Fibroblasts In Omentum Activated By Tumor Cells Promote Ovarian Cancer Metastases

PartⅠThe presence and character of CAFs in omentum tissues of patients with ovarian cancerObjectiveTo explore the presence of cancer-associated fibroblasts (CAFs) in the omentum tissues of epithelial ovarian cancer (EOC).Methodsα-smooth muscle actin (a-SMA) levels in omentum were determined by immunohistochemistric staining from 26 patients, including 7 benign ovarian diseases; 9 ovarian cancers without omentum metastasis; and 10 ovarian cancers with omentum metastasis.ResultsNormal omentum tissues of benign ovarian diseasesα-SMA expression was detected only in vascular pericytes but not in the mesothelial cells or stroma fibroblasts. In the advanced-stage ovarian cancers with omentum metastases, the tumor cells invaded the omental adipose tissue forming a solid, invading tumor cell nest, and surrounded with abundant stroma CAFs. Some cases of omentum tissues lacking detectable EOC metastasis also had CAFs.ConclusionOur research indicated that CAFs exist not only in omentum with EOC metastasis but also in omentum of patients without metastasis. PartⅡThe mechanism of fibroblast activation of NFsObjectiveTo investigate the origin of CAFs in the omentum tissuesMethods1. NFs were direct and indirect cocultured with EOC cell lines SKOV3, OVCAR3, CAOV3, ES2 for 4 days, then theα-SMA expression were assayed by PCR, Western blot and immunofluorescence staining, and the proliferation of NFs was evaluated by EdU incorporation assay.2. NFs were treated with medium containing different concentrations of transforming growth factor-β1(TGF-β1) for 2 days, the expression levels ofα-SMA and phosphorylation level of SMAD2 were determined by PCR and Western blotting, and the proliferation of NFs was evaluated by EdU incorporation assay.3. A specific inhibitor of TGF-β1(A83-01) was used to block the biological activity of TGF-β1, and then the level ofα-SMA and phosphorylated SMAD2 were measured in NFs treated with EOC cells or TGF-β1.Results1. The increasedα-SMA expression was observed in NFs followed by cocultured with EOC cell lines (P<0.05). Hyperproliferation of NFs began to be evident after coculturing with SKOV3 cells for 96 hours (P<0.05).2. The expression level ofα-SMA and phosphorylation level of SMAD2 in NFs were dose-dependently increased by TGF-β1 treatment (P<0.001). The activated fibroblasts induced by TGF-β1 also had high proliferation ability than NFs (P=0.002).3. Pretreating the NFs with A83-O1 (5μmol/L) attenuated theα-SMA expression induced by EOC cells (P=0.009 and P=0.012) and TGF-β1 (P=0.003 and P=0.005). TGF-β1 inhibition also reduced the pro-proliferation effect of TGF-β1 (P=0.003) and SKOV3 cells (P0.001) on NFsConclusionOmentum fibroblasts can be activated by EOC cells even if a direct cancer cell-fibroblasts contact is not present, and TGF-β1 is a main factor to mediate the effect.PartⅢThe role of activated fibroblasts in ovarian cancer cell adhesion and invasion to the omentumObjectiveTo. determine whether the activity level of fibroblasts play key roles in promoting the EOC cells invasion and adhesion, and identify factors expressed in the fibroblasts that promoted metastases.Methods1. NFs cultured on different exposure were seeded in omentum 3D culture matrices and adhesion and invasion assays were used to quantify their ability to promote adhesion and invasion of SKOV3 cells.3D culture matrices without fibroblasts were used as control.2. Add the A83-01 to the pretreating step of NFs or to the 3D culture matrices, and then quantify the adhesion and invasion ability of SKOV3 cells.3. NFs were cocultured with SKOV3 cells or treated with TGF-β1 with or without A83-01, and then the hepatocyte growth factor (HGF) expression were assayed by Western blot.4. Add HGF inhibitory antibody to the 3D coculture system, and then quantify the adhesion and invasion ability of SKOV3 cells.5. The matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP9) expression in NFs in different culture model including cultured alone, indirectly or direct cocultured with SKOV3 cells and treated with TGF-β1.6. Add MMP-2 inhibitor to the 3D coculture system, and then quantify the adhesion and invasion ability of SKOV3 cells.Results 1. When the 3D culture containing CAFs, NFs cocultured with SKOV3 cells and NFs exposed to TGF-β1, the adhesive SKOV3 cells increased 4.77,3.08 and 3.14-fold respectively than the control (P=0.007, P=0.022 and P=0.018), and the invaded SKOV3 cells increased 6.81,3.80, and 6.06-fold respectively (P=0.0001, P=0.03 and P=0.0005).2. Addition of the A83-01 to the pretreating step attenuated the adhesive promotion ability of NFs stimulated by SKOV3 cells or exogenous TGF-β1 (P<0.05), but had no effect on SKOV3 cell adhesion if added only to the 3D culture system.3. The NFs cocultured with SKOV3 cells indirectly expressed significantly higher levels of HGF compared with NFs cultured alone (3.6-fold, P=0.008). Exogenous TGF-β1 also increased the HGF expression in NFs (4.3-fold, P=0.002). Both of these increases were attenuated by adding A83-01 to the co-culture system that inhibited the activation of NFs (P=0.005).4. Inactivation of HGF significantly reduced the adhesion and invasion of SKOV3 cells promoted by NFs cocultured by SKOV3 and treated by TGF-β1 (P<0.01).5. When cultured alone, NFs and SKOV3 cells both expressed small amount of MMP-2. When NFs indirectly cocultured with SKOV3 cells, neither NFs nor SKOV3 cells expressed increased MMP-2. Exogenous TGF-β1 could not induce the MMP-2 expression in NFs either. Whereas in direct coculture system, increased expression of MMP-2 was found in the mixture of SKOV3 cells and NFs after 3 days (7.76-fold) (P<0.001).6. The addition of the MMP-2 inhibitor to the 3D culture decreased the adhesion and invasive potential of SKOV3 cells promoted by activated fibroblasts in a dose-dependent fashion (P<0.05).ConclusionThe activation level of fibroblasts used in the organotypic culture is critical in fostering the extent of ovarian tumor adhesion and invasion. EOC cells regulates HGF expression in fibroblasts through TGF-β1, and metastases promoting effect of activated NFs was at least partly mediated by HGF. Although MMP-2 expression and activation were not mediated by TGF-β1, MMP-2 was important for mediating the metastases of EOC cells to the 3D culture.PartⅣThe role of NFs activation in EOC cells peritoneal metastasis in vivoObjectiveTo determine the role of NFs activation in EOC cells peritoneal metastasis in vivo.Methods1. SKOV3 cells were injected i.p into BALB/c nude mice either alone, admixed with NFs or inoculation with A83-01 respectively.2. Mice were sacrificed 8 weeks after tumor inoculation, or if showing signs of distress. After sacrifice, the mice were weighed, the number and location of macroscopic lesions were recorded, and the tumor colonies were dissected, collected, and weighed.3. The tumor tissues were used for hematoxylin-eosin staining and histopathological study forα-SMA and MMP2. Image Proplus 5.1 software was used to analyze the positive area percentage of a-SMA and staining intensity of MMP2 in every region, and then the average value was the amount of every field.Result1. SKOV3 cells coinjected with NFs produced many tumor nodules in the peritoneal cavity in the mice. The tumor weight was 4.12-fold higher than mice injected with SKOV3 cells alone (P<0.001), while A83-01 can abolish this increase (P=0.0002). However, A83-01 had no influence on the weight of peritoneal metastasis in mice injected with SKOV3 cells alone.2. Microscopically, tumors injected with NFs were significantly more fibrosis, and this was reduced significantly with A83-01 administration, as assessed by both H&E stain and by immunostaining for the activated fibroblasts markerα-SMA (P<0.001). However, the activated fibroblasts also present in SKOV3 tumor. And this can be inhibited by A83-01 (P=0.015).3. Stable upregulation of MMP2 was also found in xenografts with NFs (P<0.001), which can be attenuated by A83-01 administration (P<0.001).ConclusionThe adhesion and invasion promotion ability of activated fibroblasts was completely inhibited by A83-01 in vitro and in vivo that fibroblasts activation was abrogated.