Supplementary Materialsjnm234542SupplementalData. 800CW binds GLP-1R having a half-maximal inhibitory concentration of 3.96 nM. The tracer accumulates in CHL-GLP-1R xenografts. Subcutaneous CHL-GLP-1R xenografts were visualized using in vivo NIR fluorescence imaging. The tracer accumulates specifically in the pancreatic islets of mice, and a clear fluorescent signal was detected in the pancreas of mini pigs. Conclusion: These data provide the first in vivo evidence of the feasibility of targeted fluorescence imaging of GLP-1RCpositive lesions. Intraoperative lesion delineation using exendin-4-IRDye 800CW could benefit open as well as laparoscopic surgical procedures for removal of insulinomas and focal lesions in CHI. = 6 mice per group; 3, 10, 30, and 100 g of exendin-4-IRDye 800CW). Control mice (= 4) were injected with PBS with 0.5% bovine serum albumin only. After 4 h, the mice were Mercaptopurine sacrificed by CO2 asphyxiation, and blood and CDKN2B organs were removed and collected in MagNA Lyser tubes (F. Hoffmann- La Roche Ltd.), which were weighed before and after organ collection. The circulation time of 4 h was based on our previous experience with radiolabeled exendin tracers (10). Radioimmunoprecipitation assay lysis buffer (500 L; 50 mM [hydroxymethyl]aminomethane-hydrochloride, pH 7.4, with 150 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid, 1% Triton X-100 [Dow Chemical Co.], and 1% sodium dodecyl sulphate) was added to each tube. Organs were then homogenized using a MagNA Lyser with repeated cycles of 6,000 rpm for 25 s and cooling on ice for 1 min after each cycle. Organ homogenates of control mice were used to produce standard curves for each organ. Organ homogenates (100 L) and the standards were transferred in triplicate to a black flat-bottom 96-well plate. Fluorescence was measured using an Infinite Pro M200 (excitation, 750 nm; emission, 795 nm). Standard curves were created, and tracer uptake in the various organs was calculated using Excel. Tracer uptake in each organ type was corrected for the weight of the dissected organ. To determine the specificity of tumor uptake, an additional biodistribution experiment was performed with 2 groups of mice (= 6 per group), which were injected with 3 g of exendin-4-IRDye 800CW, with coinjection of 150 g of unlabeled exendin-4 in 1 of the groups. We validated the biodistribution of the fluorescent tracer using a dual-labeled version of the exendin tracer DTPA-exendin-4 (piCHEM), labeled with both 111In and Cy5.5, as previously described (21). We calculated the tracer uptake in the organs using fluorescence as well as radioactive steps and compared the resulting values with each other (Supplemental Fig. 1). In Vivo Fluorescence Imaging of GLP-1RCPositive Tumors To show the feasibility of visualizing GLP-1RCpositive tumors using in vivo fluorescence imaging, BALB/c nude mice bearing subcutaneous CHL-GLP-1R xenografts were injected intravenously with 3 g of exendin-4-IRDye 800CW (= 3 per group). One group of mice was coinjected with an excess (150 g) of unlabeled exendin-4. After 4 h, fluorescence imaging was performed using an IVIS Lumina closed-cabinet fluorescence scanner (Caliper LifeSciences) (excitation, 745 nm; autofluorescence correction excitation, 640 nm; both measured with the indocyanine green filter). After resection of the tumor lesions, the mice were imaged again. Subsequently, the mice were Mercaptopurine dissected to remove the pancreas. Pancreata had been fixed right away Mercaptopurine in 4% formalin and inserted in paraffin for fluorescence microscopy and immunohistochemistry. Fluorescence Immunohistochemistry and Microscopy Parts of 4 m had been trim at 2 amounts, 100 m aside. One portion of each known level was deparaffinated in xylene for 2 min, and fluorescence imaging was performed using an Odyssey CLx flatbed fluorescence scanning device (800-nm channel; documenting period, 1C5 min; concentrate, 1.0 mm; Li-COR Biosciences). Subsequently, these areas had been stained for insulin as previously defined (22). Consecutive areas had been employed for fluorescence microscopy. After deparaffination Mercaptopurine in xylene (two times for 5 min), the cell nuclei had been stained with.