ObjectivesDifferent methods for obtaining tumor neovascularity parameters based on immunohistochemical markers were compared to contrast‐enhanced subharmonic imaging (SHI).MethodsEighty‐five 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, North Billerica, MA) was injected, and SHI was performed using a modified Sonix RP scanner (Analogic Ultrasound, Richmond, British Columbia, Canada) with a L9‐4 linear array (transmitting/receiving frequencies, 8/4 MHz). Afterward, specimens were stained for endothelial cells (CD31), vascular endothelial growth factor (VEGF), and cyclooxygenase 2 (COX‐2). Tumor neovascularity was assessed in 4 different ways using a histomorphometry system (×100 magnification: (1) over the entire tumor; (2) in small sub–regions of interest (ROIs); (3) in the tumor periphery and centrally; and (4) in 3 regions of maximum marker expression (so‐called hot spots). Results from specimens and from SHI were compared by linear regression.ResultsFifty‐four rats (64%) showed tumor growth, and 38 were successfully imaged. Subharmonic imaging depicted the tortuous morphologic characteristics of tumor neovessels and delineated small areas of necrosis. The immunohistochemical markers did not correlate with SHI measures over the entire tumor area or over small sub‐ROIs (P > .18). However, when the specimens were subdivided into central and peripheral regions, COX‐2 and VEGF correlated with SHI in the periphery (r = −0.42; P = .005; and r = −0.32; P = .049, respectively).ConclusionsWhen comparing quantitative contrast measures of tumor neovascularity to immunohistochemical markers of angiogenesis in xenograft models, ROIs corresponding to the biologically active region should be used to account for tumor heterogeneity.

This project compared quantifiable measures of tumor vascularity obtained from contrast-enhanced high frequency (HF) and low frequency (LF) subharmonic ultrasound imaging (SW) 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 x 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: 180 p1/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 8/4 MHz 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 24 MHz. The radiofrequency data was filtered using a 4th order IIR Butterworth bandpass filter (11-13 MHz) 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. (C) 2015 Elsevier B.V. All rights reserved.

Ultrasound imaging of tumor angiogenesis are often conducted with pathological markers as the reference standard. There is however, no standard for how to analyze specimen markers and compare them to imaging parameters. Hence we compared different methods for obtaining tumor neovascularity parameters based on immunohistochemical markers to contrast-enhanced subharmonic ultrasound imaging (SHI). Eighty-five athymic, nude, female rats were implanted with 5x106 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 36μl) and SHI was performed using a modified Sonix RP scanner (Analogic Ultrasound, Richmond, Canada) with a L9-4 linear array (transmitting/receiving frequencies 8/4MHz). After the experiments, specimens were stained for endothelial cells (CD31), vascular endothelial growth factor (VEGF), and cyclooxygenase-2 (COX-2). Tumor neovascularity was assessed in 4 different ways using a histomophometry system based on a Labophot-2 microscope (Nikon, Melville, NJ) at 100x magnification 1) over the entire tumor 2) in small sub-regions of interest (ROIs), 3) in the tumor periphery (within 2 mm of the margin) and centrally and 4) in 3 regions of maximum marker expression (so called hotspots). Results from specimens and from SHI were compared using linear regression. Of the 85 rats implanted 54 (64%) exhibited tumor growth and 38 were successfully imaged. SHI depicted the tortuous morphology of tumor neovessels and delineated small areas of necrosis. The immunohistochemical markers did not correlate with SHI measures of tumor vascularity when assessed over the entire tumor area or over small sub-ROIs (p>0.18). However when the specimens were sub-divided into a central and a peripheral region, COX-2 and VEGF correlated with SHI in the periphery (r=-0.42; p=0.005 and r=-0.32; p=0.049, respectively). When comparing quantitative contrast measures of tumor neovascularity to immunohistochemical markers of angiogenesis in xenograft models sub-ROIs corresponding to the biologically active region (i.e., the tumor periphery) should be used to account for tumor heterogeneity.

Ultrasound imaging studies of tumor angiogenesis are often conducted with pathological markers as the reference standard. There is, however, no established standard for how to analyze specimen markers and compare them to imaging parameters. Hence, this study compared different methods for obtaining tumor neovascularity parameters based on immunohistochemical markers to contrast-enhanced subharmonic ultrasound imaging (SHI). Breast cancer cells (MDA-MB-231) were implanted into the mammary fat pad of 85 athymic, nude, female rats. An ultrasound contrast agent was injected and SHI was performed using a Sonix RP scanner (Analogic Ultrasound, Richmond, BC, Canada) transmitting and receiving at 8 and 4 MHz, respectively. Tumor specimens were sliced corresponding to the imaging planes and stained for endothelial cells (CD31), vascular endothelial growth factor (VEGF), and cyclooxygenase-2 (COX-2). Tumor neovascularity was assessed in 4 different ways 1) over the entire tumor, 2) in small sub-ROIs, 3) in the tumor periphery and centrally, and 4) in regions of maximum marker expression (so called hotspots). Of the 85 rats implanted 54 (64 %) exhibited tumor growth and 38 were successfully imaged. SHI depicted the tortuous morphology of tumor neovessels and delineated small areas of necrosis. SHI measures of tumor vascularity did not correlate with the immunohistochemical markers when assessed over the entire tumor area or over small sub-ROIs (p > 0.18). However, when the specimens were sub-dived into a central and a peripheral region, COX-2 and VEGF correlated with SHI in the periphery (r = -0.42; p = 0.005 and r = -0.32; p = 0.049, respectively). In conclusion, when comparing quantitative contrast measures of tumor neovascularity to immunohistochemical markers of angiogenesis in xenograft models it appears that sub-ROIs corresponding to the biologically active region (i.e., the tumor periphery) should be used to account for tumor heterogeneity and development.