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

ABSTRACT: 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. By comparing monocyte-derived macrophages (MDM) differentiated in ascites to TAM-like asc-MDM, by LPS/IFN to inflammatory m1-MDM and by IL-10 to alternatively activated m2c-MDM, we found that conditioned media from both, asc-MDM and m2c-MDM, stimulated migration of patient-derived tumor cells, while m1-MDM failed to do so. Secretome analysis by mass spectrometry identified an overlapping set of 9 proteins secreted by both asc-MDM and m2c-MDM, but not by m1-MDM, in all tested donors. Of these, three proteins, namely transforming growth factor beta induced protein (TGFBI), 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 may be 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Ascites, Macrophage

DISEASE(S): Malignant Neoplasm Of Ovary

SUBMITTER: Johannes Graumann

LAB HEAD: Rolf Müller

PROVIDER: PXD016555 | Pride | 2020-04-28

REPOSITORIES: Pride

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

Steitz Anna Mary AM Steffes Alina A Finkernagel Florian F Unger Annika A Sommerfeld Leah L Jansen Julia M JM Wagner Uwe U Graumann Johannes J Müller Rolf R Reinartz Silke S

Cell death & disease 20200420 4

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 obser ...[more]

PMID: 32312959

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Project description: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 tumor 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 tumor cells, tumor-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 analyzing clinical correlations and potentially pro-metastatic functions, including tumor 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 tumor cells, are the major source of most cytokines and growth factors in the omental microenvironment (n=176 versus n=13). Many of these factors target tumor cells, are linked to metastasis and are associated with a short survival. Likewise, tumor stroma cells play a major role in both extracellular-matrix-triggered and lipid-mediated signaling. We have verified the functional significance of our observations for three exemplary instances. We show that the omental microenvironment (i) stimulates tumor cell migration via WNT4 which is highly expressed by CAF; (ii) induces pro-tumorigenic 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 – NFkB 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.

Project description:To compare tumor associated macrophage (TAM) from naïve and sepsis surviving mice we have employed Agilent microarrays slides with almost 60,000 genes (39,430 mRNA and 16,251 long non coding RNAs). Other experiments we conducted demonstrated TAM accumulation was increased in post-sepsis subjects. For this reason, we asked if TAM from post-sepsis mice could also exhibit a different gene expression profile. Sepsis was induced by cecal and ligation puncture. Naïve mice were used as control group. All animals were treated with ertapenem (20 mg/kg, i.p., 6 hours after surgery, and then each 12 hours for 3 days). B16-F10 melanoma (30,000 cells) were injected subcutaneously at day 14 after sepsis induction. Fourteen days after tumor inoculation, animals were killed and tumors were harvested and digested (collagenase and DNAse). TAM was isolated by a Percoll gradient (70/30) followed by a 1-hour adhesion protocol, reaching a purity of ~75%. For comparative reasons, we assessed TAM from post-sepsis (n = 4), TAM from naïve mice (n = 4), bone marrow derived macrophage from naïve (n = 4) and from post-sepsis (n = 4), M1-polarized macrophage (n = 4) and M2-polarized macrophage (n = 4). We found only minor gene expression differences between TAM from naïve and from post-sepsis mice (61 genes were up-regulated and 98 genes were down-regulated, fold-change > 0.58 or < -0.58, and p < 0.01). We found genes related to leukocyte activation were down-regulated in TAM from post-sepsis mice (e.g. Ccr7, Cd86, H2-Ab1), as well as genes related to antigen processing and presentation of peptide or polysaccharide antigen via MHC class II (H2-DMb1, Cd74, H2-Eb1, H2-Ob). A gene related to M2 polarization was up-regulated (Marco). Also, we found a down-regulation of Nfkbid in post-sepsis-derived TAM. This led us to hypothesize TAM from post-sepsis mice exhibit a more M2-like phenotype, which may in part contribute to post-sepsis tumor expansion. Three independent experiments were conducted for TAM obtaining, each experiment using n = 4 for naïve and n = 4 for post-sepsis. We selected the 4 best within a group of 12 samples, following A260/280 and A260/230 ratios. For bone marrow derived macrophage from naïve and from post-sepsis, and for M1 and M2-polarized macrophage, we conducted two independent experiments using n = 3 per group. The best 4 samples in each group was selected to microarray processing and analysis.

Project description:Tumor-associated macrophages (TAMs) are key components of the tumor microenvironment (TME) which can either promote tumor progression (M2) or induce antitumor immunity (M1). The hypothesis of our study is that the expression of FOLR2 is associated with an M2-like macrophage profile. The main goal of this study is to compare the transcriptomic profile of FOLR2 positive and FOLR2 negative TAMs obtained from the ascites of C57BL/6 mice bearing ovarian ID8 intraperitoneal tumors. Abstract: The immunosuppressive tumor microenvironment (TME) represents a major barrier for effective immunotherapy. Tumor-associated macrophages (TAMs) are highly heterogeneous and plastic cell components of the TME which can either promote tumor progression (M2-like) or boost antitumor immunity (M1-like). Selective targeting of the pro-tumorigenic subset of TAMs represents an attractive therapeutic strategy. Here, we demonstrate that a subset of TAMs that expressing folate receptor β (FRβ) possess an immunosuppressive, tumor-promoting M2-like profile, while FRβ negative TAMs feature pro-inflammatory M1 macrophage properties. In syngeneic tumor mouse models, the administration of FRβ-targeted chimeric antigen receptor (CAR) T-cells mediated elimination of FRβ+ TAMs in the TME, which resulted in an enrichment of pro-inflammatory monocytes, an influx of endogenous tumor-specific CD8+ T-cells, delayed tumor progression, and prolonged survival. Preconditioning of the TME with FRβ-specific CAR T-cells also improved the effectiveness of tumor-directed anti-mesothelin CAR T-cells, while simultaneous co-administration of both CAR products did not. Thus, CAR T-cell-mediated depletion of immunosuppressive M2-like TAMs incites a pro-inflammatory TME that broadens endogenous antitumor immunity and limits tumor progression, highlighting the pro-tumor role of FRβ+ TAMs in the TME and the therapeutic implications of TAM-depleting agents as preparative adjuncts to conventional immunotherapies that directly target tumor antigens.

Dataset Information

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

Publications

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

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