Monitoring antivascular therapy in head and neck cancer xenografts using contrast-enhanced MR and US imaging.

BACKGROUND: The overall goal of this study was to non-invasively monitor changes in blood flow of squamous cell carcinoma of the head and neck (SCCHN) xenografts using contrast-enhanced magnetic resonance (MR) and ultrasound (US) imaging. METHODS: Experimental studies were performed on mice bearing FaDu tumors and SCCHN xenografts derived from human surgical tissue. MR examinations were performed using gadofosveset trisodium at 4.7T. Change in T1-relaxation rate of tumors (ΔR1) and tumor enhancement parameters (amplitude, area under the curve-AUC) were measured at baseline and 24 h after treatment with a tumor-vascular disrupting agent (tumor-VDA), 5,6-dimethylxanthenone-4-acetic acid (DMXAA; ASA404) and correlated with tumor necrosis and treatment outcome. CE-US was performed using microbubbles (Vevo MicroMarker®) to assess the change in relative tumor blood volume following VDA treatment. RESULTS: A marked decrease (up to 68% of baseline) in T1-enhancement of FaDu tumors was observed 1 day after VDA therapy indicative of a reduction in blood flow. Early (24 h) vascular response of individual tumors to VDA therapy detected by MRI correlated with tumor necrosis and volume estimates at 10 days post treatment. VDA treatment also resulted in a significant reduction in AUC and amplitude of patient tumor-derived SCCHN xenografts. Consistent with MRI observations, CE-US revealed a significant reduction in tumor blood volume of patient tumor-derived SCCHN xenografts after VDA therapy. Treatment with VDA resulted in a significant tumor growth inhibition of patient tumor derived SCCHN xenografts. CONCLUSIONS: These findings demonstrate that both CE-MRI and CE-US allow monitoring of early changes in vascular function following VDA therapy. The results also demonstrate, for the first time, potent vascular disruptive and antitumor activity of DMXAA against patient tumor-derived head and neck carcinoma xenografts.

In vivo high-frequency, contrast-enhanced ultrasonography of uveal melanoma in mice: imaging features and histopathologic correlations.

PURPOSE: To evaluate the usefulness of in vivo imaging of uveal melanoma in mice using high-frequency contrast-enhanced ultrasound (HF-CE-US) with 2D or 3D modes and to correlate the sonographic findings with histopathologic characteristics. METHODS: Fourteen 12-week-old C57BL6 mice were inoculated into their right eyes with aliquots of 5 × 10(5)/2.5 µL B16LS9 melanoma cells and were randomly assigned to either of two groups. At 7 days after inoculation, tumor-bearing eyes in group 1 (n = 8) were imaged using HF-CE-US to determine the 2D tumor size and relative blood volume; eyes in group 2 (n = 6) were imaged by 3D microbubble contrast-enhanced ultrasound, and the tumor volume was determined. Histologic tumor burden was quantified in enucleated eyes by image processing software, and microvascular density was determined by counting von Willebrand factor-positive vascular channels. Ultrasound images were evaluated and compared with histopathologic findings. RESULTS: Using HF-CE-US, melanomas were visualized as relatively hyperechoic regions. The intraobserver variability of sonographic measurements was 9.65% ± 7.89%, and the coefficient of variation for multiple measurements was 7.33% ± 5.71%. The correlation coefficient of sonographic volume or size and histologic area was 0.71 (P = 0.11) and 0.79 (P = 0.32). The relative blood volume within the tumor demonstrated sonographically correlated significantly with histologic tumor vascularity (r = 0.83; P < 0.001). CONCLUSIONS: There was a positive linear correlation between sonographic tumor measurements and histologic tumor burden in the mouse ocular melanoma model. Contrast-enhanced intensity corresponded with microvascular density and blood volume. HF-CE-US is a real-time, noninvasive, reliable method for in vivo evaluation of experimental intraocular melanoma tumor area and relative blood volume.

Murine echocardiography and ultrasound imaging.

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics. Echocardiography provides a powerful, non-invasive tool to serially assess cardiac morphometry and function in a living animal. However, using this technique on mice poses unique challenges owing to the small size and rapid heart rate of these animals. Until recently, few ultrasound systems were capable of performing quality echocardiography on mice, and those generally lacked the image resolution and frame rate necessary to obtain truly quantitative measurements. Newly released systems such as the VisualSonics Vevo2100 provide new tools for researchers to carefully and non-invasively investigate cardiac function in mice. This system generates high resolution images and provides analysis capabilities similar to those used with human patients. Although color Doppler has been available for over 30 years in humans, this valuable technology has only recently been possible in rodent ultrasound. Color Doppler has broad applications for echocardiography, including the ability to quickly assess flow directionality in vessels and through valves, and to rapidly identify valve regurgitation. Strain analysis is a critical advance that is utilized to quantitatively measure regional myocardial function. This technique has the potential to detect changes in pathology, or resolution of pathology, earlier than conventional techniques. Coupled with the addition of three-dimensional image reconstruction, volumetric assessment of whole-organs is possible, including visualization and assessment of cardiac and vascular structures. Murine-compatible contrast imaging can also allow for volumetric measurements and tissue perfusion assessment.

Real-time quantitation of renal ischemia using targeted microbubbles: in-vivo measurement of P-selectin expression.

INTRODUCTION: Ischemia-reperfusion injury (IRI) results in cellular damage, production of free oxygen radicals, and subsequent apoptosis. During partial nephrectomy, the renal hilum is temporarily clamped to optimize resection of renal masses in a bloodless field. The trade-off for this maneuver is IRI. It is not known definitively how long a period of renal artery clamping will result in irreversible cellular death, nor in which region of the kidney the injury primarily occurs. An established marker of inflammation and ischemic injury is P-selectin, a cell adhesion molecule expressed on endothelial cells and activated platelets. The goal of this project was to use targeted microbubbles outfitted with anti-P-selectin antibodies to quantitate for the first time the microvascular reperfusion injury and regional blood flow in the kidney during IRI. MATERIALS AND METHODS: Using the protocol approved by the Institutional Animal Care and Use Committee (IACUC), 20 renal units obtained from C57/BL6J female mice were studied. The left renal artery and vein were ligated for 30 minutes. Microbubbles coated with anti-P-selectin antibodies were injected after hilar unclamping, and both kidneys were scanned. As a control, perfusion and re-perfusion of the left kidney was performed using pulse-wave Doppler mode. Subsequently, a Vevo 770 micro-ultrasound system with a resolution of 40 mum was used to noninvasively measure the microvascular flow and quantitate targeted microbubbles bound to P-selectin. Negative controls consisted of sham animals and microbubbles coated with isotype serum. Customized software produced digital subtraction video intensity units (VIU), which allowed comparison of the different regions of the kidney. RESULTS: Regional blood flow was measured in three areas: cortex, medulla, and corticomedullary junction (CMJ). In the sham left kidney, the CMJ had the highest blood volume and flow (141.1) compared with renal medulla (43.7) and cortex (100.6) VIU (p < 0.01). After hilar unclamping, blood flow rate to the left kidney decreased from 554 mm/s to 182 mm/s, despite improvement in the color of the kidney from cyanotic to pink. After 30 minutes of ischemia, P-selectin expression increased by 41%, 25%, and 14% in the CMJ, cortex, and the medulla, respectively, compared to controls. P-selectin expression, and therefore the greatest area of ischemic injury, was highest in the CMJ region (432.1) compared with the cortex (369.4) and medulla (86.5) (p < 0.01). CONCLUSION: This pilot study quantitates for the first time in an in-vivo model of IRI that the CMJ region sustains the highest degree of nephron and microvascular damage. This region is the most susceptible to ischemic injury as indicated by 41% increase in expression of P-selectin immediately postunclamping.

Cigarette smoke drives small airway remodeling by induction of growth factors in the airway wall.

BACKGROUND: Small airway remodeling (SAR) is an important cause of airflow obstruction in cigarette smokers with chronic obstructive pulmonary disease, but the pathogenesis of SAR is not understood. OBJECTIVE: To determine whether smoke causes production of profibrotic growth factors in the airway wall. METHODS: We exposed C57Bl/6 mice to cigarette smoke for up to 6 mo and examined growth factor/procollagen gene expression in laser-capture microdissected small airways by real-time reverse transcription-polymerase chain reaction. RESULTS: With a single smoke exposure, increases in procollagen, connective tissue growth factor (CTGF), transforming growth factor (TGF)-beta(1), platelet-derived growth factor (PDGF)-A and -B expression were seen 2 h after the start of smoking and declined to baseline by 24 h. With repeated exposures and at killing of animals 24 h after the last exposure, increases in procollagen, CTGF, PDGF-B, and (minimally) PDGF-A expression persisted through 1 wk, 1 mo, and 6 mo. TGF-beta(1) gene expression declined over time; however, increased immunochemical staining for phopho-Smad 2 was present at all time points, indicating continuing TGF-beta downstream signaling. Morphometric analysis showed that the small airways in smoke-exposed mice had more collagen at 6 mo. CONCLUSIONS: These findings suggest that smoke can induce growth factor and procollagen production in small airways in a time frame that initially is too short for a significant inflammatory response and that profibrotic growth factor and procollagen gene expression become self-sustaining with repeated smoke exposures. These results imply that the pathogenesis of and possible treatment approaches to emphysema and small airway remodeling might be quite different.