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.

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.

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.

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.