¹⁸F-DPA-714and¹⁸F-Alfatide â…¡ MicroPET For Inflammation Imaging And Differentiation With Tumor

Project1Longitudinal microPET imaging of muscular inflammation using TSPO targeted tracer18F-DPA-714Aim:This project aims at evaluating the value of PET inflammation imaging using the macrophage translocator protein (TSPO) targeted PET imaging tracer18F-DPA-714, and to study the longitudinal uptake changes of18F-DPA-714into the inflammatory muscles after inflammation modeling.Methods:Mouse macrophage cell line RAW264.7was used to study cell-up take and cell efflux of18F-DPA-714with or without blocking agent PK11195. Muscular inflammation models were developed using FVB mice by intramuscular injection of20μl turpentine oil. Longitudinal microPET imaging using18F-DPA-714was performed in inflammation model mice1,3,6,10,14,19and26day(s) after turpentine oil injection, and region of interest (ROI) methods were used to analysis the inflammatory muscle uptake values in different times of inflammation. On day3after turpentine oil injection,1-h dynamic PET imaging acquisition was performed with PK11195injected at25min during data acquisition, and the dynamic-curve of the inflammatory muscle uptake of18F-DPA-714was studied to test its specificity to inflammation. Inflammatory muscle tissues of day1and day6after turpentine injection was collected and staining for CD68makers expressed on macrophages. Autoradiography was performed on on inflammation tissues collected on day1,6,22after turpentine oil injection. The correlation of microPET imaging and immuno fluorescence staining and autoradiography was analyzed.Results:Macrophage uptake of18F-DPA-714increased with time until1hour of incubation with tracer, and this uptake could be blocked by co-incubation with K11195. Cell efflux of18F-DPA-714experiment saw21.55±2.05%AD cell retention1hour after medium change, and this number dropped to9.04±1.12%AD after2 hours. For microPET imaging of inflammation model mice using18F-DPA-714, inflammatory uptake of tracer reached peak level at day6after turpentine oil injection (4.02±0.64%ID/g), and it decreased to2.77±0.64%ID/g at day26after inflammation modeling. For the displacement dynamic imaging at day3after turpentine oil injection, inflammatory uptake of18F-DPA-714could be displaced by PK11195injection at25-minute during PET acquisition. Autoradiography of inflammatory tissue binding of18F-DPA-714showed higher level on tissues obtained on day6compared to day1and day22. Immunofluorescence staining of macrophage marker CD68indicated more CD68expression on inflammation samples collected on day6than day1. Staining results were in accordance with PET imaging results.Conclusions:18F-DPA-714is a macrophage TSPO specific PET imaging tracer, and could be used for PET imaging of macrophage infiltration into inflammatory sites. Chronic inflammation had higher18F-DPA-714accumulation than acute inflammation, indicating more macrophage infiltration in the chronic phase of inflammation. Project2Longitudinal microPET imaging of muscular inflammatory angiogenesis using18F-Alfatide IIAim:By using18F-Alfatide Ⅱ (18F-A1F-NOTA-E[PEG4-c(RGDflc)]2), a PET tracer targeting integrin αvβ3for angiogenesis, this project aims at evaluating the value of18F-Alfatide Ⅱ microPET imaging of inflammatory angiogenesis, and the dynamic changes of tracer uptake by inflammations during acute phase and chronic phase of inflammatory pro gression.Methods:Mouse macrophage cell line RAW264.7was used to study cell uptake of18F-Alfatide II. Muscular inflammation models were developed in FVB mice by intramuscular injection of20μ1turpentine oil Longitudinal microPET imaging using18F-Alfatide II was performed in inflammation model mice on day1,2,4,8,12,16and26after turpentine oil injection, and region of interest (ROT) methods were used to analysis the inflammatory muscle uptake values in different times of inflammation. Blocking PET imaging by pre-injection of unradiolabled RGD peptide (10mg/kg) was performed on day9after inflammation model development to test the specificity of tracer uptake in the inflammatory foci. Inflammatory muscle tissues of day1and day12after turpentine oil injection was collected and stained for CD31makers expressed on vessel endothelial cells. Staining results were compared with PET imaging results.Results:Macrophage uptake of18F-Alfatide Ⅱ was at low level, and the2-hour uptake was only0.18±0.03%AD. For microPET imaging of inflammatory angiogenesis,18F-Alfatide II uptake of inflammation increased level with time from day2to day12after turpentine oil injection, and the uptake peaked at day12with uptake value of1.87±0.35%ID/g. For blocking imaging, mice with cold RGD pre-injection showed significant lower uptake level at the inflammatory foci compared with mice without cold RGD pre-injection (1.66±0.04v.s.0.77±0.16%ID/g, P=7.33x10-4). Vasculature staining of CD31on inflammatory muscle samples obtained on day12after turpentine oil injection showed higher immunofluorescence signal compared to samples collected on day1, which was in accordance with microPET imaging result. Conclusions:There were few expression of integrin αvβ3on macrophages, and inflammation uptake of18F-Alfatide Ⅱ was due to inflammatory angiogenesis. PET imaging using18F-Alfatide Ⅱ could dynamically and accurately reflect the process of inflammatory angiogenesis. Chronic inflammation had higher uptake level of18F-Alfatide Ⅱ than acute phase, indicating more angiogenesis formation during chronic stage of inflammation than acute stage. Project318F-DPA-714and18F-Alfatide Ⅱ PET imaging of inflammation after macrophage depletion and its effect on inflammatory angiogenesisAim:Using18F-DPA-714and18F-Alfatide II PET imaging, this project aims at evaluating the tracers’uptake changes after macrophage depletion intervention, and analyzing the effect of macrophage depletion on inflammatory angiogenesis on a mouse model of muscular inflammation.Methods:Muscular inflammation models were developed using FVB mice by intramuscular injection of20μl turpentine oil. Model mice were divided into three groups randomly (n=4in each group). Control mice received no macrophage depletion intervention. Mice in group1and group2received1.4mg (200μl) Clophosome i.v. injection on day3after turpentine injection and then0.7mg(100μl) iv. injection every2-3days (Plan1), while mice in group3received1.4mg (200μl) Clophosome iv. injection2days before turpentine oil injection and then0.7mg (100μl) iv. injection every2-3days (Plan2).18F-DPA-714microPET imaging was performed on day6and day10after turpentine injection for group1and was compared to model mice without macrophage depletion intervention to see the difference of inflammation uptake of18F-DPA-714. Mice in group2and group3went through18F-Alfatide II microPET imaging on day12after turpentine injection to compare the difference of inflammation up take of18F-Alatide Ⅱ. Inflammatory tissue samples with or without macrophage depletion intervention were collected on day6and day12, and were analyzed for CD68or CD31makers for macrophages or vasculature using immuno fluorescence staining. Staining results were compared with PET imaging results.Results:Compared with model mice without macrophage depletion intervention, model mice under macrophage depletion plan1showed significant lower18F-DPA-714uptake in the inflammatory muscles on day6and day10after turpentine injection (Day6:3.30±0.18v.s.4.02±0.64%ID/g, P=0.02; Day10:3.16±0.32v.s.3.70±0.43%ID/g, P=0.03). This difference was not significant between inflammation control mice and mice receiving macrophage depletion Plan1on day14 after turpentine oil injection (3.03±0.69v.s.3.50±0.43%ID/g, P=0.19). For model mice receiving macrophage depletion Plan2, the inflammatory uptake of18F-Alfatide Ⅱ on day12was significantly lower compared to mice receiving macrophage depletion Plan1(0.98±0.19v.s.1.49±0.13%ID/g, P=2.97×10-4). Immunofluorescence staining of CD68and CD31both showed lower levels of markers expression on inflammation samples from mice receiving macrophage depletion strategies, which were in accordance with microPET imaging results.Conclusions:Macrophage infiltration into inflammatory muscles was decreased after Chlophosome intervention, and could be evaluated using18F-DPA-714PET imaging. Macrophage depletion could inhibit inflammatory angiogenesis in inflammation foci, and could be analyzed by18F-Alfatide II PET imaging. Project418F-DPA-714microPET imaging and differentiation of tumor and inflammationAim:This project aims at evaluating the value of18F-DPA-714microPET imaging in differentiating tumor and inflammation using animal models of tumors and muscular inflammation.Methods:Mice xenograft tumor models of HT29, A549, U87MG, INS-1,4T1were developed by subcutaneous injection of5×106cells (n=4-6). Muscular inflammation models were developed using FVB mice by intramuscular injection of20μl turpentine oil.18F-DPA-714microPET was performed when tumors grew to100-300mm3about3-5weeks after inoculation. Inflammation model mice also went through18F-DPA-714microPET imaging on different time-points following turpentine injection (day1,3,6,10,14,19and26). ROIs were drawn on tumors as well as inflammation muscles of model mice and its uptake values were compared. TSPO staining of macrophages were performed in tumor tissue sections and inflammation muscle samples. Staining results were compared with microPET imaging results.Results:Tumor uptakes of18F-DPA-714were at low levels, and the uptake value of18F-DPA-714for HT29, A549, U87MG, INS-1,4T1tumors were0.91±0.08%ID/g,0.46±0.28%ID/g,1.69±0.67%ID/g,1.13±0.33%ED/g,1.22±0.55%ID/g, respectively. Inflammatory muscle uptakes of18F-DPA-714were higher than the average tumor uptake on day3,6,10,14,19and26, but not day1, after turpentine oil injection TSPO immunofluorescence staining showed higher level of TSPO expression on inflammatory muscle sample on day6than on all five tumor sections, which was in accordance with microPET imaging resultConclusions:Inflammation uptake of18F-DPA-714was significantly higher than tumor uptake except for acute inflammation on day1.18F-DPA-714owns the potential of differentiating tumors and inflammation uptakes from PET images.