| Malignant tumor is one of the refractory diseases that seriously endanger human health.The efficacy of anti-tumor therapy is closely related to the tumor and the microenvironment of the tumor.At present,the commonly used clinical tumor efficacy prediction index gene detection,programmed death ligand 1 and tumor mutation burden are the molecular characteristics of tumor cells,and point sampling is needed to obtain detection samples.There are some problems,such as trauma,replacing surface with points,sampling error and poor repeatability,ignoring the specific detection of tumor microenvironment.Only through a comprehensive analysis of the molecular characteristics of tumor cells and the characteristics of tumor microenvironment is it possible to comprehensively analyze the role of tumor therapy and make individual predictions.Therefore,it is an urgent scientific problem to find sensitive biomarkers and effective detection methods to target tumor microenvironment.Fibroblast activation protein(FAP)directly promote the proliferation,migration and invasion of interstitial fibroblasts and other types of cells,including tumor cells,endothelial cells and immune cells,resulting in invasiveness,tumor neovascularization and evasion of immune surveillance.It is a very potential biomarker to reflect tumor microenvironment characteristics,tumor treatment targets and predict efficacy.Radionuclide labeled fibroblast activation protein inhibitor(FAPI)positron emission tomography(PET)/computer tomography(CT)imaging can realize three-dimensional,non-invasive and dynamic molecular imaging of FAP in tumor microenvironment.It is an ideal method to evaluate the individual characteristics of FAP positive stromal cells in tumor microenvironment.There are few researches on 18F marker FAPI at home and abroad.18F has a long half-life(110min)and positron energy has higher spatial resolution than 68Ga nuclide,so it is an ideal radionuclide for labeling imaging agents.The structure of this study includes three parts:"construction-verification-application"of 18F-labeled FAPI small molecule imaging agent.In the first part,we constructed Al[18F]labeled small molecule PET imaging agent targeting FAP,and completed the quality control work,verified the feasibility of imaging by cell and animal experiments,and applied for invention patent.The second part,taking animals and tumor patients as research objects,verified the accuracy and safety of[18F]AIF-NOTA-FAPI-04 imaging through accurate comparison of images,tissue samples and molecular pathology.The third part,taking the animal model of radiation-induced heart injury and tumor patients as the research object,through the research on the imaging and diagnostic value of different tumors,the potential exploration of pathological classification of lung cancer,the detection value of radiation-induced heart injury and so on,to explore the application value of[18F]AlF-NOTA-FAPI-04.The first part:construction research.Methods:NOTA-FAPI precursor,acetate buffer solution,anhydrous acetonitrile,AlCl3 acetate buffer and 18F fluorine ion solution were added to the micro reaction device with precise control of reaction process,and the reaction was sealed;the color change was accurately detected by the sensor,the reaction solution was continuously cooled into the cooling module in time,and the reaction system was diluted with deionized water.The diluted reaction solution was passed into the purification module,separated and purified by C18 column,then eluted with 50-80%ethanol and filtered with 0.22 μm filter to obtain the target compound[18F]AIF-NOTA-FAPI-04 with stable performance.U87MG tumor-bearing mice were constructed,the biological distribution of[18F]AlF-NOTA-FAPI-04 in different tissues and organs of mice was evaluated,and PET/CT imaging of small animals was performed to verify the feasibility of in vivo imaging.Results:The radiochemical purity of the final product was measured by HPLC,and the retention time was at 6.3min.The radiochemical purity of[18F]AlF-NOTA-FAPI-04 was more than 99.5%,and the specific activity was about 20GBq/μmol.PET/CT imaging was used to evaluate the distribution of[18F]AlF-NOTA-FAPI-04 in U87MG transplanted tumor model mice.It was observed that radioactive tracer was rapidly absorbed in the tumor and cleared rapidly through the kidney and bladder,and the uptake value was very low in most normal organs,including brain,heart,blood,bone,liver,spleen,lung,stomach,small intestine,large intestine and muscle.In the imaging after injection of 60min,the uptake level of[18F]AlF-NOTA-FAPI-04 in the transplanted tumor was the highest(15.48±1.82%ID/g).The high uptake level of the tracer in the tumor tissue lasted for 2 hours,and the uptake value of the tracer in the gallbladder was significantly higher than that in other normal tissues,followed by the kidney and small intestine.Blocking experiment showed that the uptake value of tumor after blocking was significantly lower than that of unblocked,which indicated that[18F]AlF-NOTA-FAPI-04 could specifically bind to FAP.Conclusion:Using Al[18F]labeling strategy,the synthesis of[18F]AIF-NOTA-FAPI-04 is simple and efficient;the quantitative kit of FAPI and medicinal excipients further improves the preparation efficiency;the prepared[18F]AlF-NOTA-FAPI-04 has more than 99%radiochemical purity and achieves the goal of ready-to-use,which is beneficial to clinical transformation.Part two:verification research.Methods:The colocalization experiment was used to verify the consistency of the binding site of FAPI-04 and FAP on the U87MG cell membrane.[18F]AlF-NOTA-FAPI-04 and 68Ga-DTOTA-FAPI-04 tracer reported in previous literature were constructed,and the imaging accuracy of[18F]AlF-NOTA-FAPI-04 in tumor-bearing mice was analyzed by in vivo PET/CT imaging,dynamic scanning,blocking experiment,autoradiography and multi-color immunofluorescence experiment.Patients with different types of tumors were prospectively enrolled,on the one hand,to evaluate the accuracy and safety of[18F]AlF-NOTA-FAPI-04 PET/CT imaging in patients,and on the other hand,to obtain surgical or biopsy tissue samples of patients,and the correlation between[18F]AlF-N OT A-F API-04 uptake and FAP-α expression was verified by FAP-αimmunohistochemical staining.Results:Both in vitro and in vivo experiments showed that FAPI-04 and FAP had high binding specificity.In the mouse model of xenotransplantation and tumor patients,high uptake and rapid clearance of radioactive tracer in tumor were observed.High contrast images and negligible radiation exposure to normal tissues were observed on[18F]AlF-NOTA-FAPI-04 PET/CT in 28 patients with 8 different types of cancer.Among them,5 cases were scanned by PET/CT at 1 hour,2 hours and 4 hours after intravenous injection of[18F]AlF-NOTA-FAPI-04.The tracer was highly uptake in primary,lymph node,liver and bone metastases,and the maximum standard uptake value(SUVmax)was 19.37(range 3.4-19.37).The distribution of[18F]AlF-NOTA-FAPI-04 in the normal organs of the patient was further studied by PET/CT imaging.The tracer was quickly removed from the blood and excreted mainly through the kidney and bladder.Other normal organs(blood,liver,muscle,pancreas and spleen)have low uptake of radioactive tracers,while tumors show high uptake,resulting in high-contrast PET images.There was a positive correlation between[18F]AlF-NOTA-FAPI-04 uptake and the expression of FAP in biopsy+operation specimens(r=0.439,P=0.012)and simple operation specimens(r=0.938,P=0.005).Conclusion:As a FAP imaging agent,[18F]AlF-NOTA-FAPI-04 has high specificity and can be used for high contrast and rapid imaging of tumors.After[18F]AlF-NOTA-FAPI-04 PET/CT imaging,no patients reported symptoms of discomfort,and the patients were well tolerant to the radioactive tracer.Part Ⅲ:applied research.Patients with primary lung cancer confirmed by histopathology were prospectively enrolled.Static[18F]AlF-NOTA-FAPI-04 and[18F]FDG PET/CT scans were performed within one week before treatment to evaluate the accuracy of different tracers in the diagnosis of mediastinal metastatic lymph nodes and distant metastases.Results:Prospective double-tracer imaging patients evaluated 45 metastatic foci and 26 stations of mediastinal metastatic lymph nodes.The results showed that 44 and 37 metastases were detected by[18F]AlF-NOTA-FAPI-04 PET/CT and[18F]FDG PET/CT,respectively.Overall,80.0%of the metastases were detected by both[18F]AlF-NOTA-FAPI-04 and[18F]FDG,and 17.8%through[18F]AlF-NOTA-FAPI-04 PET/CT,2.2%only through[18F]FDG PET/CT.Among them,3 stations of inflammatory lymph nodes were not detected on[18F]AlF-NOTA-FAPI-04 PET,but could be seen on[18F]FDG PET,and 5 stations of metastatic lymph nodes could be seen on[18F]AlF-NOTA-FAPI-04 PET,but not on[18F]FDG PET.Accordingly,the diagnostic accuracy of[18F]AlF-NOTA-FAPI-04 and[18F]FDG for thoracic metastatic lymph nodes was 100%and 69.23%,P=0.002.Conclusion:[18F]AlF-NOTA-FAPI-04 is superior to[18F]FDG in identifying metastatic lymph nodes and metastatic foci in patients with advanced lung cancer,and its use may contribute to tumor staging.61 patients with primary lung cancer confirmed by histopathology were prospectively enrolled.[18F]AlF-NOTA-FAPI-04 PET/CT static scan was performed before treatment to evaluate the uptake level of[18F]A1F-NOTA-FAPI-04,and to explore the expression characteristics of FAP in different pathological types,primary lesions and metastatic lesions.Results:61 patients with lung cancer,including adenocarcinoma(ADC,n=30),squamous cell carcinoma(SCC,n=17)and small cell lung cancer(SCLC,n=14),were evaluated with 61 primary lesions and 199 metastatic lesions.The results showed that there was no significant difference in the uptake of[18F]AlF-NOTA-FAPI-04 by primary lesions of ADC,SCC and SCLC(P=0.198),and there was no significant difference between primary and metastatic lesions of the same pathological type(P>0.05).However,there were significant differences in uptake among different pathological types of metastatic tumors(P<0.001).The SUVmax of metastatic lymph nodes was the highest in SCC,followed by ADC and SCLC(P<0.001).The SUVmax of bone metastasis was also the highest in SCC,followed by ADC and SCLC,but there was no significant difference between ADC and SCLC.The SUVmax of other organ metastases in SCC group was higher than that in ADC group,but there was no significant difference between SCC group and SCLC group,ADC group and SCLC group.The uptake rate of bone metastasis in SCC and ADC was significantly higher than that in lymph nodes and other organs(P<0.05),but not in SCLC.Conclusion:[18F]AlF-NOTA-FAPI-04 PET/CT imaging shows that there is a difference in the expression of FAP in metastatic lesions of lung cancer,especially in bone metastases,so it is considered to be of potential value in differential diagnosis of different pathological types of lung cancer.Retrospective analysis of myocardial[18F]AlF-NOTA-FAPI-04 uptake in 13 patients with esophageal squamous cell carcinoma(ESCC)before and during radiotherapy(36～50Gy).The diagnostic value of[18F]AlF-NOTA-FAPI-04 PET/CT in radiation-induced heart injury was verified by constructing a rat model of radiation-induced heart injury.Female Wistar rats were used to give a single high dose of 50Gy to the apical region.The rats with radiation-induced heart injury were scanned by[18F]AlF-NOTA-FAPI-04 PET/CT every week until the 12th week,and the left ventricular ejection fraction(LVEF)was measured by MRI at the 3rd,5th and 8th week.At the fifth week of radiotherapy,the rats with radiation-induced heart injury were examined by dynamic PET scanning,autoradiography,blocking test and 18F-FDG PET/CT,and pathologically verified by HE,Masson and IHC tissue staining.Results:In patients with ESCC who received concurrent chemoradiotherapy,the maximum uptake of[18F]AlF-NOTA-FAPI-04 in the myocardium irradiated in the middle of radiotherapy was higher than that before radiotherapy,4.01(2.17 to 4.86)vs 1.87(1.30 to 2.67),P=0.109.The uptake of[18F]AlF-NOTA-FAPI-04 in the myocardium of rats with radiation-induced heart injury was significantly higher than that in the myocardium without radiotherapy at 2 weeks(0.77±0.04%ID/mL vs 0.62±0.03%ID/mL,P<0.01).The myocardial uptake of[18F]AlF-NOTA-FAPI-04 reached the highest level at the 5th week after radiotherapy,and the uptake in the involved myocardium was significantly higher than that in the distal myocardium without radiotherapy(1.38±0.15%ID/mL vs 0.44±0.03%ID/mL,P<0.01).At the same time,no obvious[18F]FDG uptake was found in the injured myocardium,which was confirmed by autoradiography,Masson staining and IHC staining.There was no significant difference in left ventricular ejection fraction measured by MRI between the radiotherapy group and the control group at the 3rd week(71.2±1.29%vs 72.3±2.47%,P>0.05).Until the 8th week,the left ventricular ejection fraction of the radiotherapy group was lower than that of the control group(56.4±2.72%vs 65.0±3.14%,P<0.05).Conclusion:Myocardial uptake of[18F]AlF-NOTA-FAPI-04 in patients with ESCC tends to increase during radiotherapy compared with that before radiotherapy,and is consistent with the area of high dose cardiac irradiation.It is proved by animal experiments that[18F]AlF-NOTA-FAPI-04 PET/CT can noninvasively detect radiation-induced cardiac injury,and it is earlier than the beginning of the decrease of left ventricular ejection fraction.This study suggests that[18F]AlF-NOTA-FAPI-04 might be used as a PET imaging agent for the diagnosis of radiation-induced cardiac injury. |
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