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 5×106 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 low frequency 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 high frequency 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.

Human glandular organoid formation in murine engineering chambers after collagenase digestion and flow cytometry isolation of normal human breast tissue single cells

“…While there are now patient‐derived tumor xenograft models to study BC growth and metastasis using either human (Rowan et al, ; Dahibawkar et al, ; Fritsche et al, ; Sp et al, ) or mouse‐derived cancer cell lines (Singh et al, ; Zou et al, ), there is no mouse model for studying normal breast tissue biology such as that required for examining high MD‐associated BC risk. The complex cellular interactions that underpin MD and BC cannot be easily created in an in vitro setting, thus our pilot study demonstrated the value of our murine biochamber as a model to assess altered cellular function in HMD and LMD human mammary tissues.…”

Recent Experiences and Advances in Contrast-Enhanced Subharmonic Ultrasound

“…This setup was then used to estimate tumor interstitial pressure in swine [ 17 ] and angiogenic marker expression in a murine breast cancer model [ 18 ] with Definity. Rat tumors were also scanned with a Vevo 2100 (Visualsonics, Toronto, Ontario, Canada) using a 24 MHz probe during injection of Definity [ 19 ]. Similar to work by Needles et al [ 6 ], subharmonic images were constructed by the postprocessing acquired radiofrequency data on the Vevo 2100 using filters at the subharmonic [ 19 ].…”

“…Rat tumors were also scanned with a Vevo 2100 (Visualsonics, Toronto, Ontario, Canada) using a 24 MHz probe during injection of Definity [ 19 ]. Similar to work by Needles et al [ 6 ], subharmonic images were constructed by the postprocessing acquired radiofrequency data on the Vevo 2100 using filters at the subharmonic [ 19 ]. Tumor fractional vascularity was then calculated using both high frequency ( f 0 = 24 MHz) and low frequency ( f 0 = 8 MHz) SHI and compared to angiogenic marker expression on pathology, with the strongest correlation observed between high frequency SHI and vascular endothelial growth factor expression [ 19 ].…”

“…Similar to work by Needles et al [ 6 ], subharmonic images were constructed by the postprocessing acquired radiofrequency data on the Vevo 2100 using filters at the subharmonic [ 19 ]. Tumor fractional vascularity was then calculated using both high frequency ( f 0 = 24 MHz) and low frequency ( f 0 = 8 MHz) SHI and compared to angiogenic marker expression on pathology, with the strongest correlation observed between high frequency SHI and vascular endothelial growth factor expression [ 19 ]. These results demonstrate the feasibility of SHI on a variety of ultrasound scanners using a variety of frequency pairings.…”

Noninvasive Estimation of Tumor Interstitial Fluid Pressure from Subharmonic Scattering of Ultrasound Contrast Microbubbles

“…Since tissues can also generate the second, third harmonic signals, etc., tissue signals by extracting higher harmonics cannot be completely suppressed [11]. While in the tissue it is usually difficult to generate a subharmonic scattering, subharmonic imaging and three-dimensional subharmonic imaging with a higher CTR have been developed and applied to multiple organs and tumor imaging [12][13][14][15].…”