Nystatin facilitates endostatin uptake by tumors and tumor blood vessels. (A-B) Bioluminescence imaging of tumors from A549 tumor–bearing mice treated with 20 mg/kg Rh-endostatin combined with or without 4 mg/kg nystatin. Rh-endostatin fluorescence imaging of excised tumors at 3 hours was shown (n = 4/group). Rhodamine fluorophore excitation (532 nm) and emission (575 nm) filter sets were used. Rh-endostatin signal was quantified as the average fluorescence intensity (B). (C) A549 tumor–bearing mice (n = 4/group) were injected with ¹²⁵I-endostatin [specific radioactivity, 1 μCi/μg (0.037 Bq/μg); 0.25 mg/kg] combined with or without 4 mg/kg nystatin. ¹²⁵I-endostatin uptake levels in indicated tissues at 3 hours were calculated as the percentage of the injected dose per gram of tissue wet weight (%ID/g). (D-E) Biotin-endostatin (4 mg/kg) was intravenously injected into A549 tumor–bearing mice combined with or without 4 mg/kg nystatin. Tumors (n = 4/group) were resected at 3 hours and examined by immunofluorescence using a Nikon A1 microscope (20×/0.75 NA air objective). Biotin-endostatin and tumor blood vessels were detected by TRITC-streptavidin and anti-CD31 staining (FITC), respectively. Colocalization percentage of endostatin with CD31 (E) was defined as the area of colocalized biotin-endostatin (red pixels) within CD31-possitive blood vessels (green pixels) divided by the total area of blood vessels. *P < .05 and **P < .01. Error bars represent SEMs.