Experimental Study Of [⁶⁸Ga]FAPI PET/CT In The Diagnosis And Therapy Monitoring Of Pulmonary Fibrosis

Objectives:Idiopathic pulmonary fibrosis（IPF）is a progressive pulmonary disease with poor prognosis.Due to the lack of specific biomarkers for disease evaluation and insidious onset of disease symptoms,it is difficult to realize timely intervention.Fibroblast activation protein（FAP）is a non-classical serine protease up-regulated in fibrotic remodeling sites.The purpose of this study was to use CT,[¹⁸F]FDG PET,⁶⁸Ga labeled fibroblasts activating protein inhibitor([⁶⁸Ga]FAPI)PET to image bleomycin-constructed pulmonary fibrosis mice and pirfenidone treated pulmonary fibrosis mice,and to compare the results of the three imaging means.The relationships between imaging results and pathological results were analyzed to explore the advantages of[⁶⁸Ga]FAPI PET imaging in the diagnosis and treatment of pulmonary fibrosis monitoring.Methods:[⁶⁸Ga]FAPI was prepared and evaluated for radiochemical purity and stability.Male C57BL/6 mice aged 8～12 weeks were used.In the first part of the study,mice were randomly divided into the experimental group and control group.In the experimental group,bleomycin（BLM,2 mg/kg）was injected into IPF mice to induce IPF model;in the control group,normal saline（NS）was given to mice through the air tube.After modeling,the mice underwent weekly[¹⁸F]FDG PET/CT and[⁶⁸Ga]FAPI PET/CT scans for 4 weeks to assess disease progression.After weekly[⁶⁸Ga]FAPI PET/CT imaging,some mice were sacrificed for biological distribution study,and lung tissues were collected for hydroxyproline quantitative detection,HE staining,picrosirius red staining,PSR staining,glucose transporter 1（GLUT1）and FAP immunohistochemistry staining.The expression levels of GLUT1 and FAP in pathological sections were analyzed semi-quantitatively,and the correlations between imaging parameters and pathological semi-quantitative values were evaluated.In the second part of the study,mice were randomly divided into NS control group,BLM non-treatment group and BLM+PFD treatment group.Mice in the control group were given NS through endovascular intubation;Mice in the treatment group and non-treatment group were injected BLM（2 mg/kg）through endovascular intubation to induce IPF mouse model;Mice in the treatment group were orally given PFD 400 mg/kg/d from the 9～28th day after the establishment of the model.In the second part,after modeling,all mice underwent[¹⁸F]FDG PET/CT and[⁶⁸Ga]FAPI PET/CT scans once a week for 4 weeks to evaluate disease progression and therapeutic effect.At the 28th day,the mice were sacrificed after the imaging completed and the biological distribution was conducted.The same pathological staining was performed on lung tissues as in the first part,and the expression levels of GLUT1 and FAP in the pathological sections were semi-quantitatively analyzed.The correlation between pathological parameters and imaging parameters was analyzed.Results:CT imaging and[¹⁸F]FDG PET imaging can reflect the changes of lung anatomy and GLUT1-positive inflammatory cells before and after pulmonary fibrosis treatment,respectively.[⁶⁸Ga]FAPI PET/CT can reflect the expression level of FAP in pulmonary and the degree of fibrosis lesions before and after pulmonary fibrosis treatment.In the first part,no significant changes were observed in the three imaging methods in the control group.CT imaging in the experimental group showed that lung density increased from 7 days to 28days after modeling,and the degree of lung consolidation showed a trend of first increasing and then decreasing with time.Lung consolidation was the most significant in mice at 21days after modeling,and the mean lung density（MLD）of CT semi-quantitative index was consistent with the change trend of images.[¹⁸F]FDG PET imaging showed that the radioactivity distribution of[¹⁸F]FDG in the lung increased from the 7～28th day after modeling,demonstrating a trend of first increasing and then decreasing with time.The radioactivity distribution in the lung of mice was the highest at the 14th day after modeling,and the change trend of[¹⁸F]FDG uptake in the lung was consistent with the change trend of images.[⁶⁸Ga]FAPI PET imaging showed that the radioactivity distribution of[⁶⁸Ga]FAPI in the lungs of mice increased from the 7 to 28th day after modeling,demonstrating a trend of first increasing and then decreasing with time.The radioactivity distribution in the lungs of mice was the highest at the 21st day after modeling,and the change trend of[⁶⁸Ga]FAPI in the lungs was consistent with the change trend of images.In the pathological study results of the first part,no significant changes were observed in the staining of sections in the control group,while HE staining in the experimental group showed that the damage of lung structure was first aggravated and then alleviated from the7～28th day after modeling.PSR staining and semi-quantitative analysis showed that the distribution of lung red-stained collagen first increased and then decreased from the 7～28th day after modeling,and the positive PSR staining area reached the maximum at the 21st day,which was consistent with the change trend of lung MLD and[⁶⁸Ga]FAPI lung uptake.GLUT1 staining and semi-quantitative analysis showed that the infiltration of GLUT1-positive inflammatory cells in the lungs worsened first and then decreased from the 7～28th day after modeling,and the positive area of GLUT1 staining reached the maximum at the14th day,which was consistent with the change trend of[¹⁸F]FDG uptake in the lungs.FAP staining and semi-quantitative analysis showed that the number of FAP positive fibroblasts in the lungs increased first and then decreased from the 7～28th day after modeling,and the FAP positive area reached the maximum at the 21st day after modeling,which was consistent with the change trend of[⁶⁸Ga]FAPI lung uptake.In addition,[¹⁸F]FDG lung uptake and[⁶⁸Ga]FAPI lung uptake in the experimental group were positively correlated with semi-quantitative values of GLUT1 immunohistochemical staining and FAP immunohistochemical staining,respectively.Lung MLD,[¹⁸F]FDG lung uptake,[⁶⁸Ga]FAPI lung uptake correlated well with PSR positive area percentage and lung hydroxyproline content.Among them,[⁶⁸Ga]FAPI lung uptake had the best correlation with indexes reflecting the degree of fibrosis.In the second part,the trend of changes in the imaging results of mice in the control and non-treatment groups was consistent with the control and experimental groups in the first part of the study.The degree of lung consolidation in the treatment group was lighter than that in the experimental group,showing a trend of first aggravation and then stabilization.At the 14th day,the lung consolidation was more significant,and then remained stable.MLD was lower than that of the non-treatment group from day 14 to 28,with the maximum value occurring at day 14 and remaining stable thereafter.The radioactivity distribution of[¹⁸F]FDG in the lung of mice in the treatment group was lower than that in the non-treatment group,showing a trend of first increasing and then decreasing,peaking at the 14th day,and then decreasing.[¹⁸F]FDG lung uptake was lower than that of non-treatment group mice at the 14～28th day,and its maximum value decreased and remained stable after the 14th day.The radioactivity distribution of[⁶⁸Ga]FAPI in the lung of mice in the treatment group was lower than that in the non-treatment group,showing a trend of increasing first and then decreasing.The radioactivity distribution in the lung of mice in the treatment group was similar at day 14 and 21,and decreased significantly from day 21 to day 28.[⁶⁸Ga]FAPI lung uptake of was lower than that of mice in the non-treatment group at the 14～28th day.In the pathological results of the second part of the study,no significant changes were observed in staining of all sections in the control group.At the end of 28 days of treatment,HE staining showed that the lung structure of the non-treatment group was severely damaged,while that of the treatment group was less damaged.PSR staining showed that the lung collagen deposition was heavier in the non-treatment group and lighter in the treatment group.Semi-quantitative analysis showed that compared with the control group,the difference in the non-treatment group was statistically significant,while the difference between the treatment group and control group was not statistically significant.GLUT1staining showed that non-treatment GLUT1-positive inflammatory cell infiltration was more severe,while the GLUT1-positive inflammatory cell infiltration was less in the treatment group.Semi-quantitative analysis showed that the difference between the non-treatment group and control group was statistically significant,while the difference between the treatment group and the control group was not statistically significant.FAP staining showed that there was an obvious proliferation of FAP-positive fibroblasts in the lungs of the non-treatment group,but a light proliferation of FAP-positive fibroblasts in the treatment group.Semi-quantitative analysis showed that the difference in the non-treatment group and control group was statistically significant,while the difference between the treatment group and control group was not statistically significant.The[¹⁸F]FDG lung uptake and[⁶⁸Ga]FAPI lung uptake in the treatment group was positively correlated with semi-quantitative values of GLUT1 immunohistochemical staining and FAP immunohistochemical staining,respectively,and the lung MLD,[¹⁸F]FDG lung uptake,[⁶⁸Ga]FAPI lung uptake were positively correlated with percentage of PSR positive area and lung hydroxyproline content.Conclusion:This study suggests that CT,[¹⁸F]FDG PET and[⁶⁸Ga]FAPI PET can monitor the disease progression of animal models of pulmonary fibrosis and the therapeutic effect of pirfenidone on animal models of pulmonary fibrosis from different angles.Traditional CT imaging and[¹⁸F]FDG PET imaging can reflect the anatomy and glucose metabolism of pulmonary fibrosis.[⁶⁸Ga]FAPI PET can reflect the expression level of FAP in pulmonary fibrosis,which can more accurately reflect the degree of pulmonary fibrosis than CT imaging and[¹⁸F]FDG PET imaging.It provides a non-invasive imaging method for monitoring the progression of pulmonary fibrosis and evaluating the therapeutic effect of pulmonary fibrosis.