[¹⁸F]AlF-ND-bisFAPI PET Of Fibroblast Activation Protein As A Non-invasive Biomarker To Monitor The Progression Of Liver Fibrosis

BackgroundHepatic fibrosis is a progressive disease.However,the current monitoring methods have notable limitations that are a challenge to early detection.Fibroblast activation protein is from active hepatic stellate cells(aHSC)and influenced ECM production which make it have profibrotic and possibly proinflammatory effects and is closely related to the severity of liver fibrosis.Therefore,FAP has been seem as a valuable potential biomarkers of liver fibrosis progression.ObjectivesTo evaluate the utility of[18F]AIF-ND-bisFAPI positron emission tomography(PET)imaging of fibroblast activation protein(FAP)to monitor the progression of liver fibrosis.Methods1.The radioactive probe[18F]AIF-ND-bisFAPI was labeled by 18F-AlF chelating method,and the yield and radiochemical purity were determined.2.Assessment of combination of ability and the junction of[18F]AlF-ND-bisFAPI with rat fibroblasts HSC-T6 after LPS treated through cell uptake and blocking test,and analysis of FAP expression in active stellate cells through immunofluorescence and western blot test.3.Built BDL and CCl4 hepatic fibrosis animal models,and ’modest’ group was weeks CCl4 and 10 days BDL,’serious’ group was 12 weeks CCl4 and 21 days BDL.4.Analysis and qualification of sirius red and H&E staining were used to different times of hepatic fibrosis models.5.Analysis and qualification of FAP expression in WB and immunofluorescence were used in different times of livers.6.[18F]AIF-ND-bisFAPI micro-pet imaging for different liver fibrosis models and inhibition assay for 10 days BDL.To Evaluate the relationship between uptake of[18F]AIF-ND-bisFAPI and severity of hepatic fibrosis and verify in biodistribution.7.Collected different degree of liver fibrosis of patients with liver paraffin section to evaluate the relationship between the expression of[18F]AIF-ND-bisFAPI and the severity of hepatic fibrosis was analyzed by Masson’s staining and autoradiography.Result1.[18F]Alf-ND-bisFAPI was successfully labeled and prepared by 18F-AlF chelating method.The yield of[18F]Alf-ND-bisFAPI was 9.7±1.2%.And the peak of the product was single,the peak time of the product was about the same as that of the unlabeled precursor verified by HPLC.2.In vitro cell uptake experiments showed that[18F]AIF-ND-bisFAPI uptake was significantly higher in activated rat fibroblast HSC-T6 after LPS stimulation than in unstimulated HSC-T6 cells;Uptake of[18F]AIF-ND-bisFAPI by concurrently active HSC-T6 cells could be inhibited by FAPI-04,demonstrating the specificity of[18F]AIF-ND-bisFAPI cell uptake.3.Western blotting and cell fluorescence showed that the expression of FAP in HSCT6 increased after LPS stimulation,and the expression of FAP was not only expressed in the cell membrane but also in the cytoplasm.4.Animal models of liver fibrosis construction:both CCl4 and BDL surgery stimulate hepatic fibrocyte activation in mice,leading to the development of liver fibrosis,which is exacerbated over time.H&E staining showed a gradual increase in the number of damaged hepatocytes and inflammatory cells over time,and Sirius red staining showed a gradual increase in collagen fibers over time.5.PET imaging in small animals showed that in both animal models,[18F]AlF-NDbisFAPI uptake increased with exacerbation of fibrosis which has statistically significant.And after supersaturation inhibition of FAPI-04,the uptake of[18F]AlF-ND-bisFAPI was significantly reduced by the 10 days BDL model,illustrating the uptake specificity of[18F]Alf-ND-bisFAPI and ruling out the possibility of false positives due to cholestasis,the biodistribution verified the results.6.The autoradiography and Masson showed that the uptake of[18F]AlF-ND-bisFAPI increased with the degree of liver fibrosis,however,the uptake value of[18F]AlFND-bisFAPI was not completely positively correlated with collagen fiber density.Conclusion[18F]AlF-ND-bisFAPI PET imaging is a promising noninvasive imaging method that can be used to monitor the progression of early hepatic fibrosis and is worthy of further study in clinical application.