| Backgrounds and Purpose In the last years,more and more evidence have confirmed that inflammation plays an important role in tumor initiation,promotion,and progression,especially chronic inflammation.On one hand,tumor can be triggered by infection and chronic inflammatory disease,such as chronic gastritis caused by Helicobacter pylori(H.pylori)developed to gastric cancer,sustained inflammatory bowel diseases(IBD)to colorectal cancer,primary sclerosing cholangitis(PSC)to cholangiocarcinoma.Inflammation can increase the risk of cancer by release of bioactive molecules(growth factors?cytokines?chemokines)to form the tumor microenvironment,the neoangiogenes,maintained cell survival signals and extracellular metallo proteinases that promote epithelil-mesenchymal transition(EMT),gene mutation,and immune evasion facilitate carcinogenesis programs.On the other hand,the bioactive molecules that exist in tumor microenvironment in turn lead to the inflammation called tumor-associated inflammation(TAI),such as TNF-?,IL-6.It is now widely accepted that immune responses have emerged as crucial players to demonstrate the strong association between inflammation and tumor,owing to the dual role of immune cells and cytokines.The effective diagnosis and treatment of chronic inflammation is the key to decrease incidence and mortality of tumor.TSPO-formerly known as the Peripheral Benzodiazepine Receptor(PBR)-is an ubiquitous 18 k Da molecule located on the outer mitochondrial membrane(OMM)and has a high evolutionnary conservation,it can be expressed in CNS microglia and correlates with the inflammatory states of the CNS(Central Nervous System),because of which it is widely applied in the detection of diseases in CNS.In peripheral tissues,it exists in monouclear cells?lymphocyte?multinucleargiant cell and bronchial and bronchiole epithelium,pneumocytes and alveolar macrophages in lung,so it also been usd in some peripheral inflammation.Besides it,TSPO is also expressed in tumor cell,which is thought positively assosiated with tumor progression and malignancy.Many radioligands have been explored for imaging the 18-k Da translocator protein(TSPO),a diagnostic and therapeutic target for inflammation and tumor.Here,we aimed to evaluate the TSPO novel radioligand18F-VUIIS1008 for positron emission tomography(PET)imaging of peripheral inflammation and cancer by the estimation of physicochemicalcharacteristic,the perform of cell assays and immunohistology,as well as Micro PET imaging to achieve the specific imaging of inflammation and tumor.Procedures1.The preparation of 18F-VUIIS1008 was made by making 18F-radiolabeled on the tosylate substrate and the physicochemical characteristic was evaluated including the stability in saline snd mouse serum in vitro and Log P.2.RAW264.7 mouse macrophage cells were used for cell uptake and efflux assays of 18F-VUIIS1008.The affinity of 18F-VUIIS1008 for TSPO was also measured in RAW264.7 by competitive binding assay.3.Mouse left hind limb inflammation model was established by subcutaneous injection of CFA(complete Freund's adjuvant).Inflammation model was first evaluated by CBB(coomassie brilliantblue),HE(Hematoxylin-eosin),IHC(Immunohistochemistry),IFC(Immunofluorescence)in histology to evaluate the TSPO expression and macrophage in inflamed and contralateral tissues.4.Mouse biodistribution was performed before Micro PET imaging to explore18F-VUIIS1008 distributionin in vivo.Then inflammation model was imaged at different days(in model 3,7,12,15,19,24,29,35,38,45days)using18F-VUIIS1008 by Micro PET-CT to assess whether it can be applied in inflammation imaging and reflect the process.Blocking or inhibition experiments involving co-injection with PK11195(1mg/kg)and with VUIIS1008(1mg/kg)were conducted to evaluate the specificity and reversibility of radioligand binding.5.PET imaging using 18F-VUIIS1008 was performed in human lung adenocarcinoma cell A549,human hepatoma cell Hep G2,human breast cancer cell line MDA-MB231,MCF7,human gastric carcinoma cell MGC-803 and human glioma cell U87 subcutaneous tumor models to evaluate potential of 18F-VUIIS1008 in tumor imaging.Results1.The radioproductivity and radiochemical purity of 18F-VUIIS1008 was 41±5%,98% after radiolabellment and purity,respectively.The radioactivity was more than 4107Ci/.The radiochemical purity of 4h was still more than 98% both in saline and serum,which showed 18F-VUIIS1008 had a good stabiliity in vitro.The Log P value was 1.58±0.03,showing it was lipid solubility.All of this made it feasibility in imaging in vivo.2.Cell assays confirmed 18F-VUIIS1008 has a high affinity to TSPO(IC50=1.287e-007)and can be uptaked well in RAW264.7(the uptake percentage of 1h is 14±0.3% of total activity which can be inhibited to 4±0.7% of total activity in uptake assay,the internalization rate of 18F-VUIIS1008 after 2 hours was still 12±1 % in efflux assay),which showed 18F-VUIIS1008 can be uptaked well in cell.3.CBB,HE,and Immunohistochemistry showed the TSPO expression was significantly more in inflamed tissues than that in contralateral tissues.Immunofluorescence staining confirmed the TSPO expression in macrophage in inflamed tissues was more than that in the contralateral.4.The biodistribution showed 18F-VUIIS1008 was concentrated in heart,lung,live and kidney,which had a correlation with the TSPO expression and Log P.The uptake of 18F-VUIIS1008 PET imaging showed different uptake levels by days in inflammation models,with a peak of inflammation at day7 and 29,suppose it was associated with the mechanism that the inflammation model was performed by CFA,which was similar with RA(Rheumatoid Arthritis).The different peak could be attributed to different kinds of inflammatory cells,new blood vessels and local edema,delayed type hypersensitivity in later period.PET results were correlated with CBB,HE,Immunohistochemistry and Immunofluorescence staining performance.Tracer uptakes could be blocked by PK11195(1.85±0.19 % ID/g v.s 1.06±0.14 % ID/g and VUIIS1008(1.85±0.19 %ID/g v.s 0.5 ± 0.08 %ID/g,which demonstrated the specificity and reversibility of 18F-VUIIS1008 binding.5.The uptakes of 18F-VUIIS1008 in different tumors(human breast cancer MDA-MB-231:0.52 ± 0.14 %ID/g,human hepatoma Hep G2:0.6 ± 0.06%ID/g,human glioma U87:0.5 + 0.08 %ID/g,human lung adenocarcinoma A549:0.46 ± 0.16 %ID/g,human gastric carcinoma MGC803:0.43 ± 0.14%ID/g,human breast cancer MCF7:0.32±0.1%ID/g)were significantly lower than that in inflammation uptake(2.79+0.03 %ID/g).Conclusion Appropriate physicochemical characteristic made 18F-VUIIS1008 possible for imaging in vivo.The performances in cell assays,CBB,HE,IHC,IFC,biodistribution confirmed it had a high affinity for TSPO and can be uptaked well in RAW264.7 mouse macrophage cells and inflamatory tissues.18F-VUIIS1008 Micro PET imaging in inflammation and tumor models confirmed the specificity of18F-VUIIS1008 in inflammation imaging.These preliminary results demonstrated that the new TSPO targeted radioligand 18F-VUIIS1008 can be adapted for the study of inflammation,suggesting strong potential to be a specific PET tracer for inflammation and better differentiation between peripheral inflammation and tumor. |
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