Mechanism Research And Clinical Application Of The Effects Of Platelets On The Metastasis Of Ovarian Cancer

BackgroundOvarian cancer is one of malignant tumors in female genital organs.Regardless of its relatively low incidence,ovarian cancer is the leading cause of death among all gynecological malignancies.The annual mortality rate of ovarian cancer continues to rise,especially in developing countries.The high death rate mainly attributes to its metastasis,such as intraperitoneal dissemination.Therefore,clinical management of ovarian cancer patients is challenging.Platelets may be involved in the progression of metastasis in ovarian cancer.Clinical data show that ovarian cancer patients with thrombocytosis are more likely to progress to advanced stages,with shorter survival time and higher incidence of metastasis.It was also demonstrated that platelets could promote invasive ability of ovarian cancer cells.However,the underlying mechanism remains unknown.Epithelial to mesenchymal transition(EMT)is mainly responsible for the changes of invasive ability in malignant cancer cells.The EMT process can be initiated by many extracellular factors,such as transforming growth factor-beta(TGF-β).Studies have shown that TGF-β could trigger EMT in carcinoma cells and increase their metastatic seeding in distant organs via Smad-dependent pathway.As platelets harbor high levels of TGF-β,we proposed that platelet might induce EMT in human ovarian cancer cells by activating TGF-β signaling pathway to increase their invasion.Thus,in the current study,we investigated whether platelets could promote invasion in ovarian cancer cells by inducing EMT,and whether TGF-β signaling pathway was activated.TGF-β type Ⅰ receptor(TβR I)inhibitor was used to test whether platelet-induced invasion and EMT could be reversed by blocking the pathway.Moreover,we also assessed the clinical performance of platelet counts and other coagulation parameters in the auxiliary diagnosis of tumors,such as ovarian tumor and colorectal cancer.Materials and MethodsThe study included three parts.In the first part,blood samples were collected in 85 patients with ovarian tumor,which includes 16 patients with benign ovarian tumor and 69 ovarian cancer patients.Clinico-pathological data of the patients were collected from their electronic medical files.SK-OV-3 and OVCAR-3 ovarian cancer cells were treated with isolated platelets.Transwell assays were used to analyze the change of invasive capacity.Total RNA was extracted from controls or platelet-treated SK-OV-3 cells,and transcriptome in each group was analyzed by microarray.EMT caused by platelets was confirmed by measuring the levels of EMT-related markers using quantitative real-time PCR and Western blot.In the second part,TGF-P levels in serum and cell culture media were measured by commercial ELISA kit.The activation of Smad2,a TGF-β signaling downstream effector was examined using Western blotting.TβR I inhibitor A83-01 was used in the vitro and vivo study to confirm the role of TGF-β/Smad pathway in platelet-induced EMT and invasion of ovarian cancer cells.In the third part,we included 156 ovarian tumor patients and tested their PLT,WBC,Hb,PT,APTT,TT,D-dimer,FDP,FIB and CA125 to compare the performance of PLT and other coagulation parameters in distinguishing benign from malignant or distinguishing early stage from advanced stage in ovarian cancer.178 colorectal cancer patients were also enrolled and their PT,APTT,TT,D-dimer,FDP,FIB,CEA and LDH were obtain to evaluate the performance of these coagulation parameters in distinguishing early stage from advanced stage or detecting distant organ metastasis in colorectal cancer.ResultsIn the first part,we found that ovarian cancer patients with platelet counts of more than 350x109/1 had a higher incidence of metastasis to the omentumand mesentery.Co-culture with platelets markedly increased the invasive properties of SK-OV-3 and OVCAR-3 cells.Microarray,real-time PCR and Western blot demonstrated that invasion related molecules were up-regulated and adhesion related molecule were down-regulated in the platelet-treated cells.In the second part,significantly higher TGF-β levels were seen in patients with increased platelet counts compared to those with normal platelet counts.Active TGF-P levels in the ovarian cancer cells and platelets co-culture conditioned medium was markedly increased compared to the medium from tumor cell cultures alone.SK-OV-3 and OVCAR-3 cells treated with platelets also showed increased phosphorylation of the TGF-P signaling effector Smad2 compared to the untreated cells.Exposure of the platelets-treated SK-OV-3 and OVCAR-3 cells to A83-β1 resulted in their invasive abilities compared to those incubated with platelets alone.Upon addition of the TβR I inhibitor A83-01 to the platelets-treated cells,the EMT-like alterations were inhibited at the mRNA and protein level.Concordantly,A83-01 treatment also restored EMT-like alterations and inhibited platelet-induced invasion in vivo.In the third part,we found that PLT,D-dimer and FDP in ovarian cancer patients were significantly higher than those in patients with benign tumor.And for their identification,either AUC of D-dimer or AUC of FDP is larger than that of PLT.Moreover,PLT,D-dimer and FDP in ovarian cancer patients with advanced stage were higher than those in patients with early stage.For their identification,either AUC of D-dimer or AUC of FDP is larger than that of PLT.In the colorectal cancer patients,the level of PT,APTT,TT,D-dimer,FDP,FIB,CEA and LDH were not significantly different.However,D-dimer levels were significantly increased when colorectal patients developed distant organ metastasis.ConclusionHigher platelet counts were significantly associated with a higher incidence of intraperitoneal dissemination.Platelets could induce EMT in ovarian cancer cells and then promote their invasive ability.Platelets activated the TGF-β/Samd pathway in the ovarian cancer cells,and TβR I inhibitor A83-01 could inhibit the effects of platelets on the ovarian cells.Therefore,platelets may induce EMT in ovarian cancer cells and increase their invasion via TGF-βsignaling.D-dimer and FDP were superior to PLT in distinguishing benign from malignant or distinguishing early stage from advanced stage in ovarian cancer.Combining with CEA and/or LDH,D-dimer could be a useful surveillance marker for distant organ metastasis in colorectal cancer.