Ultrosound Molecular Imaging Of Tumor Angiogenesis With BST2Targeted Microbubble Contrast Agent

Objective: Ultrasound molecular imaging is an emerging technique forsensitive detection of intravascular targets, Molecular imaging ofangiogenesis has strong potential for both clinical use and as a researchtool in tumor biology and the development of anti-angiogenic therapies.Our objective is to investigate the feasibility of bone marrow stromalantigen2(BST2) as a potential target for tumor angiogenesis, BST2targeted microbubbles as ultrasound contrast agents were prepared and invitro targeting ability were evaluated in mouse vascular endothelial cell(bEnd.3); The microbubbles and the imaging methods were evaluated in amouse model of prostate cancer in vivo, to find new targets for thedetection of tumor angiogenesis and tumor therapy.Methods: By immunocytochemical staining the expression level ofBST2protein in the mouse vascular endothelial cells was analyzed, andCD31as positive control. The biotinylated anti-BST2monoclonalantibody was used to prepare the targeted microbubbles through the biotin-avidin bridge. The resulting BST2-targeted microbubbles wereobserved under the light microscope and characterized by AccuSizer780Aparticle size analyzer. The targeting specificity and attachment capabilityof the BST2targeted microbubbles to the bEnd.3cells were assessed invitro, and non-targeted microbubbles as control. In vivo, twenty male micewere inoculated subcutaneously with the mice prostate cancer cells RM-1to establish the mice tumor angiogenesis models; The experimental micewere divided into: the BST2targeted microbubbles group andnon-targeted microbubbles group; The two groups were injected with thesame concentration of microbubbles, and then Ultrasound imaging wasperformed with a Sequoia512system, to investigate the capability ofBST2-targeted microbubbles adhere to the vascular endothelial cells;Fluorescein-labeled Lycopersicon esculentum Lectin injected into thetail vein of mice with prostate RM-1tumors, After10minutes,Fluorescein-labeled BST2microbubbles were injected intravenously(50μg/mouse) in order to delineate the vascular endothelium. Animalswere sacrificed5minutes after lectin administration, and tumors wereharvested and snap frozen. Control tumors were harvested from animalsthat were treated with lectin, but did not receive injection of fluorescencelabeled BST2microbubbles；Used the immunohistochemical staining todetect the BST2protein expression in mice tumor angiogenesis. Results: Immunocytochemical staining showed that the expression ofBST2protein in the mouse vascular endothelial cells, and its expressionlocation and intensity is very similar to CD31; The in vitro cell adhesiondemonstrate that the BST2-targeted microbubbles were able to specificallybind to the surface of vascular endothelial cells, and significantly higherthan that of non-targeted microbubbles. The ultrasound molecular imagingquantitative analysis showed that in the tumors of mice injected withBST2-targeted microbubbles, the signal intensity at7min, remaining49.75%of that at30s, while the injected non-targeted microbubblesremaining12.28%. So, the BST2-targeted microbubbles group thesignificant enhancement of signal intensity was4-fold higher than that ofnon-targeted microbubbles group (P<0.01). Representative Fluorescencemicroscopy images of the vasculature in tumors from Fluorescence labeledBST2microbubble treated animals demonstrate the accumulation oftargeted within the vasculature. No microbubbles were observed outsidethe vasculature demarcated by FITC-lectin staining, suggesting thatmicrobubbles are binding a target on the luminal endothelium of the tumor.Immunohistochemical staining showed that the expression of BST2proteinin the mouse vascular endothelial cells.Conclusion: The BST2targeted microbubbles were successful preparedthrough the biotin-avidin bridge and able to specifically adhere to vascularendothelial cells, meanwhile the adhesion rate is significantly higher than that of non-targeted microbubbles. The ultrasound molecular imagingquantitative analysis showed that the BST2-targeted microbubbles groupthe significant enhancement of signal intensity was4.05-fold higher thanthat of non-targeted microbubbles group. This finding was furtherconfirmed through Immunohistochemical staining assay. Therefore, theBST2-targeted microbubbles can be used to detect mice tumor vascularendothelial cells, and is expected to be used as a new target of ultrasonicimaging to detect tumor angiogenesis, for clinical tumor diagnosis andprognosis treatment.