, ifnγ can prevent the inhibitory effect of ascites on the inducibility of il12b in macrophages , consistent with a previous report that ifnγ was able to shift monocyte differentiation from tam-like cells to pro-inflammatory macrophages  <cit> .
it is currently unclear if, and if so how, tlr pathways, ifnγ triggered stat signaling, chromatin remodeling and p <dig> accumulation functionally interact in the regulation/dysregulation of the il12b gene in either normal macrophages or tams.
understand these connections will be crucial to be able to explore the potential of the ifnγ – il- <dig> axis in stimulating cytotoxic immune responses and assess potential applications.
CONCLUSIONS
in the present study, we have address the question as to whether associated-derived tams from different patients with ovarian cancer represent a continuum of overlapping transcriptomes or can be categorized into phenotypically distinct groups on the basis of their global gene expression patterns.
the results of both principal component analysis  and coexpression analysis clearly demonstrated that the latter is the case, and lead to the definition of two highly distinct subgroups of patients differing in the expression of genes associated with cytokine signaling, immune regulation and extracellular matrix reorganization.
one of the two subgroups identified  is associated with a high expression of protumorigenic, immunosuppressive and clinically unfavorable markers, including il- <dig>  il- <dig>  cd <dig> and pcolce <dig>  by contrast, the second subgroup is characterized by the upregulation of genes linked to ifn signaling and associated with a longer survival.
expression of this ifn-related signature also showed a striking link to a longer survival, and ifnγ abrogated the inhibitory effect of ovarian cancer ascites on the inducibility of il- <dig> in cultured macrophages.
as il- <dig> is a key mediator of a cytotoxic immune response, this finding provides a possible explanation for the link of the ifn signaling-associated signature b to ovarian cancer survival.
additional files
additional file 1: supplemental table.
table s1: patients data .
additional file 2: supplemental datasets s1-s <dig>
dataset s1: complete rna-seq data.
dataset s2: edger results.
dataset s3: signature a expression data.
dataset s4: signature b expression data.
dataset s5: cluster i expression data.
dataset s6: cluster ii expression data.
dataset s7: cluster iii expression data.
dataset s8: common cluster i / signature a genes.
dataset s9: common cluster iii / signature b genes.
dataset s10: precog z-scores for signature a and b genes.
additional file 3: supplemental figures s1-s <dig>
figure s1: inverse association of pcolce <dig> expression with high-grade serous ovarian cancer survival .
figure s2: hierarchial clustering of coexpressed high variance genes.
figure s3: venn diagram showing the overlaps of the the upstream regulators gene sets identified in fig.
3c.
figure s4: subgroup-selective expression of signature genes.
figure s5: association of signature a  and signature b  with ovarian cancer survival .
figure s6: association of the ecm remodeling-linked genes of signature a with high-grade serous ovarian cancer survival.
figure s7: association of tumor-infiltrating host cells with high-grade serous ovarian cancer survival .
figure s8: expression of type i ifn genes in different cell types present in ovarian cancer ascites.
abbreviations
gogene ontology
ifninterferon
ilinterleukin
lpslipopolysaccharide
mdmmonocyte-derived macrophage
osoverall survival
pcaprincipal component analysis
pcolce2procollagen c-endopeptidase enhancer 2
rfsrelapse-free survival
rna-seqrna sequencing
tamtumor-associated macrophage
tnftumor necrosis factor α
tpmtranscripts per million

 ovarian tumors ranks fifth as the cause of cancer-related death in women and represents the deadliest of all gynecological tumors. more than 90% of ovarian cancers are carcinomas that originate from the ovarian surface or fallopian tube epithelium. high grade serous adenocarcinoma is the most common ovarian carcinoma subtype, with most patients presenting with advanced stage disease and disseminated tumor masses at the time of diagnosis. although most ovarian cancers are highly sensitive to first-line adjuvant chemotherapy, the disease has a dire prognosis with an overall 5-year survival rate of less than 40%. several characteristic features contribute to the fatal nature of high grade serous ovarian adenocarcinoma, including the shedding of tumor cells at a very early stage of the disease, their spreading via the peritoneal fluid to form transcoelomic metastases and the tumor-promoting and immune suppressive effect of the peritoneal tumor environment, frequently formed by the malignancy-associated effusion within the peritoneal cavity, commonly referred to as malignant ascites. this tumor microenvironment, consisting to a large extent of host-derived cells, is crucial for the growth, progression, therapy resistance and immune escape of malignant tumors, including ovarian cancer. the most common cell types in ovarian hgsc-associated ascites are macrophages and t lymphocytes. tumor-associated macrophages can be derived from both blood monocytes or resident tissue macrophages, with the latter most likely representing the major origin of tams in ovarian cancer. tam activation is skewed by factors of the tumor microenvironment to adopt a spectrum of phenotypes that represent mixed forms of alternatively activated and pro-inflammatory macrophages, which has also been clearly demonstrated for tams in ovarian cancer ascites. tams do not possess tumoricidal activity, but are rather thought to promote immune suppression and various aspects of cancer growth and progression, including tumor cell invasion, angiogenesis and metastasis. consistent with these tumor-promoting functions of tams, expression of the alternative activation marker cd in tams from malignancy-associated ascites showed a strong correlation with early relapse of serous ovarian carcinoma after first-line therapy. among the soluble factors contributing to tam polarization, tumor progression and a poor clinical outcome, interleukin, il- transforming growth factor β and arachidonic acid play a prominent role. to date, transcriptional signatures of human tams that distinguish subgroups of patients have not been described. in the present study, we address this issue by determining the transcriptome of tams from different patients in conjunction with principal component analysis and coexpression analysis to define distinct subgroups. these analyses lead to the identification of two subgroups differing in the expression of genes associated with cytokine signaling, immune regulation, extracellular matrix reorganization and overall survival. of note, an interferon related signature showed a striking association with a favorable clinical outcome. furthermore, ifnγ counteracted repression by ovarian cancer ascites of il- a key mediator of an anti-tumor response mediated by natural killer cells and t lymphocytes, consistent with the observed association of an ifn signaling-associated signature with ovarian cancer survival. methods patient samples ascites was collected from patients undergoing primary surgery at the university hospital in marburg. patient characteristics are presented in additional file 1: table s isolation and immunophenotyping of cells from ovarian cancer ascites mononuclear cells were isolated from ascites by lymphocyte separation medium density gradient centrifugation and further purified by magnetic cell sorting using cd microbeads. tams were analyzed by flow cytometry for surface expression of cd cd cd cd cd and cd as described. tumor cell spheroids and t cells were analyzed as previously published. isolation and culture of monocyte-derived macrophages monocyte-derived macrophages were generated from monocytes from healthy donors as described and cultured in rpmi medium with 5% human male ab serum or cell-free ascites from ovarian cancer patients, as indicated. t cell from peripheral blood were isolated as described for ascites. buffy coats were obtained from the blood bank at ukgm giessen, germany. rt-qpcr isolation of rna and rt-qpcr were carried out as described. the following primers were used:rpl27_fw: 5′aaagctgtcatcgtgaagaac rpl27_rv: 5′gctgtcactttgcgggggtag il12b_fw: 5′gcgaggttctaagccattcg il12b_rev: 5′actccttgttgtcccctctg cxcl10_fw: 5′aagcagttagcaaggaaaggtc cxcl10_rv: 5′gacatatactccatgtagggaagtga gbp4_fw: 5′ttcaaaggcattagagattcttga gbp4_rv: 5′gtggagcccagagggaag gpnmb_fw: 5′ctatgagaagaactgcagaaatg gpnmb_rv: 5′gttatgatggctttggccgg lgals2_fw: 5′ccacgagttgagccctgag lgals2_rv: 5′cggcttcatgtccatgttc kitlg_fw: 5′gccaagtcttacaagggcag kitlg_rv: 5′gaaactctctctctttctcttgc mrc1_fw: 5′cct cgg acc tgg tta ggg mrc1_rv: 5′ggatgtgtggtcctccttgg elisa concentrations of p in cell culture supernatants were determined using an elisa kit from biolegend/biozol according to the instructions of the manufacturer. ifnγ, il- and il- levels in ascites were quantified by elisa kits purchased from ebioscience. rna sequencing rna isolation and rna-seq was carried out on an illumina hiseq as described. genome assembly and gene model data were retrieved from ensembl revision sequencing data were deposited at ebi arrayexpress. statistical analysis of experimental data paired and unpaired t tests were carried out with the python functions scipy.stats.ttest_rel () and scipy.stats.ttest_ind (), respectively. results were expressed as follows: *p < ; **p < ; ***p < ; ****p < . confidence intervals were calculated using the bootstrap method. further statistical analyses were performed using the python functions numpy.percentile () and pandas.dataframe.boxplot (). analysis of rna-seq data rna-seq data was aligned to ensembl v using star and processed as reported. the number of mapped reads was in the range of – million. tpm values were calculated based on the total gene read counts and length of merged exons and corrected for contamination by tumor cells as described. the source code for implementing the algorithm for tpm correction has also been deposited at github. genes were considered expressed if they had a minimum tpm value of tam samples with tpm > for epcam or msln were excluded due to presumed tumor cell contamination. all genomic sequence and gene annotation data were retrieved from ensembl release genome assembly hg pca and delineation of differentially expressed gene clusters pca was carried out on using the sklearnpca and sklearn_pca.fit_transform () functions on rna-seq data. pearson correlation coefficients were determined with scipy.stats.pearsonr (). the bioconductor package edger was used for the delineation of high variance gene clusters differentially regulated in subgroups of tams identified by pca. coexpression analysis genes with the greatest expression variance were identified by pandas.dataframe.var (). pearson correlation coefficients were determined for the top genes using scipy.stats.pearsonr (). sets of genes with r >  and n >  were combined and analyzed by hierarchical clustering using the scipy.cluster.hierarchy functions linkage and dendrogram. the resulting clusters were analyzed for intersections the signatures identified by pca, which revealed close relationships of cluster i with signature b and cluster iii with signature a. pathway analyses gene sets were analyzed for upstream regulators using the ingenuity pathway analysis database as described. functional annotations were performed by gene ontology enrichment analysis. survival analyses overall survival data were retrieved from precog. associations between gene expression and relapse-free survival were analyzed by the web-based tool “km plotter” using the following settings: ‘auto select best cutoff’, probe set option: ‘jetset best probe’, histology: serous, datasets: all; other settings: default.  clustering of ovarian carcinoma tam samples the transcriptomes determined for independent samples of tam isolated from the ascites of ovarian cancer patients were analyzed for potential similarity patterns by different approaches, as schematically summarized in fig.  these samples were selected for very low contamination with tumor cells, as indicated by low tpm values for the epithelial marker genes epcam and msln. furthermore, we excluded all genes highly expressed in tumor cells or t cells versus tams to minimize interference by contaminating cells.fig. schematic representation of data analysis and summary of results. nomenclature for designation of clusters: tam samples clustered by pca: letters; genes clustered by coexpression analysis: roman numbers. genes identified by edger and upregulated in tam subgroup a or b were defined as signature a and b, respectively genewise normalized tam transcriptomes were used for the bioinformatic delineation of similarity patterns. principal component analysis did not yield a clear partitioning of tam samples into subgroups, although eigenvalues suggested that the first two components can explain most of the data. we therefore additionally grouped samples according to the expression of cd previously established as a tam marker associated with a poor clinical outcome of ovarian cancer. combining pca with the the expression pattern of this surrogate marker revealed two discernible subgroups, which was confirmed by pearson correlation and distance-based multidimensional scaling analysis. two other markers associated with pro-tumorigenic functions, i.e., il and pcolce, showed a very similar pattern of expression, except for il expression in tam. based on this data we defined subgroup a as tam tam tam tam tam and tam expressing cd pcolce and il at high levels relative to the subgroup b samples tam tam tam tam tam and tam these subgroups were confirmed by flow cytometry showing a significantly higher fraction of cd163+ and cd163+cd206+ cells in subgroup a versus subgroup b tams, which was not observed for other macrophage markers.fig. clustering of ovarian carcinoma tam samples based on rna-seq data. a principal component analysis of tam transcriptomes. samples with high expression of cd are shown in red, samples with low expression of cd in blue. b heatmap based on pearson correlation coefficients calculated for the tam transcriptomes identified by pca. c-e expression of il pcolce and cd in tam samples of clusters a and b. dotted lines show the quantiles used in panel. f flow cytometry analysis of cluster a and b samples. the plot shows the fraction of cd16+, cd32+, cd64+, cd163+, cd206+ and cd163+cd206+ cells. g concentrations of il- and il in the ascites of cluster a and b patients determined by elisa. boxes show the upper and lower quartiles, whiskers the 95% confidence intervals, and horizontal lines the median. asterisks indicate the statistical significance determined by unpaired t test. n/a, not applicable since all values >97%; ns: not significant taken together, these data indicate that cluster a tams are skewed towards alternative activation, extracellular matrix remodeling and promotion of tumor growth. as these markers are associated with a short relapse-free survival, it is likely that cluster b is linked to a favorable clinical outcome. this conclusion is supported by the observation that the ascites concentrations of il- highly predictive of a poor survival of ovarian cancer was consistently and dramatically increased in subgroup a versus subgroup b patients. a similar pattern was observed with il-, also associated with a short time to relapse. cluster-specific gene expression to gain more insight into the specific phenotypes of subgroup a and b tams, we analyzed the rna-seq data sets with edger, a bioconductor package for reliable gene-specific dispersion estimation in small datasets. the edger tool identified a group of genes differentially expressed in the two subgroups of tams with an fdr of <. of these, genes were upregulated in subgroup a versus b, and genes showed the opposite pattern. these gene sets were termed signature a and signature b. in contrast to these protein coding rnas, edger did not identify any differentially expressed lncrnas in our rna-seq data set.fig. identification of differentially expressed genes in subgroup a versus b tams by edger. a scatter plot showing the expression of genes identified by the edger tool in tam subgroups a or b identified by pca in fig.  data represent the ratio of median tpm values for subgroup a versus subgroup b. b functional annotation of genes upregulated in subgroup a or subgroup b by gene ontology enrichment analysis. p values are plotted against fold enrichment. only specific non-redundant terms with p values < and enrichment > are shown. c upstream regulator analysis of upregulated genes with p < 10− d expression of the ifn signaling-associated genes of signature b identified by go enrichment analysis. boxes show the upper and lower quartiles, whiskers the 95% ci, and horizontal lines the median. e validation of rna-seq data. analysis by rt-qpcr of signature a and b genes in tam samples from subgroup a and b. error bars show the standard deviation and horizontal lines the mean. red: cluster a samples; blue: cluster b samples. asterisks indicate the statistical significance determined by unpaired t test; ns: not significant gene ontology enrichment analysis identified significant associations of signature a with diverse biological processes, including ecm remodeling, locomotion, endocytosis as well as lipid and carbohydrate catabolism. in contrast, innate immune defense mechanisms, t cell activation and ifn signaling were strongly associated with signature b. consistent with these findings, ingenuity upstream regulator analysis indicated that signature b genes are major targets of proinflammatory pathways triggered by lipopolysaccharide, tumor necrosis factor α and infγ, with all three target gene sets showing strong overlaps. the differential expression patterns of signature a and b genes are shown in fig. 3d and additional file 3: figure s for the ifn and ecm gene sets identified by go enrichment analysis as representative examples. as expected, these gene sets showed opposite patterns of regulation and the subgroup-selective expression was clearly significant for the vast majority of genes. the rna-seq data for several genes identified by edger were confirmed by rt-qpcr. taken together, these observations point to a relatively high complexity of biological functions affected by signature a genes, while the role of signature b genes appears to be specifically associated with ifn-stimulated immune defense mechanisms. confirmation of clustering by coexpression analysis to obtain independent evidence for the robustness of the clusters defined by pca and edger we performed coexpression analysis the genes showing the highest variance across all tam samples. pearson correlation and hierarchical clustering yielded three large clusters of coregulated genes. hierarchical clustering of tam samples using a combined set of these genes  yielded the identical partitioning into subgroups a and b as the pca-based approach in fig. 2a. consistent with this observation, two of these gene clusters showed substantial and specific overlaps with signatures a and b, respectively, i.e., cluster i with signature a; cluster iii with signature b, as shown in fig. 4c as well as additional file 2: datasets s and s fig. coexpression analysis of all tam samples. a correlation based heatmap of gene clusters defined by coexpression analysis of genes with the highest variance across all tam samples, followed by hierarchical clustering. b hierarchical clustering of patients based on the genes identified in panel. c overlap of genes in clusters i - iv with signatures a and b identified by pca. d functional annotation of cluster i, cluster ii and cluster iii genes by gene ontology enrichment analysis. p values are plotted against fold enrichment. only specific non-redundant terms with p values < and enrichment > are shown. e expression of cluster i genes in subgroup a and b samples. f as panel, but for cluster ii genes. g as panel, but for cluster iii genes. boxes show the upper and lower quartiles, whiskers the 95% confidence intervals, and horizontal lines the median functional annotations revealed endocytosis and chemotaxis for cluster i, chromatin modification and splicing for cluster ii, and immune defense and interferon signaling for cluster iii. consistent with the overlaps of described in the preceding paragraph, the go terms for cluster i were also found for signature b, and the terms for cluster iii correspond to those for signature a. finally, expression of cluster i genes was higher in subgroup b versus subgroup a, and vice versa, expression of cluster iii genes was higher in subgroup a relative to subgroup b. in contrast, no differential expression was observed for cluster ii genes. taken together, these findings clearly suggest that cluster iii corresponds to signature a, while cluster i corresponds to signature b. thus, both strategies, pca of patient samples followed by edger and coexpression analysis of high variance genes yielded very similar results, and identified ifn signaling as a hallmark of signature b upregulated in subtype b tams. association of signature b, interferon signaling and survival in order to identify potential associations between the expression of this genes in ovarian cancer with clinical outcome we made use of the precog database, which contains the results of a meta-analysis of patients from studies. these studies used solid ovarian tumor tissue containing substantial amounts of myeloid cells for transcriptome analysis, suggesting that the precog data are also suitable for testing survival associations for genes expressed in abundant tumor-associated host cells, such as tams. signature a, cluster iii and their overlap comprised similar fractions of genes associated with poor or favorable os. only when genes with distinct functional annotations were analyzed separately a clear os association was detectable. thus, signature a genes linked to ecm remodeling were strongly associated with a short os. in contrast, signature b, cluster i and their overlap were clearly linked to a favorable clinical outcome. this applied in particular to the ifn signaling-associated genes of signature b, as shown in detail in fig. 5b. a similar inverse association with relapse-free survival was also found based on data from the km plotter database, as exemplified for irf and tap in fig. 5c and d. all precog z-scores for signature a and b genes are also listed in additional file 2: dataset s fig. association of cluster-specific gene expression with ovarian cancer survival. a mean z-scores for signature a and b genes; the ecm-related and ifn signaling-associated genes of signature a and b, respectively; cluster iii and i genes; and genes representing the intersection of signature a with cluster iii or of signature b with cluster i. survival data were obtained from the precog database with ovarian cancer patients. b os z-scores for signature b genes that are associated with ifn signaling. significant associations with a favorable clinical outcome are shown in blue. the corresponding data for the complete signatures a and b are shown in additional file 3: figure s c-f kaplan-meier plots analyzing the association of irf tap cd and cd with rfs of high-grade serous ovarian cancer determined by km plotter. g expression of the signature b genes hla-dpb hla-dra and kynu in tams, tumor cells and cd3+ tats isolated from ovarian cancer ascites these associations with a favorable clinical course are not simply consequence of the extent of myeloid cell infiltration. first, activation state-independent myeloid marker genes, such as cd or cd did not show any significant association with ovarian cancer os or rfs. second, tumor infiltration by myeloid cells inferred from rna expression data showed no significant association with os, with a trend towards a worse clinical outcome for monocytes. third, a number of the ifn signaling-associated genes of signature b are expressed at high levels selectively in tams compared to tumor cells or tumor-associated cd3+ lymphocytes isolated from ovarian cancer ascites, as exemplified by hla-dra, hla-dpb and kynu in fig. 5g). expression of ifn-encoding genes is linked to ovarian cancer survival analysis of the precog database also revealed a significant association of ifng expression with a longer survival. similar associations were also found for several genes coding for type i ifns, i.e., ifna ifna ifna and ifnb evaluation of rna-seq data for different cell types showed that tumor-associated t cells express ifng at relatively high levels, while tams and tumor cells do not. interestingly, all tat samples expressed ifng at higher levels than normal cd3+ t cells isolated from the blood of healthy donors, with >10-fold higher levels observed with three out of the five patients analyzed. it is therefore likely, that partially activated tats are a major source of ifnγ within the tumor microenvironment and the malignancy-associated ascites. this is consistent with the presence of readily detectable ifnγ levels in the ascites of a subgroup of patients. by contrast, all type i ifn genes associated with a favorable os were expressed a very low levels by all three cell types, if at all.fig. associations of ifn gene expression with survival and abrogation by ifnγ of the ascites-induced il12b activation block. a z-scores for the association of ifn genes with os. blue bars: significant associations with a favorable clinical outcome. b expression of ifng in tams, tumor cell and tats samples from ovarian carcinoma ascites, and in cd3+ t cells from healthy donors. each dot represents an individual sample. c ifnγ concentrations in the ascites from n =  ovarian cancer patients determined by elisa. d il12b expression in mdms differentiated for d either in rpmi plus 5% human a/b serum or in ovarian cancer ascites in the absence or presence of ifnγ. cultures were stimulated with lps plus ifnγ or solvent only for 24 h and rna was analyzed by rt-qpcr. the experiment was performed with independent samples. e p protein concentrations in the culture medium of the experiments in panel. each dot represents an independent sample. horizontal lines: median. significance was determined by t-test between unstimulated and ifnγ + lps-stimulated cells ifnγ prevents the ascites-induced il12b activation block in macrophages interleukin- is a particularly interesting cytokine in the context of ovarian cancer due to its immune stimulatory anti-tumor effects and its inverse associations with disease progression patients. a hallmark of tams in ovarian cancer ascites is their defect to release il- in response to inflammatory stimuli, which results from a transcriptional block of the il12b gene encoding the p subunit. another cytokine with beneficial immune stimulatory and anti-tumor effects in ovarian cancer patients is ifnγ, consistent with the observation that ifnγ can prevent the skewing of monocyte differentiation by ovarian cancer ascites from immunostimulatory il-10lowil-12high macrophages to tam-like il-10highil-12low cells. to assess the relevance of the ifn signaling-associated signature b we explored the relationship between il- and ifnγ in further detail. toward this end, we measured the inducibility of il12b mrna by lipopolysaccharide plus ifnγ in monocyte-derived macrophages that were differentiated either in the presence of regular cell culture medium or in ascites, both in the absence or presence of ifnγ during macrophage differentiation. as expected, all ascites samples tested blocked il12b induction, which, however, was almost completely prevented by ifnγ. in full agreement with these findings, secretion of p was strongly induced by lps plus fnγ in r which was blocked by ascites only in the absence of ifnγ during differentiation. these results clearly point to a clinically beneficial ifnγ – p40/il12b axis in differentiating myeloid cells in the ovarian cancer microenvironment, thus providing a potential explanation for the association of subgroup b tams with clinically favorable parameters. discussion delineation of subgroups of tams transcriptional signatures of tams distinguishing subgroups of patients in a biologically or clinically meaningful way have not been reported to date. we have used the ovarian cancer-associated ascites as an experimental system to address this issue by an unbiased approach. by applying pca to data derived by next generation sequencing we were able to split tam samples from different patients into subgroups characterized by distinct gene expression patterns. remarkably, cluster b is basically congruent with the subgroup of patients with low expression of the il pcolce and cd genes, a low fraction of anti-inflammatory cd163+/cd206+ tams and low ascites concentrations of il- and il-. these features are known as negative prognostic factors for different tumor entities, including ovarian cancer. the same subgroups were also identified by an another unbiased approach, i.e., coexpression analysis of all tam samples. therefore, these findings clearly suggest that subgroups a and b represent patients with a poor and favorable clinical outcome, respectively. ifn signaling is a hallmark of signature b tams intriguingly, a hallmark of subgroup b is the upregulation of target genes of the ifn signaling network. consistent with this finding, our analysis of the precog database revealed a strong association of these genes with a longer os. this is in agreement with previous studies which associated a high protein expression of several ifn signaling components with a favorable clinical outcome, including irf. furthermore, ifnγ inclusion in the first-line therapy of ovarian cancer resulted in an effector immune cell response and a prolongation of progression-free survival, while type i ifns had no benefit. in keeping with these observations, elevated ifng expression in ovarian cancer tumor tissue correlated with an improved clinical outcome in patients. our upstream regulator and functional annotations yielded ifnγ and type i ifn as activated signaling pathways. we attribute this apparent ambiguity to the fact that type i and ii target genes show substantial overlaps. type i ifns signal through their common receptor complex via jak1/tyk to the heterotrimer isgf and, to a lesser extent, through stat homodimers, whereas ifnγ, the single type ii ifn, uses only stat homodimers phosphorylated by ifnγ receptor-associated jak1/jak. furthermore, stat-independent pathways could be activated differentially by the different receptor complexes. taken together, this explains the overlapping, but not identical, effects of the two types of ifns. interestingly, jak is among the signature b genes identified in this study and might thus contribute to ifnγ-mediated effects. taken together with our observation that, in contrast to ifng, none of the type i ifn genes associated with a longer os is expressed at significant levels by tams, tats or tumor cells it is conceivable that upregulation of the ifn target genes of signature b is due to activated ifnγ signaling. collectively, these findings suggest that the increased expression of ifn target genes in cluster b tams results, at least in part, from an elevated level of ifnγ in the tumor microenvironment. of note, a substantial fraction of the ifn signature genes upregulated in cluster b tams are also target genes of pro-inflammatory pathways. this suggests that other cytokines present in ascites, notably tnfα, might contribute to the induction of these genes. this would be consistent with the prevailing opinion that pro-inflammatory macrophages inhibit tumor progression. since rna-seq measures mean transcript levels within a cell population, the fraction of tams expressing a given signature cannot be determined. it is therefore possible that elevated expression of ifn genes reflects the higher relative occurrence of a subpopulation of cells. these might be a newly recruited, cd163-negative, monocyte-like tam subset similar to the macrophage fraction that is able to elicit inflammatory responses in immune privileged reproductive organs. this hypothesis is in agreement with the flow cytometry data in fig. 2f, indicating that cd163+ and cd163− cells occur in both subgroup a and b, even though their ratio is clearly different in both subpopulations. likewise, it is possible that a few tam samples could not be fitted into either subgroup due to the presence of similar fractions of functionally different subpopulations. role of t cells ascites contains substantial numbers of different types of t cells, in particular cd4+ and cd8+ cells, known as important ifnγ producers under physiological conditions. a functional role of t cells in ovarian cancer is supported by many published observations strongly associating infiltrating t cells with a longer survival, with a high ratio of cd8+ versus regulatory t cells having a strong impact. consistent with these findings, transcriptome analyses defined distinct high-grade serous ovarian carcinoma subtypes, of which the immune reactive subtype was associated with the best prognosis. transcriptome profiling also identified several genes contributing to cytotoxic t lymphocyte recruitment as being differentially expressed in tumors with high versus low cd t cell infiltration, including irf, providing another link between t cells, ifnγ and a favorable clinical outcome. furthermore, the analysis in fig. 6a revealed a positive impact of ifng expression on ovarian cancer os. it is thus possible that ifnγ in the tumor microenvironment and the expression of ifn target genes in tams are indicators of the presence of activated, ifnγ secreting t and/or nk cells mediating anti-tumor immune responses. our rna-seq data support the conclusion that tats are a major source of ifnγ in ascites. since tats showed a considerably higher level of ifng expression compared to normal t cells, it is likely that the former are partially activated, at least in a subset of patients. this would be consistent with the observed clonal expansion of t cell subpopulations of unknown biological relevance in ovarian cancer ascites. however, tats apparently are not functional with respect to an anti-tumor response, as suggested by progression of the disease, presumably due the inhibitory effect of ascites on t cell activation. in line with this conclusion other makers of an activated th th th or th response were only weakly upregulated in a subset of tats relative to normal t cells, expressed at similar levels in both or not expressed at all. inflammatory signaling and il- induction there is a large body of evidence to suggest that ifnγ and il- are key determinants of a beneficial immune response in many cancers. physiologically, il- is released by macrophages and other antigen-presenting cells in response to proinflammatory signals, including toll-like receptor ligands and ifnγ from t or nk cells. il- in turn stimulates a cytotoxic response by inducing multiple immune stimulatory processes, including the differentiation of naive t cells into th cells, the activation of cd8 + t cells and the maturation or activation of nk cells. multiple observations strongly support the hypothesis that il- and probably its induction by ifnγ, are crucial determinants of ovarian cancer outcome. for example, il- locally produced significantly delayed peritoneal disease development in a mouse model, engineered tumor-targeted t cells ectopically expressing a fused il12a/il12b cdna have been reported to eradicate ovarian tumors in vivo and a highly significant association was found between high ifng and il12b expression and a more favorable clinical outcome of advanced stage ovarian carcinoma. in tams, the acquisition of a proinflammatory phenotype, including the secretion of il- in response to proinflammatory signals is non-functional. in lps-stimulated murine macrophages, il12b induction is selectively dependent on the nfκb family member rel and on tlr signaling-induced chromatin remodeling which is independent of rel. furthermore, ifnγ has been shown to enhance the synthesis of il- by priming macrophages for lps-mediated induction of the il12b gene. finally, the nuclear accumulation of a nfκb p homodimer with presumed inhibitory function has been suggested for the acquisition of a tam phenotype characterized by the defective production of il-. intriguingly, ifnγ can prevent the inhibitory effect of ascites on the inducibility of il12b in macrophages, consistent with a previous report that ifnγ was able to shift monocyte differentiation from tam-like cells to pro-inflammatory macrophages. it is currently unclear if, and if so how, tlr pathways, ifnγ triggered stat signaling, chromatin remodeling and p accumulation functionally interact in the regulation/dysregulation of the il12b gene in either normal macrophages or tams. understand these connections will be crucial to be able to explore the potential of the ifnγ – il- axis in stimulating cytotoxic immune responses and assess potential applications.  in the present study, we have address the question as to whether associated-derived tams from different patients with ovarian cancer represent a continuum of overlapping transcriptomes or can be categorized into phenotypically distinct groups on the basis of their global gene expression patterns. the results of both principal component analysis and coexpression analysis clearly demonstrated that the latter is the case, and lead to the definition of two highly distinct subgroups of patients differing in the expression of genes associated with cytokine signaling, immune regulation and extracellular matrix reorganization. one of the two subgroups identified is associated with a high expression of protumorigenic, immunosuppressive and clinically unfavorable markers, including il- il- cd and pcolce by contrast, the second subgroup is characterized by the upregulation of genes linked to ifn signaling and associated with a longer survival. expression of this ifn-related signature also showed a striking link to a longer survival, and ifnγ abrogated the inhibitory effect of ovarian cancer ascites on the inducibility of il- in cultured macrophages. as il- is a key mediator of a cytotoxic immune response, this finding provides a possible explanation for the link of the ifn signaling-associated signature b to ovarian cancer survival. additional files additional file 1: supplemental table. table s1: patients data. additional file 2: supplemental datasets s1-s dataset s1: complete rna-seq data. dataset s2: edger results. dataset s3: signature a expression data. dataset s4: signature b expression data. dataset s5: cluster i expression data. dataset s6: cluster ii expression data. dataset s7: cluster iii expression data. dataset s8: common cluster i / signature a genes. dataset s9: common cluster iii / signature b genes. dataset s10: precog z-scores for signature a and b genes. additional file 3: supplemental figures s1-s figure s1: inverse association of pcolce expression with high-grade serous ovarian cancer survival. figure s2: hierarchial clustering of coexpressed high variance genes. figure s3: venn diagram showing the overlaps of the the upstream regulators gene sets identified in fig. 3c. figure s4: subgroup-selective expression of signature genes. figure s5: association of signature a and signature b with ovarian cancer survival. figure s6: association of the ecm remodeling-linked genes of signature a with high-grade serous ovarian cancer survival. figure s7: association of tumor-infiltrating host cells with high-grade serous ovarian cancer survival. figure s8: expression of type i ifn genes in different cell types present in ovarian cancer ascites. abbreviations gogene ontology ifninterferon ilinterleukin lpslipopolysaccharide mdmmonocyte-derived macrophage osoverall survival pcaprincipal component analysis pcolce2procollagen c-endopeptidase enhancer 2 rfsrelapse-free survival rna-seqrna sequencing tamtumor-associated macrophage tnftumor necrosis factor α tpmtranscripts per million 
the observed ifnγ-mediated restoration of the inducibility of il- in the presence of ascites provides a possible explanation for the association of an interferon signaling-associated signature with a favorable clinical outcome. consistent with these results, ifnγ was able to abrogate the suppressive effect of ovarian cancer ascites on the inducibility of il12b expression and il- secretion, a key determinant of a cytotoxic immune response. intriguingly, analysis of published data for ovarian cancer patients revealed a strong association of this transcriptional signature with a longer overall survival. a hallmark of subgroup a is a high expression of clinically unfavorable markers, including  high cd expression, a surface receptor characteristic of an anti-inflammatory activation state, increased pcolce expression, indicative of enhanced extracellular matrix organization, and elevated ascites levels of il- and il- linked to the aggressiveness of ovarian cancer and immune suppression. in contrast, subgroup b tams are characterized by the upregulation of genes linked to immune defense mechanisms and interferon signaling. we have addressed this issue by analyzing global expression patterns of tams isolated from the ascites of ovarian cancer patients. tams isolated from different ovarian cancer patients can be stratified by coexpression or principal component analysis into subgroups with specific biological features and associated with distinct clinical outcomes. the survival of ovarian cancer patients is linked to the presence of tams with a transcriptional signature that is characterized by a low expression of protumorigenic and immunosuppressive markers and an upregulation of genes linked to interferon signaling. ovarian cancer ascitestumor-associated macrophagesinterferon signalingcd163interleukin 6interleukin 10pcolce2http://dx.doi.org/ /501100001659deutsche forschungsgemeinschaftre 1590/1-1mu 601/17-1reinartz silke müller rolf http://dx.doi.org/ /100008672wilhelm sander-stiftungissue-copyright-statement© the author 2017
 although tumor-associated macrophages are essential for cancer progression, connections between different clinical outcomes and transcriptional networks have not been reported. we have addressed this issue by analyzing global expression patterns of tams isolated from the ascites of ovarian cancer patients.  tams isolated from different ovarian cancer patients can be stratified by coexpression or principal component analysis into subgroups with specific biological features and associated with distinct clinical outcomes. a hallmark of subgroup a is a high expression of clinically unfavorable markers, including  high cd expression, a surface receptor characteristic of an anti-inflammatory activation state, increased pcolce expression, indicative of enhanced extracellular matrix organization, and elevated ascites levels of il- and il- linked to the aggressiveness of ovarian cancer and immune suppression. in contrast, subgroup b tams are characterized by the upregulation of genes linked to immune defense mechanisms and interferon signaling. intriguingly, analysis of published data for ovarian cancer patients revealed a strong association of this transcriptional signature with a longer overall survival. consistent with these results, ifnγ was able to abrogate the suppressive effect of ovarian cancer ascites on the inducibility of il12b expression and il- secretion, a key determinant of a cytotoxic immune response.  the survival of ovarian cancer patients is linked to the presence of tams with a transcriptional signature that is characterized by a low expression of protumorigenic and immunosuppressive markers and an upregulation of genes linked to interferon signaling. the observed ifnγ-mediated restoration of the inducibility of il- in the presence of ascites provides a possible explanation for the association of an interferon signaling-associated signature with a favorable clinical outcome. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords ovarian cancer ascitestumor-associated macrophagesinterferon signalingcd163interleukin 6interleukin 10pcolce2http://dx.doi.org/ /501100001659deutsche forschungsgemeinschaftre 1590/1-1mu 601/17-1reinartz silke müller rolf http://dx.doi.org/ /100008672wilhelm sander-stiftungissue-copyright-statement© the author 2017 