The Effect Of Pancreatic Cancer Cells Overexpressed VNN1 On Paraneoplastic Islets And The Role Of USPIO-VNN1 In Pancreatic Cancer Cells Imaging

Background:Pancreatic cancer is one of common malignant digestive diseases,and prognosis of this disease is very poor.Early diagnosis of pancreatic cancer is very difficult,mainly because of the late onset of disease-specific symptoms and a shortage of good biomarkers for the disease.By the time of diagnosis,less than 20%of patients can be salvaged by surgical resection,which remains the only chance for cure.Therefore,early detection of pancreatic cancer is crucial for patients to receive surgical treatment and improve the prognosis.Epidemiological studies have shown that new-onset diabetes(ND)can be an early clinical manifestation of pancreatic cancer,this diabetes is known as pancreatic cancer associated new-onset diabetes(PCAND).So,ND can be regarded as a first filter for early-stage pancreatic cancer screening.However,the incidence of normal ND is much higher than pancreatic cancer,it's still difficult to screen a relatively small number of PCAND from a large number of patients with normal ND.Our previous study reported that Vanin-1(VNN1)was a novel biomarker of PCAND,which can be used to differentiate PCAND from normal ND and detect the early-stage pancreatic cancer.However,the complete functional mechanisms of VNN1 in the pathogenesis of PCAND are still unknown.In this study,we analyzed the effect of pancreatic cancer cells overexpressed VNN1 on paraneoplastic islets and the underlying mechanism.Meanwhile,we tested whether the downstream molecules of VNN1 could be used for early detection of pancreatic cancer.Furthermore,we synthesized VNN1 functionalized ultrasmall superparamagnetic iron oxide nanopaticles(USPIO-VNN1).Then we investigated whether the USPIO-VNN1 could be used for tracking the pancreatic cancer cells with VNN1 overexpression by MRI scanning,and discussed the possibility of USPIO-VNN1 for early-stage pancreatic cancer imaging.Methods:The lentiviral vector encoding human VNN1 cDNA was transfected into pancreatic cancer cell lines(PANC-1 and CFPAC-1).PANC-1 and CFPAC-1 cells with stably overexpression of VNN1 were termed as PANC-VNN1 and CFPAC-VNN1.Primary islets were isolated from C57BL/6 mice,then pancreatic cancer cells and primary islets co-culturing system was constructed using transwell chamber(6-well,0.4?m pore size).After co-culturing for 24 hours,the viability of islets was determined by Trypan blue staining and flow cytometry analysis,the function of islets was determined by insulin secretion assay.The co-cultured islets were transplanted under the kidney capsule of diabetic B6 rag1-/-mice,and their blood glucose levels were monitored for more than 14 weeks.The immunohistological staining was used for evaluating the insulin expression of primary islets and the VNN1 expression of pancreatic caner specimens.The GSH levels in the co-cultured islets were measured using a Total Glutathione Quantification Kit.The protein expressions of cleaved caspase,3,cleaved caspase-9 and PPARy were assessed by western blot.The cysteamine concentrations in conditioned medium,islets and pancreatic caner serum samples were measured by HPLC assay.The USPIO was synthesized using ferric acetylacetonate,oleic acid,oleylamine and so on,then the USPIO was coated with PEG to synthesize the PEG-USPIO.The physiochemical properties of PEG-USPIO were characterized by transmission electron microscope(TEM),Dynamic Light Scattering(DLS),magnetic property measurement system(MPMS)and Fourier Transform Infrared spectra(FTIR).Next,the VNN1 antibody-functionalized USPIO(USPIO-VNN1)was achieved by the N-hydroxysuccinimide(NHS)/ethyl diethylaminopropyl carbodiimide(EDC)method.The PANC-VNN1 and control cells were treated with 40 ?g Fe/ml PEG-USPIO or USPIO-VNN1 for 24 hours,then the PEG-USPIO or USPIO-VNN1 labeled cells was detected by Prussian blue staining and MRI scanning.The subcutaneous tumor models in nude mice were established by implanted PANC-VNN1 and control cells respectively.20 mg Fe/kg PEG-USPIO or USPIO-VNNlwas injected into the tumor-bearing mice via tail vein,24 hours later,the accumulation of nanoparticles in tumors was detected by MRI scanning.Results:Trypan blue staining and flow cytometry analysis showed that primary islets co-cultured with PANC-VNN1 or CFPAC-VNN1 cells had lower viability compared to the untreated and control cells co-cultured islets.Besides,after co-cultured with PANC-VNN1 or CFPAC-VNN1 cells,the protein expressions of cleaved caspase-3 and cleaved caspase-9 were increased significantly in islets,and there were no intact islets detected.The basal insulin secretion of islets was significantly decreased afterco-cultured with pancreatic cancer cells,and the decrease was particularly apparent in PANC-VNN1 or CFPAC-VNN1 cells co-cultured group.The untreated,control cells co-cultured and PANC-VNN1 cells co-cultured islets were transplanted under the kidney capsule of diabetic B6 ragl-/-mice respectively.All mice that received 200 or 400 IEQ islets achieved normoglycemia,and the mice in PANC-VNN1 cells co-cultured group achieved normoglycemia later than the mice in untreated and control cells co-cultured groups.HPLC assay showed that the cysteamine(downstream molecule of VNN1)concentration in conditioned medium of PANC-VNN1 or CFPAC-VNN1 cells was increased more significant than control cells.Compared to untreated and control cells co-cultured islets,cysteamine and ROS concentrations were increased dramatically while GSH level and PPARy expression were decreased obviously in islets co-cultured with PANC-VNN1 or CFPAC-VNN1 cells.After pre-incubation with 10?M GSH or 10?M thiazolidinedione(TZD,PPARy agonist)for 2 hours,the ROS content was decreased while the viability and basal insulin secretion were increased in islets co-cultured with PANC-VNN1 cells.The serum concentration of cysteamine was measured in 225 candidates(52 PCAND patients,61 pancreatic cancer patients without ND,57 normal ND patients and 55 healthy subjects)by HPLC assay.PCAND patients had higher serum concentration of cysteamine compared to pancreatic cancer patients without ND,normal ND patients and healthy controls.The ROC methodology was conducted to determine whether the serum concentration of cysteamine could be used to differentiate PCAND from normal ND,and the AUC was 0.828 ± 0.039(P<0.05).TEM showed that the core size of USPIO was about 3-7nm and these nanoparticles displayed good dispersibility.USPIO was coated with PEG to synthesize the PEG-USPIO,the average size of PEG-USPIO was about 20nm and the saturation magnetization intensity of PEG-USPIO was nearly 35 emu/g.PEG-USPIO was combined with VNN1 monoclonal antibody to synthesize USPIO-VNN1 by NHS/EDC method.The PANC-VNN1 and control cells were treated with 40 ?g Fe/ml PEG-USPIO or USPIO-VNN1 for 24 hours,prussian blue staining revealed that nanoparticles was accumulated more obvious in PANC-VNN1 cells treated with USPIO-VNN1 than other 3 groups(control cells treated with PEG-USPIO,PANC-VNN1 cells treated with PEG-USPIO,control cells treated with USPIO-VNN1),MRI scanning showed that PANC-VNN1 cells labeled by USPIO-VNN1 displayed hypointense signals in T2-weighted MR images compared to other 3 groups.20 mg Fe/kg PEG-USPIO or USPIO-VNNlwas injected into the tumor-bearing mice via tail vein,24 hours later,MRI scanning indicated that USPIO-VNN1 could selectively accumulated in the tumors derived from PANC-VNN1 cells(T2-weighted signal decreased in tumor region),whereas there was no significant signal change in the tumors of the mice from other 3 groups.And this result was further confirmed by prussian blue staining using tumor sections.Toxicity assessments demonstrated that USPIO-VNN1 had good biocompatibility in vivo.Conclusion:Our results demonstrated that pancreatic cancer cells with VNN1 overexpression could increase oxidative stress response of paraneoplastic islets through promoting secretion of cysteamine,which further aggravated paraneoplastic islets dysfunction.Cysteamine could serve as a novel biomarker of PCAND,and determination of serum cysteamine level could be used to differentiate early-stage pancreatic cancer patients from the ND population.The newly synthesized USPIO-VNN1 was capable of targeting pancreatic cancer cells with VNN1 overexpression in.vivo by MRI scanning,which suggested that USPIO-VNN1 might be served as a promising diagnostic probe for direct visualization of early-stage pancreatic cancer with secondary ND.