title: High and low frequency subharmonic imaging of angiogenesis in a murine breast cancer model.
abstract: This project compared quantifiable measures of tumor vascularity obtained from contrast-enhanced high frequency (HF) and low frequency (LF) subharmonic ultrasound imaging (SHI) to 3 immunohistochemical markers of angiogenesis in a murine breast cancer model (since angiogenesis is an important marker of malignancy and the target of many novel cancer treatments). Nineteen athymic, nude, female rats were implanted with 5×10(6) breast cancer cells (MDA-MB-231) in the mammary fat pad. The contrast agent Definity (Lantheus Medical Imaging, N Billerica, MA) was injected in a tail vein (dose: 180μl/kg) and LF pulse-inversion SHI was performed with a modified Sonix RP scanner (Analogic Ultrasound, Richmond, BC, Canada) using a L9-4 linear array (transmitting/receiving at 8/4MHz in SHI mode) followed by HF imaging with a Vevo 2100 scanner (Visualsonics, Toronto, ON, Canada) using a MS250 linear array transmitting and receiving at 24MHz. The radiofrequency data was filtered using a 4th order IIR Butterworth bandpass filter (11-13MHz) to isolate the subharmonic signal. After the experiments, specimens were stained for endothelial cells (CD31), vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2). Fractional tumor vascularity was calculated as contrast-enhanced pixels over all tumor pixels for SHI, while the relative area stained over total tumor area was calculated from specimens. Results were compared using linear regression analysis. Out of 19 rats, 16 showed tumor growth (84%) and 11 of them were successfully imaged. HF SHI demonstrated better resolution, but weaker signals than LF SHI (0.06±0.017 vs. 0.39±0.059; p<0.001). The strongest overall correlation in this breast cancer model was between HF SHI and VEGF (r=-0.38; p=0.03). In conclusion, quantifiable measures of tumor neovascularity derived from contrast-enhanced HF SHI appear to be a better method than LF SHI for monitoring angiogenesis in a murine xenograft model of breast cancer (corresponding in particular to the expression of VEGF); albeit based on a limited sample size.
authors.affiliation: Department of Radiology, Thomas Jefferson University, Philadelphia, PA 19107, USA; School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA 19104, USA.
authors.forename: Manasi
authors.initials: M
authors.lastname: Dahibawkar
authors.affiliation: Yale University, New Haven, CT 06520, USA.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Forsberg
authors.affiliation: Department of Radiology, Thomas Jefferson University, Philadelphia, PA 19107, USA; School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA 19104, USA.
authors.forename: Aditi
authors.initials: A
authors.lastname: Gupta
authors.affiliation: University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Samantha
authors.initials: S
authors.lastname: Jaffe
authors.affiliation: University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Kelly
authors.initials: K
authors.lastname: Dulin
authors.affiliation: Department of Radiology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Eisenbrey
authors.affiliation: Department of Radiology, Thomas Jefferson University, Philadelphia, PA 19107, USA; School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA 19104, USA.
authors.forename: Valgerdur G
authors.initials: VG
authors.lastname: Halldorsdottir
authors.affiliation: Plymouth-Whitemarsh High School, Plymouth Meeting, PA 19462, USA.
authors.forename: Anya I
authors.initials: AI
authors.lastname: Forsberg
authors.affiliation: Department of Radiology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
authors.forename: Jaydev K
authors.initials: JK
authors.lastname: Dave
authors.affiliation: Department of Radiology, Thomas Jefferson University, Philadelphia, PA 19107, USA; School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA 19104, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Marshall
authors.affiliation: Department of Radiology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
authors.forename: Priscilla
authors.initials: P
authors.lastname: Machado
authors.affiliation: Department of Radiology, Thomas Jefferson University, Philadelphia, PA 19107, USA; Department of Radiologic Sciences, Jefferson College of Health Professions, Thomas Jefferson University, Philadelphia, PA 19107, USA.
authors.forename: Traci B
authors.initials: TB
authors.lastname: Fox
authors.affiliation: Department of Radiology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
authors.forename: Ji-Bin
authors.initials: JB
authors.lastname: Liu
authors.affiliation: Department of Radiology, Thomas Jefferson University, Philadelphia, PA 19107, USA. Electronic address: flemming.forsberg@jefferson.edu.
authors.forename: Flemming
authors.initials: F
authors.lastname: Forsberg
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor / Q000032:analysis; D003287:Contrast Media; D051546:Cyclooxygenase 2 / Q000378:metabolism; D004195:Disease Models, Animal; D005260:Female; D005466:Fluorocarbons; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D008325:Mammary Neoplasms, Experimental / Q000000981:diagnostic imaging*; D051379:Mice; D008819:Mice, Nude; D009389:Neovascularization, Pathologic / Q000000981:diagnostic imaging*; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D015203:Reproducibility of Results; D012815:Signal Processing, Computer-Assisted; D014463:Ultrasonography; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; D003287:Contrast Media; D005466:Fluorocarbons; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D042461:Vascular Endothelial Growth Factor A; D051546:Cyclooxygenase 2; C042852:perflutren
keywords: Breast cancer; Murine xenografts; Signal processing; Subharmonic imaging; Ultrasound contrast agent