Study Of The Expression Of VWF In Osteosarcoma And Its Role In The Process Of Migration And Invasion

Background:Von Willebrand factor(VVF)is a large multimeric glycoprotein,which is produced by vascular endothelial cells(ECs)and megakaryocytes.The intracellularly synthesized VWF can be secreted eontinuously to the plasma immediately,or it ean be stored in the form of large multimeric species(ultra-large VWF,UL-VWF)in α-granules of platelets and Weibel-Palade bodies of ECs.Once the blood vessel walls are damaged or stimulated by some inflammatory factors,VWF will be released into the blood circulation.VWF can bind to the Collagen α-1 chain of type Ⅰ and type Ⅲ collagen(via its A3 domain)and bind to platelet glycoprotein Iba(GPIba)(via its A1 domain).Therefore,VWF functions as a bridge between platelets and endothelial matrix,and plays an important role in the mechanism of hemostasis and thrombus formation.Recent years,in addition to its major role in hemostasis,VWF has been reported to participate in many pathophysiological processes,such as inflammation,tissue damage and repair,angiogenesis,immune response and cancer metastasis.Regarding the role of VWF in tumor metastasis,it was hypothesized that VWF participates in adhesion of cancer cells to platelets and endothelial surfaces,thus facilitating extravasation and promoting metastasis.A role for platelets in eancer metastasis is well established.Tumor cells can participant in promoting platelet aggregation and forming heteroaggregates to protect tumor cells from immune surveillance.Moreover,VWF can contribute to the metastatic process through association of heteroaggregates with the vascular endothelium.Thus,VWF as a major participant in promoting the interactions between platelet and endothelium,presents itself as a highly likely candidate to promote metastasis.For a long time,most studies have demonstrated that levels of VWF in cancer patients are signifieantly increased,and may be positively correlated with the malignancy of tumors.In contrast,other investigations suggested that VWF exerts a pro-apoptotic effect on the tumor cells and acts as an anti-metastatic protein.Discordant observations regarding the role of VWF in cancer metastasis may be attributed to the focus of investigations on ECs and platelets as the source of VWF.However,recent studies have reported VWF detection in cancer cells of non-endothelial origin,including osteosarcoma cells,glioma cells and hepatocellular carcinoma cells.Thereore,more and more researches focus on tumor cell-derived VWF as a potential anti-metastasis target.Osteosarcoma,which is a highly metastatic cancer,is the most common malignant bone tumor,and pulmonary metastasis is the most common cause of death in osteosarcoma patients.With the advancement of medical technology in recent years,the long-term survival rate of patients with osteosarcoma has significantly improved.However,once metastasis occurs,the 5-year survival rate is only approximately 20%.Therefore,the development of exploring new therapeutic targets for inhibiting the metastasis of osteosarcoma has become a topic of considerable interest.Objective:This study was aimed to explore the difference in VWF expression levels between orthotopic and metastatic tumor tissues of osteosarcoma patients.To compare the discrepancy in the expression patterns of VWF in ECs and osteosarcoma cells.Preliminary demonstration of fuctions and mechanisms that osteosarcoma cell-derived VWF mediates platelet adhesion and the process of migration and invasion of osteosarcoma cells.Methods:(1)Plasma samples from 66 patients with osteosarcoma and 60 normal volunteers used as control group(Control,CTL)from the First Affiliated Hospital of Soochow University were collected.Enzyme-linked immunosorbent assay(enzyme-linked)was used to detect the expression levels of VWF in plasma of osteosarcoma patients and CTL groups.(2)The pathological specimens of 12 patients with osteosarcoma from the First Affiliated Hospital of Soochow University were collected.Hematoxylin-Eosin staining(HE)and immunohistochemical analyses(IHC)were performed on paraffin-embedded pathological sections.Mouse anti-human VWF monoclonal antibody(mAb)SZ34 was used to compare the expression levels of VWF in different osteosarcoma pathological specimens.(3)The expression of VWF in human osteosarcoma cell line SAOS2 was detected by western blot(Wb)and immunofluorescence assay.This discrepancy in the expression patterns of VWF in human microvascular endothelial cells(HMEC-1)and SAOS2 was analyzed by VWF multimer analysis.(4)Phorbol-12-Myristate-13-Acetate(PMA)was used to obtain VWF-overexpressing SAOS2 cells.Immunofluorescence assay,flow cytometry(FCM)and VWF multimer analysis were used to detect the different levels of VWF in SAOS2 cells with or without the treatment by PMA.(5)Platelet adhesion assay was performed under static and shear flow conditions to determine the adhesion between VWF-overexpressing SAOS2 cells and platelets.Three VWF-GPIba pathway blocking antibodies,SZ123,SZ2 and AVW3 were used to study the role of this pathway in the adhesion of SAOS2 cells to platelets.(6)Cell Counting Kit-8(CCK8)assay was used to detect the proliferation of VWF-overexpressing SAOS2 cells after co-culture with different concentrations of washed platelets.(7)Using human embryonic kidney cells(HEK293)as a negative control,transwell migration and invasion experiments were used to investigate the migration and invasion capacities of VWF-overexpressing SAOS2 cells after adhesion to platelets.The above three VWF-GPIbα pathway blocking antibodies were used to study the effect of this pathway on the migration and invasion capacities of SAOS2 cells.(8)Levels of platelet-derived growth factor-BB(PDGF-BB)and transforming growth factor-β1(TGF-β1)in the supernatant of VWF-overexpressing SAOS2 cells co-cultured with platelets were detected by ELISA kit.The above three VWF-GPIba pathway blocking antibodies were used to study the effects of the inhibition of the pathway on the levels of PDGF-BB and TGF-β1 in the supernatant.(9)SPSS 19.0 and GraphPad Prism 5.0 software were used to analyse the data above.The measurement data were expressed as mean ± standard deviation(x±s).P<0.05 for the difference was statistically significant.Results:(1)Levels of VWF in 66 patients with osteosarcoma and in 60 normal volunteers were detected by ELISA.Data has shown that VWF levels in orthotopic osteosarcoma group,osteosarcoma with metastasis group and CTL group were not statistically significant(P>0.05).(2)The pathological specimens of 12 patients with osteosarcoma were detected by HE and IHC.The results showed that VWF was expressed in orthotopic osteosarcoma cells,invasive osteosarcoma cells and metastatic osteosarcoma cells.VWF expression was significantly higher in the samples of invasive osteosarcoma and osteosarcoma with lung metastasis than in the samples of osteosarcoma in situ.(3)VWF expression was detected in SAOS2 cells,HMEC-1 cells and platelet lysates(positive control),but not in HEK293 cells or MG63 cells(negative control)by Western blotting.Immunofluorescence assay using FITC-RAH VWF IgG showed that a certain amount of VWF granules were present in the cytoplasm of SAOS2 cells.VWF multimer analysis showed that VWF was predominantly present in the form of medium molecular weight polymers in SAOS2 cells,whereas VWF polymers of various molecular weights were expressed in ECs.(4)The intensity of the inmunofluorescence staining for VWF expression in SAOS2 cells treated with PMA was much stronger than that in untreated cells,and the level of VWF expression was increased in the whole cell.In addition,flow cytometric analysis indicated that the VWF levels in the cells treated with PMA were significantly higher than those in untreated cells.The same results were obtained by VWF multimer analysis.(5)Platelet adhesion assay under static conditions showed that the adhesion of platelets to VWF-overexpressing SAOS2 cells(17.96±0.59%)was significantly higher than that of untreated cells(6.00±0.26%),P<0.001;After VWF-GPIba pathway blocking antibodies SZ123,SZ2 and AVW3 added to the experimental systems,AVW3(6.51±0.35%)(P<0.001)and SZ2(5.50±0.24%)(P<0.001)could reduce the adhesion between platelets and SAOS2 cells,respectively;However,the results of the experiment with SZ123(17.20±0.6%)were not statistically significant(P>0.05).After 15 minutes of induced shear flow conditions,platelet adhesion assay showed the adhesion of platelets to VWF-overexpressing SAOS2 cells(14.96±0.68%)was significantly higher than untreated cells(4.97±0.36%),P<0.01.AVW3(2.52±0.35%)(P<0.01),SZ2(1.75±0.19%)(P<0.001),and SZ123(2.11±0.20%)(P<0.001)could reduce the adhesion of platelets to SAOS2 cells,respectively.(6)CCK8 assay was used to detect the proliferation of VWF-overexpressing SAOS2 cells after co-cultured with different concentrations of washed platelets.The results showed that the difference in cell proliferation was the biggest at 36 hours.The OD value of 5×105 platelets group(1.62±0.25)significantly higher than that of 5×104 platelets group(1.45±0.29),P<0.05.The OD value of 5×104 platelets group(1.45±0.29)was significantly higher than that of 5×103 platelet group(1.31±0.35),P<0.05.The difference of OD of 5×103 platelet group(1.31±0.21)and platelet-free group(1.28±0.23)was not statistically significant(P>0.05).(7)Transwell assay was used to investigate the migration and invasion capacities of VWF-overexpressing SAOS2 cells after co-cultured with platelets.The results showed that the number of migrated cells(52.3±6.99)and invasive cells(32.2±2.55)in the platelet co-culture group were significantly higher than those in cell alone group(22.4±3.53,8.1±1.58),P<0.005.After adding SZ2 and AVW3 to the experimental systems,the number of cells migrated(14.4±1.72,14.0±1.81)and invasive cells(10.4±1.71,9.1±1.58)were significantly lower than those in the platelet co-culture group.,P<0.001.There was no significant difference in the number of migrated cells(48.6±6.62)and invasive cells(30.4±2.04)in the experimental group added with SZ123 and the platelet co-culture group,P>0.05.Moreover,there was no significant difference in the number of migrated cells and invasive cells between all of the experimental groups of the negative control HEK293 cells,P>0.05.(8)The levels of PDGF-BB and TGF-β1 in the supernatant after co-culture of VWF-overexpressing SAOS2 cells with platelets were detected by ELISA kit.The results showed that the PDGF-BB and TGF-β1 levels in the platelet co-culture group were significantly higher than those in the platelet alone group.When 0.5ug/mL,lug/mL,and 2ug/mL of SZ2 and AVW3 were added to the experimental systems,the levels of PDGF-BB and TGF-β1 were significantly lower than those of the platelet co-culture group.Moreover,the deereasement was correlated with the antibody concentration.There was no significant difference in the levels of PDGF-BB and TGF-β1 between the experimental group added with S2123 and the platelet co-culture group.Conclusion:(1)VWF synthesized in ECs and megakaryocytes is not correlated with the metastasis of osteosarcoma,but closely related to VWF derived from osteosarcoma cells.(2)VWF particles are predominantly present in the form of medium molecular weight polymers in human osteosarcoma cell line SAOS2 cells.Moreover,VWF in osteosarcoma eells differs from ECs in aspects of synthesis,storage,distribution,structure and function.(3)SAOS2 cells with high expression of VWF in the periplasm and membrane can be successfully obtained by stimulation of a certain amount of PMA,and the VWF-overexpressing cells can form aggregates by adheison to platelets through the collagen-VWF-GPIba axis.Thereby,platelets will be activated to secrete and release growth factors such as PDGF and TGF-p,further promoting the migration and invasion of osteosarcoma cells.(4)VWF expressed by osteosarcoma cells plays an important role in the development of osteosarcoma.This finding provides a theoretical basis for the development of new therapeutic drugs for the prevention of osteosarcoma metastasis,and further enriched and perfect the mechanism of osteosarcoma metastasis,thus has important clinical value.