Optimizing ultrasound molecular imaging of secreted frizzled related protein 2 expression in angiosarcoma.
Secreted frizzled related protein 2 (SFRP2) is a tumor endothelial marker expressed in angiosarcoma. Previously, we showed ultrasound molecular imaging with SFRP2-targeted contrast increased average video pixel intensity (VI) of angiosarcoma vessels by 2.2 ± 0.6 VI versus streptavidin contrast. We hypothesized that redesigning our contrast agents would increase imaging performance. Improved molecular imaging reagents were created by combining NeutrAvidin™-functionalized microbubbles with biotinylated SFRP2 or IgY control antibodies. When angiosarcoma tumors in nude mice reached 8 mm, time-intensity, antibody loading, and microbubble dose experiments optimized molecular imaging. 10 minutes after injection, the control-subtracted time-intensity curve (TIC) for SFRP2-targeted contrast reached a maximum, after subtracting the contribution of free-flowing contrast. SFRP2 antibody-targeted VI was greater when contrast was formulated with 10-fold molar excess of maleimide-activated NeutrAvidin™ versus 3-fold (4.5 ± 0.18 vs. 0.32 ± 0.15, VI ± SEM, 5 x 106 dose, p < 0.001). Tumor vasculature returned greater average video pixel intensity using 5 x 107 versus 5 x 106 microbubbles (21.2 ± 2.5 vs. 4.5 ± 0.18, p = 0.0011). Specificity for tumor vasculature was confirmed by low VI for SFRP2-targeted, and control contrast in peri-tumoral vasculature (3.2 ± 0.52 vs. 1.6 ± 0.71, p = 0.92). After optimization, average video pixel intensity of tumor vasculature was 14.2 ± 3.0 VI units higher with SFRP2-targeted contrast versus IgY-targeted control (22.1 ± 2.5 vs. 7.9 ± 1.6, p < 0.001). After log decompression, 14.2 ΔVI was equal to ~70% higher signal, in arbitray acoustic units (AU), for SFRP2 versus IgY. This provided ~18- fold higher acoustic signal enhancement than provided previously by 2.2 ΔVI. Basing our targeted contrast on NeutrAvidin™-functionalized microbubbles, using IgY antibodies for our control contrast, and optimizing our imaging protocol significantly increased the SFRP2-specific signal returned from angiosarcoma vasculature, and may provide new opportunities for targeted molecular imaging.
Joint Department of Biomedical Engineering, North Carolina State University, and The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.|Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
James K
JK
Tsuruta
Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
Nicholas P
NP
Schaub
Joint Department of Biomedical Engineering, North Carolina State University, and The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
Juan D
JD
Rojas
Joint Department of Biomedical Engineering, North Carolina State University, and The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
Jason
J
Streeter
Department of Surgery, Medical College of South Carolina, Charleston, South Carolina, United States of America.
Nancy
http://orcid.org/0000-0003-0246-9892
N
Klauber-DeMore
Joint Department of Biomedical Engineering, North Carolina State University, and The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.|Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, Chapel Hill, North Carolina, United States of America.
Paul
P
Dayton
D000818:Animals; D001360:Avidin / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000378:metabolism; D045744:Cell Line, Tumor; D003287:Contrast Media / Q000378:metabolism; D006394:Hemangiosarcoma / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism / Q000648:ultrastructure*; D008819:Mice, Nude; D045423:Microbubbles; D057054:Molecular Imaging / Q000379:methods*; D009368:Neoplasm Transplantation; D014463:Ultrasonography / Q000379:methods*; D000094742:Secreted Frizzled-Related Proteins
D001360:Avidin; D014408:Biomarkers, Tumor; D003287:Contrast Media; D008565:Membrane Proteins; C109618:SFRP2 protein, human; D000094742:Secreted Frizzled-Related Proteins; C093483:neutravidin