Sample-Treatment with the Virucidal ß-Propiolactone Does Not Preclude Analysis by Large Panel Affinity Proteomics, Including the Discovery of Biomarker Candidates.

Virus inactivation is a prerequisite for safe handling of high-risk infectious samples. ß-Propiolactone (BPL) is an established reagent with proven virucidal efficacy. BPL primarily reacts with DNA, RNA, and amino acids. The latter may modify antigenic protein epitopes interfering with binding properties of affinity reagents such as antibodies and aptamers used in affinity proteomic screens. We investigated (i) the impact of BPL treatment on the analysis of protein levels in plasma samples using the aptamer-based affinity proteomic platform SomaScan and (ii) effects on protein detection in conditioned medium samples using the proximity extension assay-based Olink Target platform. In the former setup, BPL-treated and native plasma samples from patients with ovarian cancer (n = 12) and benign diseases (n = 12) were analyzed using the SomaScan platform. In the latter, conditioned media samples collected from cultured T cells with (n = 3) or without (n = 3) anti-CD3 antibody stimulation were analyzed using the Olink Target platform. BPL-related changes in protein detection were evaluated comparing native and BPL-treated states, simulating virus inactivation, and impact on measurable group differences was assessed. While approximately one-third of SomaScan measurements were significantly changed by the BPL treatment, a majority of antigen/aptamer interactions remained unaffected. Interaction effects of BPL treatment and disease state, potentially altering detectability of group differences, were observable for less than one percent of targets (0.6%). BPL effects on protein detection with Olink Target were also limited, affecting 3.6% of detected proteins with no observable interaction effects. Thus, effects of BPL treatment only moderately interfere with affinity proteomic detectability of differential protein expression between different experimental groups. Overall, the results prove high-throughput affinity proteomics well suited for the analysis of high-risk samples inactivated using BPL.

Reciprocal crosstalk between Th17 and mesothelial cells promotes metastasis-associated adhesion of ovarian cancer cells.

BACKGROUND: IL-17A and TNF synergistically promote inflammation and tumorigenesis. Their interplay and impact on ovarian carcinoma (OC) progression are, however, poorly understood. We addressed this question focusing on mesothelial cells, whose interaction with tumor cells is known to play a pivotal role in transcoelomic metastasis formation. METHODS: Flow-cytometry and immunohistochemistry experiments were employed to identify cellular sources of IL-17A and TNF. Changes in transcriptomes and secretomes were determined by bulk and single cell RNA sequencing as well as affinity proteomics. Functional consequences were investigated by microscopic analyses and tumor cell adhesion assays. Potential clinical implications were assessed by immunohistochemistry and survival analyses. RESULTS: We identified Th17 cells as the main population of IL-17A- and TNF producers in ascites and detected their accumulation in early omental metastases. Both IL-17A and its receptor subunit IL-17RC were associated with short survival of OC patients, pointing to a role in clinical progression. IL-17A and TNF synergistically induced the reprogramming of mesothelial cells towards a pro-inflammatory mesenchymal phenotype, concomitantly with a loss of tight junctions and an impairment of mesothelial monolayer integrity, thereby promoting cancer cell adhesion. IL-17A and TNF synergistically induced the Th17-promoting cytokines IL-6 and IL-1ß as well as the Th17-attracting chemokine CCL20 in mesothelial cells, indicating a reciprocal crosstalk that potentiates the tumor-promoting role of Th17 cells in OC. CONCLUSIONS: Our findings reveal a novel function for Th17 cells in the OC microenvironment, which entails the IL-17A/TNF-mediated induction of mesothelial-mesenchymal transition, disruption of mesothelial layer integrity and consequently promotion of OC cell adhesion. These effects are potentiated by a positive feedback loop between mesothelial and Th17 cells. Together with the observed clinical associations and accumulation of Th17 cells in omental micrometastases, our observations point to a potential role in early metastases formation and thus to new therapeutic options.

TRAIL-dependent apoptosis of peritoneal mesothelial cells by NK cells promotes ovarian cancer invasion.

A crucial requirement for metastasis formation in ovarian high-grade serous carcinoma (HGSC) is the disruption of the protective peritoneal mesothelium. Using co-culture systems of primary human cells, we discovered that tumor-associated NK cells induce TRAIL-dependent apoptosis in mesothelial cells via death receptors DR4 and DR5 upon encounter with activated T cells. Upregulation of TRAIL expression in NK cells concomitant with enhanced cytotoxicity toward mesothelial cells was driven predominantly by T-cell-derived TNFα, as shown by affinity proteomics-based analysis of the T cell secretome in conjunction with functional studies. Consistent with these findings, we detected apoptotic mesothelial cells in the peritoneal fluid of HGSC patients. In contrast to mesothelial cells, HGSC cells express negligible levels of both DR4 and DR5 and are TRAIL resistant, indicating cell-type-selective killing by NK cells. Our data point to a cooperative action of T and NK in breaching the mesothelial barrier in HGSC patients.

The lysophosphatidic acid-regulated signal transduction network in ovarian cancer cells and its role in actomyosin dynamics, cell migration and entosis.

Lysophosphatidic acid (LPA) species accumulate in the ascites of ovarian high-grade serous cancer (HGSC) and are associated with short relapse-free survival. LPA is known to support metastatic spread of cancer cells by activating a multitude of signaling pathways via G-protein-coupled receptors of the LPAR family. Systematic unbiased analyses of the LPA-regulated signal transduction network in ovarian cancer cells have, however, not been reported to date. Methods: LPA-induced signaling pathways were identified by phosphoproteomics of both patient-derived and OVCAR8 cells, RNA sequencing, measurements of intracellular Ca2+ and cAMP as well as cell imaging. The function of LPARs and downstream signaling components in migration and entosis were analyzed by selective pharmacological inhibitors and RNA interference. Results: Phosphoproteomic analyses identified > 1100 LPA-regulated sites in > 800 proteins and revealed interconnected LPAR1, ROCK/RAC, PKC/D and ERK pathways to play a prominent role within a comprehensive signaling network. These pathways regulate essential processes, including transcriptional responses, actomyosin dynamics, cell migration and entosis. A critical component of this signaling network is MYPT1, a stimulatory subunit of protein phosphatase 1 (PP1), which in turn is a negative regulator of myosin light chain 2 (MLC2). LPA induces phosphorylation of MYPT1 through ROCK (T853) and PKC/ERK (S507), which is majorly driven by LPAR1. Inhibition of MYPT1, PKC or ERK impedes both LPA-induced cell migration and entosis, while interference with ROCK activity and MLC2 phosphorylation selectively blocks entosis, suggesting that MYPT1 figures in both ROCK/MLC2-dependent and -independent pathways. We finally show a novel pathway governed by LPAR2 and the RAC-GEF DOCK7 to be indispensable for the induction of entosis. Conclusion: We have identified a comprehensive LPA-induced signal transduction network controlling LPA-triggered cytoskeletal changes, cell migration and entosis in HGSC cells. Due to its pivotal role in this network, MYPT1 may represent a promising target for interfering with specific functions of PP1 essential for HGSC progression.

Basal cell adhesion molecule promotes metastasis-associated processes in ovarian cancer.

BACKGROUND: Basal cell adhesion molecule (BCAM) is a laminin α5 (LAMA5) binding membrane-bound protein with a putative role in cancer. Besides full-length BCAM1, an isoform lacking most of the cytoplasmic domain (BCAM2), and a soluble form (sBCAM) of unknown function are known. In ovarian carcinoma (OC), all BCAM forms are abundant and associated with poor survival, yet BCAM's contribution to peritoneal metastatic spread remains enigmatic. METHODS: Biochemical, omics-based and real-time cell assays were employed to identify the source of sBCAM and metastasis-related functions of different BCAM forms. OC cells, explanted omentum and a mouse model of peritoneal colonisation were used in loss- and gain-of-function experiments. RESULTS: We identified ADAM10 as a major BCAM sheddase produced by OC cells and identified proteolytic cleavage sites proximal to the transmembrane domain. Recombinant soluble BCAM inhibited single-cell adhesion and migration identically to membrane-bound isoforms, confirming its biological activity in OC. Intriguingly, this seemingly anti-tumorigenic potential of BCAM contrasts with a novel pro-metastatic function discovered in the present study. Thus, all queried BCAM forms decreased the compactness of tumour cell spheroids by inhibiting LAMA5 - integrin ß1 interactions, promoted spheroid dispersion in a three-dimensional collagen matrix, induced clearance of mesothelial cells at spheroid attachment sites in vitro and enhanced invasion of spheroids into omental tissue both ex vivo and in vivo. CONCLUSIONS: Membrane-bound BCAM as well as sBCAM shed by ADAM10 act as decoys rather than signalling receptors to modulate metastasis-related functions. While BCAM appears to have tumour-suppressive effects on single cells, it promotes the dispersion of OC cell spheroids by regulating LAMA5-integrin-ß1-dependent compaction and thereby facilitating invasion of metastatic target sites. As peritoneal dissemination is majorly mediated by spheroids, these findings offer an explanation for the association of BCAM with a poor clinical outcome of OC, suggesting novel therapeutic options.

Prostacyclin Released by Cancer-Associated Fibroblasts Promotes Immunosuppressive and Pro-Metastatic Macrophage Polarization in the Ovarian Cancer Microenvironment.

Metastasis of high-grade ovarian carcinoma (HGSC) is orchestrated by soluble mediators of the tumor microenvironment. Here, we have used transcriptomic profiling to identify lipid-mediated signaling pathways encompassing 41 ligand-synthesizing enzymes and 23 cognate receptors in tumor, immune and stroma cells from HGSC metastases and ascites. Due to its strong association with a poor clinical outcome, prostacyclin (PGI2) synthase (PTGIS) is of particular interest in this signaling network. PTGIS is highly expressed by cancer-associated fibroblasts (CAF), concomitant with elevated PGI2 synthesis, whereas tumor-associated macrophages (TAM) exhibit the highest expression of its surface receptor (PTGIR). PTGIR activation by PGI2 agonists triggered cAMP accumulation and induced a mixed-polarization macrophage phenotype with altered inflammatory gene expression, including CXCL10 and IL12A repression, as well as reduced phagocytic capability. Co-culture experiments provided further evidence for the interaction of CAF with macrophages via PGI2, as the effect of PGI2 agonists on phagocytosis was mitigated by cyclooxygenase inhibitors. Furthermore, conditioned medium from PGI2-agonist-treated TAM promoted tumor adhesion to mesothelial cells and migration in a PTGIR-dependent manner, and PTGIR activation induced the expression of metastasis-associated and pro-angiogenic genes. Taken together, our study identifies a PGI2/PTGIR-driven crosstalk between CAF, TAM and tumor cells, promoting immune suppression and a pro-metastatic environment.

Arachidonic acid, a clinically adverse mediator in the ovarian cancer microenvironment, impairs JAK-STAT signaling in macrophages by perturbing lipid raft structures.

Survival of ovarian carcinoma is associated with the abundance of immunosuppressed CD163high CD206high tumor-associated macrophages (TAMs) and high levels of arachidonic acid (AA) in the tumor microenvironment. Here, we show that both associations are functionally linked. Transcriptional profiling revealed that high CD163 and CD206/MRC1 expression in TAMs is strongly associated with an inhibition of cytokine-triggered signaling, mirrored by an impaired transcriptional response to interferons and IL-6 in monocyte-derived macrophages by AA. This inhibition of pro-inflammatory signaling is caused by dysfunctions of the cognate receptors, indicated by the inhibition of JAK1, JAK2, STAT1, and STAT3 phosphorylation, and by the displacement of the interferon receptor IFNAR1, STAT1 and other immune-regulatory proteins from lipid rafts. AA exposure led to a dramatic accumulation of free AA in lipid rafts, which appears to be mechanistically crucial, as the inhibition of its incorporation into phospholipids did not affect the AA-mediated interference with STAT1 phosphorylation. Inhibition of interferon-triggered STAT1 phosphorylation by AA was reversed by water-soluble cholesterol, known to prevent the perturbation of lipid raft structure by AA. These findings suggest that the pharmacologic restoration of lipid raft functions in TAMs may contribute to the development new therapeutic approaches.

Isolation of native EVs from primary biofluids-Free-flow electrophoresis as a novel approach to purify ascites-derived EVs.

Although extracellular vesicles (EVs) have been extensively characterized, efficient purification methods, especially from primary biofluids, remain challenging. Here we introduce free-flow electrophoresis (FFE) as a novel approach for purifying EVs from primary biofluids, in particular from the peritoneal fluid (ascites) of ovarian cancer patients. FFE represents a versatile, fast, matrix-free approach for separating different analytes with inherent differences in charge density and/or isoelectric point (pI). Using a series of buffered media with different pH values allowed us to collect 96 fractions of ascites samples. To characterize the composition of the individual fractions, we used state-of-the-art methods such as nanoflow and imaging flow cytometry (nFCM and iFCM) in addition to classical approaches. Of note, tetraspanin-positive events measured using nFCM were enriched in a small number of distinct fractions. This observation was corroborated by Western blot analysis and electron microscopy, demonstrating only minor contamination with soluble proteins and lipid particles. In addition, these gently purified EVs remain functional. Thus, FFE represents a new, efficient and fast method for separating native and highly purified EVs from complicated primary samples.

The multicellular signalling network of ovarian cancer metastases.

BACKGROUND: Transcoelomic spread is the major route of metastasis of ovarian high-grade serous carcinoma (HGSC) with the omentum as the major metastatic site. Its unique tumour microenvironment with its large populations of adipocytes, mesothelial cells and immune cells establishes an intercellular signaling network that is instrumental for metastatic growth yet poorly understood. METHODS: Based on transcriptomic analysis of tumour cells, tumour-associated immune and stroma cells we defined intercellular signaling pathways for 284 cytokines and growth factors and their cognate receptors after bioinformatic adjustment for contaminating cell types. The significance of individual components of this network was validated by analysing clinical correlations and potentially pro-metastatic functions, including tumour cell migration, pro-inflammatory signal transduction and TAM expansion. RESULTS: The data show an unexpected prominent role of host cells, and in particular of omental adipocytes, mesothelial cells and fibroblasts (CAF), in sustaining this signaling network. These cells, rather than tumour cells, are the major source of most cytokines and growth factors in the omental microenvironment (n = 176 vs. n = 13). Many of these factors target tumour cells, are linked to metastasis and are associated with a short survival. Likewise, tumour stroma cells play a major role in extracellular-matrix-triggered signaling. We have verified the functional significance of our observations for three exemplary instances. We show that the omental microenvironment (i) stimulates tumour cell migration and adhesion via WNT4 which is highly expressed by CAF; (ii) induces pro-tumourigenic TAM proliferation in conjunction with high CSF1 expression by omental stroma cells and (iii) triggers pro-inflammatory signaling, at least in part via a HSP70-NF-κB pathway. CONCLUSIONS: The intercellular signaling network of omental metastases is majorly dependent on factors secreted by immune and stroma cells to provide an environment that supports ovarian HGSC progression. Clinically relevant pathways within this network represent novel options for therapeutic intervention.

Phosphoproteomics identify arachidonic-acid-regulated signal transduction pathways modulating macrophage functions with implications for ovarian cancer.

Arachidonic acid (AA) is a polyunsaturated fatty acid present at high concentrations in the ovarian cancer (OC) microenvironment and associated with a poor clinical outcome. In the present study, we have unraveled a potential link between AA and macrophage functions. Methods: AA-triggered signal transduction was studied in primary monocyte-derived macrophages (MDMs) by phosphoproteomics, transcriptional profiling, measurement of intracellular Ca2+ accumulation and reactive oxygen species production in conjunction with bioinformatic analyses. Functional effects were investigated by actin filament staining, quantification of macropinocytosis and analysis of extracellular vesicle release. Results: We identified the ASK1 - p38δ/α (MAPK13/14) axis as a central constituent of signal transduction pathways triggered by non-metabolized AA. This pathway was induced by the Ca2+-triggered activation of calmodulin kinase II, and to a minor extent by ROS generation in a subset of donors. Activated p38 in turn was linked to a transcriptional stress response associated with a poor relapse-free survival. Consistent with the phosphorylation of the p38 substrate HSP27 and the (de)phosphorylation of multiple regulators of Rho family GTPases, AA impaired actin filament organization and inhibited actin-driven macropinocytosis. AA also affected the phosphorylation of proteins regulating vesicle biogenesis, and consistently, AA enhanced the release of tetraspanin-containing exosome-like vesicles. Finally, we identified phospholipase A2 group 2A (PLA2G2A) as the clinically most relevant enzyme producing extracellular AA, providing further potentially theranostic options. Conclusion: Our results suggest that AA contributes to an unfavorable clinical outcome of OC by impacting the phenotype of tumor-associated macrophages. Besides critical AA-regulated signal transduction proteins identified in the present study, PLA2G2A might represent a potential prognostic tool and therapeutic target to interfere with OC progression.

Upregulation of mesothelial genes in ovarian carcinoma cells is associated with an unfavorable clinical outcome and the promotion of cancer cell adhesion.

A hallmark of ovarian high-grade serous carcinoma (HGSC) is its early and massive peritoneal dissemination via the peritoneal fluid. It is generally believed that tumor cells must breach the mesothelium of peritoneal organs to adhere to the underlying extracellular matrix (ECM) and initiate metastatic growth. However, the molecular mechanisms underlying these processes are only partially understood. Here, we have analyzed 52 matched samples of spheroids and solid tumor masses (suspected primary lesions and metastases) from 10 patients by targeted sequencing of 21 loci previously proposed as targets of HGSC driver mutations. This analysis revealed very similar patterns of genetic alterations in all samples. One exception was FAT3 with a strong enrichment of mutations in metastases compared with presumed primary lesions in two cases. FAT3 is a putative tumor suppressor gene that codes for an atypical cadherin, pointing a potential role in peritoneal dissemination in a subgroup of HGSC patients. By contrast, transcriptome data revealed clear and consistent differences between tumor cell spheroids from ascites and metastatic lesions, which were mirrored by the in vitro adherence of ascites-derived spheroids. The adhesion-induced transcriptional alterations in metastases and adherent cells resembled epithelial-mesenchymal transition, but surprisingly also included the upregulation of a specific subset of mesothelial genes, such as calretinin (CALB2) and podoplanin (PDPN). Consistent with this finding, calretinin staining was also observed in subsets of tumor cells in HGSC metastases, particularly at the invasive tumor edges. Intriguingly, a high expression of either CALB2 or PDPN was strongly associated with a poor clinical outcome. siRNA-mediated CALB2 silencing triggered the detachment of adherent HGSC cells in vitro and inhibited the adhesion of detached HGSC cells to collagen type I. Our data suggest that the acquisition of a mesenchymal-mesothelial phenotype contributes to cancer cell adhesion to the ECM of peritoneal organs and HGSC progression.

Tumor-associated macrophages promote ovarian cancer cell migration by secreting transforming growth factor beta induced (TGFBI) and tenascin C.

A central and unique aspect of high-grade serous ovarian carcinoma (HGSC) is the extensive transcoelomic spreading of tumor cell via the peritoneal fluid or malignant ascites. We and others identified tumor-associated macrophages (TAM) in the ascites as promoters of metastasis-associated processes like extracellular matrix (ECM) remodeling, tumor cell migration, adhesion, and invasion. The precise mechanisms and mediators involved in these functions of TAM are, however, largely unknown. We observed that HGSC migration is promoted by soluble mediators from ascites-derived TAM, which can be emulated by conditioned medium from monocyte-derived macrophages (MDM) differentiated in ascites to TAM-like asc-MDM. A similar effect was observed with IL-10-induced alternatively activated m2c-MDM but not with LPS/IFNγ-induced inflammatory m1-MDM. These observations provided the basis for deconvolution of the complex TAM secretome by performing comparative secretome analysis of matched triplets of different MDM phenotypes with different pro-migratory properties (asc-MDM, m2c-MDM, m1-MDM). Mass spectrometric analysis identified an overlapping set of nine proteins secreted by both asc-MDM and m2c-MDM, but not by m1-MDM. Of these, three proteins, i.e., transforming growth factor beta-induced (TGFBI) protein, tenascin C (TNC), and fibronectin (FN1), have been associated with migration-related functions. Intriguingly, increased ascites concentrations of TGFBI, TNC, and fibronectin were associated with short progression-free survival. Furthermore, transcriptome and secretome analyses point to TAM as major producers of these proteins, further supporting an essential role for TAM in promoting HGSC progression. Consistent with this hypothesis, we were able to demonstrate that the migration-inducing potential of asc-MDM and m2c-MDM secretomes is inhibited, at least partially, by neutralizing antibodies against TGFBI and TNC or siRNA-mediated silencing of TGFBI expression. In conclusion, the present study provides the first experimental evidence that TAM-derived TGFBI and TNC in ascites promote HGSC progression.

Multi-platform Affinity Proteomics Identify Proteins Linked to Metastasis and Immune Suppression in Ovarian Cancer Plasma.

A central reason behind the poor clinical outcome of patients with high-grade serous carcinoma (HGSC) of the ovary is the difficulty in reliably detecting early occurrence or recurrence of this malignancy. Biomarkers that provide reliable diagnosis of this disease are therefore urgently needed. Systematic proteomic methods that identify HGSC-associated molecules may provide such biomarkers. We applied the antibody-based proximity extension assay (PEA) platform (Olink) for the identification of proteins that are upregulated in the plasma of OC patients. Using binders targeting 368 different plasma proteins, we compared 20 plasma samples from HGSC patients (OC-plasma) with 20 plasma samples from individuals with non-malignant gynecologic disorders (N-plasma). We identified 176 proteins with significantly higher levels in OC-plasma compared to N-plasma by PEA (p < 0.05 by U-test; Benjamini-Hochberg corrected), which are mainly implicated in immune regulation and metastasis-associated processes, such as matrix remodeling, adhesion, migration and proliferation. A number of these proteins have not been reported in previous studies, such as BCAM, CDH6, DDR1, N2DL-2 (ULBP2), SPINT2, and WISP-1 (CCN4). Of these SPINT2, a protease inhibitor mainly derived from tumor cells within the HGSC microenvironment, showed the highest significance (p < 2 × 10-7) similar to the previously described IL-6 and PVRL4 (NECTIN4) proteins. Results were validated by means of the aptamer-based 1.3 k SOMAscan proteomic platform, which revealed a high inter-platform correlation with a median Spearman ρ of 0.62. Likewise, ELISA confirmed the PEA data for 10 out of 12 proteins analyzed, including SPINT2. These findings suggest that in contrast to other entities SPINT2 does not act as a tumor suppressor in HGSC. This is supported by data from the PRECOG and KM-Plotter meta-analysis databases, which point to a tumor-type-specific inverse association of SPINT2 gene expression with survival. Our data also demonstrate that both the PEA and SOMAscan affinity proteomics platforms bear considerable potential for the unbiased discovery of novel disease-associated biomarkers.

Dual-platform affinity proteomics identifies links between the recurrence of ovarian carcinoma and proteins released into the tumor microenvironment.

The peritoneal fluid (ascites), replete with abundant tumor-promoting factors and extracellular vesicles (EVs) reflecting the tumor secretome, plays an essential role in ovarian high-grade serous carcinoma (HGSC) metastasis and immune suppression. A comprehensive picture of mediators impacting HGSC progression is, however, not available. Methods: Proteins in ascites from HGSC patients were quantified by the aptamer-based SOMAscan affinity proteomic platform. SOMAscan data were analyzed by bioinformatic methods to reveal clinically relevant links and functional connections, and were validated using the antibody-based proximity extension assay (PEA) Olink platform. Mass spectrometry was used to identify proteins in extracellular microvesicles released by HGSC cells. Results: Consistent with the clinical features of HGSC, 779 proteins in ascites identified by SOMAscan clustered into groups associated either with metastasis and a short relapse-free survival (RFS), or with immune regulation and a favorable RFS. In total, 346 proteins were linked to OC recurrence in either direction. Reanalysis of 214 of these proteins by PEA revealed an excellent median Spearman inter-platform correlation of ρ=0.82 for the 46 positively RFS-associated proteins in both datasets. Intriguingly, many proteins strongly associated with clinical outcome were constituents of extracellular vesicles. These include proteins either linked to a poor RFS, such as HSPA1A, BCAM and DKK1, or associated with a favorable outcome, such as the protein kinase LCK. Finally, based on these data we defined two protein signatures that clearly classify short-term and long-term relapse-free survivors. Conclusion: The ascites secretome points to metastasis-promoting events and an anti-tumor response as the major determinants of the clinical outcome of HGSC. Relevant proteins include both bone fide secreted and vesicle-encapsulated polypeptides, many of which have previously not been linked to HGSC recurrence. Besides a deeper understanding of the HGSC microenvironment our data provide novel potential tools for HGSC patient stratification. Furthermore, the first large-scale inter-platform validation of SOMAscan and PEA will be invaluable for other studies using these affinity proteomics platforms.

Cell type-selective pathways and clinical associations of lysophosphatidic acid biosynthesis and signaling in the ovarian cancer microenvironment.

The peritoneal fluid of ovarian carcinoma patients promotes cancer cell invasion and metastatic spread with lysophosphatidic acid (LPA) as a potentially crucial mediator. However, the origin of LPA in ascites and the clinical relevance of individual LPA species have not been addressed. Here, we show that the levels of multiple acyl-LPA species are strongly elevated in ascites versus plasma and are associated with short relapse-free survival. Data derived from transcriptome and secretome analyses of primary ascite-derived cells indicate that (a) the major route of LPA synthesis is the consecutive action of a secretory phospholipase A2 (PLA2 ) and autotaxin, (b) that the components of this pathway are coordinately upregulated in ascites, and (c) that CD163+CD206+ tumor-associated macrophages play an essential role as main producers of PLA2 G7 and autotaxin. The latter conclusion is consistent with mass spectrometry-based metabolomic analyses of conditioned medium from ascites cells, which showed that tumor-associated macrophages, but not tumor cells, are able to produce 20:4 acyl-LPA in lipid-free medium. Furthermore, our transcriptomic data revealed that LPA receptor (LPAR) genes are expressed in a clearly cell type-selective manner: While tumor cells express predominantly LPAR1-3, macrophages and T cells also express LPAR5 and LPAR6 at high levels, pointing to cell type-selective LPA signaling pathways. RNA profiling identified cytokines linked to cell motility and migration as the most conspicuous class of LPA-induced genes in macrophages, suggesting that LPA exerts protumorigenic properties at least in part via the tumor secretome.

The Combination of MiRNA-196b, LCN2, and TIMP1 is a Potential Set of Circulating Biomarkers for Screening Individuals at Risk for Familial Pancreatic Cancer.

Individuals at risk (IAR) of familial pancreatic cancer (FPC) are good candidates for screening. Unfortunately, neither reliable imaging modalities nor biomarkers are available to detect high-grade precursor lesions or early cancer. Circulating levels of candidate biomarkers LCN2, TIMP1, Glypican-1, RNU2-1f, and miRNA-196b were analyzed in 218 individuals with sporadic pancreatic ductal adenocarcinoma (PDAC, n = 50), FPC (n = 20), chronic pancreatitis (n = 10), IAR with relevant precursor lesions (n = 11) or non-relevant lesions (n = 5), 20 controls, and IAR with (n = 51) or without (n = 51) lesions on pancreatic imaging. In addition, corresponding duodenal juice samples were analyzed for Glypican-1 (n = 144) enrichment and KRAS mutations (n = 123). The panel miR-196b/LCN2/TIMP1 could distinguish high-grade lesions and stage I PDAC from controls with absolute specificity and sensitivity. In contrast, Glypican-1 enrichment in serum exosomes and duodenal juice was not diagnostic. KRAS mutations in duodenal juice were detected in 9 of 12 patients with PDAC and only 4 of 9 IAR with relevant precursor lesions. IAR with lesions on imaging had elevated miR-196b/LCN2/TIMP1 levels (p = 0.0007) and KRAS mutations in duodenal juice (p = 0.0004) significantly more often than IAR without imaging lesions. The combination miR-196b/LCN2/TIMP1 might be a promising biomarker set for the detection of high-grade PDAC precursor lesions in IAR of FPC families.

Chromatin Binding of c-REL and p65 Is Not Limiting for Macrophage IL12B Transcription During Immediate Suppression by Ovarian Carcinoma Ascites.

Tumors frequently exploit homeostatic mechanisms that suppress expression of IL-12, a central mediator of inflammatory and anti-tumor responses. The p40 subunit of the IL-12 heterodimer, encoded by IL12B, is limiting for these functions. Ovarian carcinoma patients frequently produce ascites which exerts immunosuppression by means of soluble factors. The NFκB pathway is necessary for transcription of IL12B, which is not expressed in macrophages freshly isolated from ascites. This raises the possibility that ascites prevents IL12B expression by perturbing NFκB binding to chromatin. Here, we show that ascites-mediated suppression of IL12B induction by LPS plus IFNγ in primary human macrophages is rapid, and that suppression can be reversible after ascites withdrawal. Nuclear translocation of the NFκB transcription factors c-REL and p65 was strongly reduced by ascites. Surprisingly, however, their binding to the IL12B locus and to CXCL10, a second NFκB target gene, was unaltered, and the induction of CXCL10 transcription was not suppressed by ascites. These findings indicate that, despite its reduced nuclear translocation, NFκB function is not generally impaired by ascites, suggesting that ascites-borne signals target additional pathways to suppress IL12B induction. Consistent with these data, IL-10, a clinically relevant constituent of ascites and negative regulator of NFκB translocation, only partially recapitulated IL12B suppression by ascites. Finally, restoration of a defective IL-12 response by appropriate culture conditions was observed only in macrophages from a subset of donors, which may have important implications for the understanding of patient-specific immune responses.

Prognosis of ovarian cancer is associated with effector memory CD8+ T cell accumulation in ascites, CXCL9 levels and activation-triggered signal transduction in T cells.

The accumulation of intratumoral CD8+ T cells is associated with the survival of high grade serous ovarian carcinoma patients, but it is unclear which CD8+ T cell subsets contribute to this effect and how they are affected by the peritoneal tumor microenvironment. Here, we provide evidence for a functional link between long relapse-free survival, accumulation of CD8+ effector memory T (TEM) cells in peritoneal effusion (ascites), and the level of the CD8+ TEM attracting chemokine CXCL9, produced by macrophages as a major source. We also propose a novel mechanism by which the tumor microenvironment could contribute to T cell dysfunction and shorter survival, i.e., diminished expression levels of essential signaling proteins, including STAT5B, PLCγ1 and NFATc2. CD8+ TEM cells in ascites, CXCL9 levels and the expression of crucial signal transduction proteins may therefore be important biomarkers to gauge the efficiency of immune therapies and potentially represent therapeutic targets.

Proteotranscriptomics Reveal Signaling Networks in the Ovarian Cancer Microenvironment.

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

Soluble NKG2D ligands in the ovarian cancer microenvironment are associated with an adverse clinical outcome and decreased memory effector T cells independent of NKG2D downregulation.

The immune receptor NKG2D is predominantly expressed on NK cells and T cell subsets and confers anti-tumor activity. According to the current paradigm, immune surveillance is counteracted by soluble ligands shed into the microenvironment, which down-regulate NKG2D receptor expression. Here, we analyzed the clinical significance of the soluble NKG2D ligands sMICA and sULBP2 in the malignancy-associated ascites of ovarian cancer. We show that high levels of sMICA and sULBP2 in ascites were associated with a poor prognosis. Ascites inhibited the activation of normal NK cells, which, in contrast to the prevailing notion, was not associated with decreased NKG2D expression. Of note, an inverse correlation of soluble NKG2D ligands with effector memory T cells and a direct correlation with pro-tumorigenic CD163+CD206+ macrophages was observed. Thus, the role of soluble NKG2D ligands within the ovarian cancer microenvironment is more complex than anticipated and does not exclusively function via NKG2D downregulation.