Mammary cancer initiation and progression studied with magnetic resonance imaging.

INTRODUCTION: Previous work from this laboratory demonstrated that magnetic resonance imaging (MRI) detects early murine mammary cancers and reliably differentiates between in situ and invasive cancer. Based on this previous work, we used MRI to study initiation and progression of murine mammary cancer, and monitor the transition from the in situ to the invasive phase. METHODS: In total, seven female C3(1) SV40 Tag mice were imaged every two weeks between the ages of 8 to 23 weeks. Lesions were identified on T2-weighted images acquired at 9.4 Tesla based on their morphology and growth rates. Lesions were traced manually on MR images of each slice. Volume of each lesion was calculated by adding measurements from individual slices. Plots of lesion volume versus time were analyzed to obtain the specific growth rate (SGR). The time at which in situ cancers (referred to as 'mammary intraepithelial neoplasia (MIN)') and invasive cancers were first detected; and the time at which in situ cancers became invasive were recorded. RESULTS: A total of 121 cancers (14 to 25 per mouse) were identified in seven mice. On average the MIN lesions and invasive cancers were first detected when mice were 13 and 18 weeks old, respectively. The average SGR was 0.47 ± 0.18 week(-1) and there were no differences (P >0.05) between mice. 74 lesions had significantly different tumor growth rates before and after ~17 weeks of age; with average doubling times (DT) of 1.88 and 1.27 weeks, respectively. The average DT was significantly shorter (P <0.0001) after 17 weeks of age. However, the DT for some cancers was longer after 17 weeks of age, and about 10% of the cancers detected did not progress to the invasive stage. CONCLUSIONS: A wide range of growth rates were observed in SV40 mammary cancers. Most cancers transitioned to a more aggressive phenotype at approximately 17 weeks of age, but some cancers became less aggressive. The results suggest that the biology of mammary cancers is extremely heterogeneous. This work is a first step towards use of MRI to improve understanding of factors that control and/or signal the development of aggressive breast cancer.

T(2)∗ relaxation times of intraductal murine mammary cancer, invasive mammary cancer, and normal mammary gland.

PURPOSE: This study investigates the feasibility of T(2)∗ to be a diagnostic indicator of early breast cancer in a mouse model. T(2)∗ is sensitive to susceptibility effects due to local inhomogeneity of the magnetic field, e.g., caused by hemosiderin or deoxyhemoglobin. In these mouse models, unlike in patients, the characteristics of single mammary ducts containing pure intraductal cancer can be evaluated. METHODS: The C3(1)SV40Tag mouse model of breast cancer (n = 11) and normal FVB∕N mice (n = 6) were used to measure T(2)∗ of normal mammary gland tissue, intraepithelial neoplasia, invasive cancers, mammary lymph nodes, and muscle. MRI experiments were performed on a 9.4T animal scanner. High resolution (117 microns) axial 2D multislice gradient echo images with fat suppression were acquired first to identify inguinal mammary gland. Then a multislice multigradient echo pulse sequence with and without fat suppression were performed over the inguinal mammary gland. The modulus of a complex double exponential decay detected by the multigradient echo sequence was used to fit the absolute proton free induction decay averaged over a region of interest to determine the T(2)∗ of water and fat signals. RESULTS: The measured T(2)∗ values of tumor and muscle are similar (∼15 ms), and almost twice that of lymph nodes (∼8 ms). There was a statistically significant difference (p < 0.03) between T(2)∗ in normal mammary tissue (13.7 ± 2.9 ms) and intraductal cancers (11 ± 2.0 ms) when a fat suppression pulse was applied. CONCLUSIONS: These are the first reported T(2)∗ measurements from single mammary ducts. The results demonstrated that T(2)∗ measurements may have utility for identifying early pre-invasive cancers in mouse models. This may inspire similar research for patients using T(2)∗ for diagnostic imaging of early breast cancer.

Concomitant proliferation and caspase-3 mediated apoptosis in response to low shear stress and balloon injury.

BACKGROUND: Arterial remodeling occurs as a response to hemodynamic change and direct vessel wall injury through the process of neointimal hyperplasia (NH). A concomitant response of vascular smooth muscle cell (VSMC) proliferation and apoptosis exists. The purpose of this study is to assess the cellular response of vessels following exposure to low shear stress (tau) and balloon injury in order to further elucidate the mechanisms underlying vascular injury. Our hypothesis is that the combination of low tau and balloon injury results in NH approximating that seen in clinical arterial restenosis, and that quantitative analysis of VSMC proliferation and apoptosis correlates with the associated increase in arterial remodeling. METHODS AND RESULTS: New Zealand White rabbits underwent surgery on the carotid artery creating low tau (n =11), balloon injury (n = 11), combined low tau and balloon injury (n =11), and sham (n = 13) groups. Experiments were terminated at 1, 3, and 28 d. Day 1 and 3 arteries were analyzed with immunohistochemistry for apoptotic markers, terminal transferase dUTP nick end labeling (TUNEL), and activated caspase-3, and a cellular proliferation marker, accumulated proliferating cell nuclear antigen (PCNA), as well as immunoblot analysis for activated caspase-3 and PCNA at day 3. There was significantly greater apoptosis in the combined group as compared with the other groups assessed by quantitative TUNEL and activated caspase-3 levels at both days 1 and 3. Similarly, an increase in cellular proliferation assessed by PCNA expression, was significantly greater in the combined group as compared with the other groups. At 28 d there was no difference in NH observed in the low tau (26 +/- 3 microm) and balloon injury (51 +/- 17 microm) groups. However, significantly more NH was observed in the combined group (151 +/- 35 microm) as compared with the other groups. CONCLUSIONS: An increase in VSMC apoptosis via a caspase-3 dependent pathway is up-regulated by 24 h in the face of combined low shear stress and balloon-induced vessel wall injury. Paradoxically, this increase in VSMC apoptosis is associated with a significant increase in neointimal thickening at 28 d. The concomitant increase of both apoptosis and proliferation are indicative of a robust arterial remodeling response.

Correlation of In Vivo and Ex Vivo ADC and T2 of In Situ and Invasive Murine Mammary Cancers.

Ex vivo MRI may aid in the evaluation of surgical specimens, and provide valuable information regarding the micro-anatomy of mammary/breast cancer. The use of ex vivo MRI to study mouse mammary cancer would be enhanced if there is a strong correlation between parameters derived from in vivo and ex vivo scans. Here, we report the correlation between apparent diffusion coefficient (ADC) and T2 values measured in vivo and ex vivo in mouse mammary glands with in situ cancers (mammary intraepithelial neoplasia (MIN)) and invasive cancers (those which spread outside the ducts into surrounding tissue). MRI experiments were performed on the Polyoma middle T oncoprotein breast cancer mouse model (n = 15) in a 9.4T scanner. For in vivo experiments, T2-weighted (T2W) images were acquired to identify abnormal regions, then ADC and T2 values were measured for nine selected slices. For ex vivo experiments, a midline incision was made along the spine, and then skin, glands, and tumors were gently peeled from the body. Tissue was fixed in formalin, placed around a mouse-sized sponge, and sutured together mimicking the geometry of the gland when attached to the mouse. The same pulse sequences used for in vivo experiments were repeated for ex vivo scans at room temperature. Regions of interest were manually traced on T2W images defining features that could be identified on in vivo and ex vivo images. The results demonstrate a strong positive correlations between in vivo and ex vivo invasive cancers for ADC (r = 0.89, p <0.0001) and T2 (r = 0.89, p <0.0001) values; and weak to moderate positive correlations between in vivo and ex vivo in situ cancers for ADC (r = 0.61, p <0.0001) and T2 (r = 0.79, p <0.0001) values. The average ex vivo ADC value was about 54% of the in vivo value; and the average ex vivo T2 was similar to the in vivo value for cancers. Although motion, fixation, and temperature differences affect ADC and T2, these results show a reliable relationship between ADC and T2 in vivo and ex vivo. As a result ex vivo images can provide valuable information with clinical and research applications.

Quantitative evaluation of internal marks made using MRgFUS as seen on MRI, CT, US, and digital color images - a pilot study.

This pilot study compared the detectability of internal thermal marks produced with MRI-guided focused ultrasound (MRgFUS) on MRI, computed tomography (CT), ultrasonography (US), and color images from digital scanning. Internal marks made using MRgFUS could potentially guide surgical, biopsy or radiotherapy procedures. New Zealand White rabbits (n = 6) thigh muscle were marked using a Philips MRgFUS system. Before and after sonications, rabbits were imaged using T1- and T2-weighted MRI. Then rabbits were sacrificed and imaging was performed using CT and US. After surgical excision specimens were scanned for color conspicuity analysis. Images were read by a radiologist and quantitative analysis of signal intensity was calculated for marks and normal muscle. Of a total of 19 excised marks, approximately 79%, 63%, and 62% were visible on MRI, CT, and US, respectively. The average maximum temperature elevation in the marks during MRgFUS was 39.7 ± 10.1 °C, and average dose diameter (i.e., the diameter of the area that achieved a thermal dose greater than 240 cumulative equivalent minutes at 43 °C) of the mark at the focal plane was 7.3 ± 2.1 mm. On MRI the average normalized signal intensities were significantly higher in marks compared to normal muscle (p < 0.05). On CT, the marked regions were approximately 10 HU lower than normal muscle (p < 0.05). The results demonstrate that MRgFUS can be used to create internal marks that are visible on MRI, CT and US.

Attenuated herpes simplex virus 1 blocks arterial apoptosis and intimal hyperplasia induced by balloon angioplasty and reduced blood flow.

Injury caused by distention of the arterial wall by balloon angioplasty can result in apoptosis and vascular smooth muscle cell proliferation. Here, we report that a brief exposure of the arterial lumen to a genetically engineered, attenuated herpes simplex virus 1 blocks activation of caspase 3-dependent apoptosis and MAPK-dependent cell proliferation induced by carotid artery balloon angioplasty and ligation to reduce blood flow. The procedure enables the restoration of the endothelial cell layer lining the lumen and prevents neointimal hyperplasia and restenosis. These findings have a broad application in prevention of balloon angioplasty-induced restenosis.

Slower onset of low shear stress leads to less neointimal thickening in experimental vein grafts.

Vein grafts respond to low flow and shear stress (tau(w)) by generating thicker walls and smaller lumens through the processes of neointimal hyperplasia and remodeling. Clinically, however, vein grafts with obviously low tau(w), such as those distal to high-grade proximal obstructions, are not infrequently found to be widely patent and pliable. One possible explanation for this phenomenon may be that vein grafts remodel more favorably in response to changes in shear that occur gradually over time compared to abruptly. This hypothesis was tested in an experimental animal model in this report. Two separate models of experimental vein graft failure were created, causing either immediate exposure to ultralow tau(w) (<1 dyne/cm2) or delayed exposure to ultralow tau(w). Under general anesthesia and using a sterile technique, the right external jugular (EJ) veins of 28 New Zealand white rabbits were surgically exposed and isolated. An end-to-side distal EJ/common carotid artery anastomosis was created, resulting in a widely patent arteriovenous fistula. For the immediate exposure group (n = 5), the EJ was suture-ligated just proximal to the thoracic inlet, distal to a small 10-50 microm venous tributary. This created a reversed vein segment immediately and abruptly exposed to high wall tension (2.0 +/- 0.3 x 10(4) dyne/cm) and ultralow tau(w) (0.15 +/- 0.08 dyne/cm2). For the delayed exposure group (n = 22), the EJ was ligated over a 0.035 guidewire, leaving a small aperture to sustain some measure of blood flow and tau(w). This predictably resulted in slightly less wall tension (1.4 +/- 0.2 x 10(4) dyne/cm) and higher tau(w) (0.68 +/- 0.21 dyne/cm2) than the immediate exposure group. During the first week, the small outflow aperture in the delayed exposure grafts thrombosed, eventually exposing them to the same low level of tau(w) as the immediate exposure grafts. Thus, the only difference in the two models was that delayed exposure grafts enjoyed a slower decline in tau(w) than immediate exposure grafts. Fourteen rabbits in the delayed exposure group were harvested over the first 7 days to define the patency curve of the restricted outflow channel. As expected, the small aperture had thrombosed in all animals by 7 days. The remaining 14 grafts were harvested after 4 weeks, and 13/14 remained patent. Examination of the hemodynamic parameters at the time of death confirmed that wall tension and tau(w) had equalized (wall tension 0.9 +/- 0.1 vs. 1.1 +/- 0.1 x 10(4) dyne/cm, tau(w) 0.45 +/- 0.12 vs. 0.30 +/- 0.08 dyne/cm2). Histological examination revealed less neointimal hyperplasia in the delayed exposure group compared to the immediate exposure group (wall thickness 266 +/- 16 vs. 180 +/- 24 microm, p = 0.025) as well as a slightly greater luminal diameter (0.30 +/- 0.02 vs. 0.40 +/- 0.02 cm, p = 0.038). The results of this experiment suggest that slow exposure to reduced tau(w) results in more favorable remodeling (less thickening) than abrupt exposure. This finding may explain the occasional clinical observation of a widely patent vein graft even in the face of proximal arterial obstruction and very low flow; the change in tau(w) presumably occurred slowly mitigating the remodeling response.

Modulation of vascular remodeling induced by a brief intraluminal exposure to the recombinant R7020 strain of Herpes simplex-1.

OBJECTIVE: Vascular remodeling in response to injury or low shear stress (or both) is characterized by neointimal hyperplasia and luminal contraction. When profound, the response leads to restenosis after percutaneous endovascular intervention as well as to de novo stenosis in vein grafts. It has recently been reported that exposure of vein patches to neurovirulence-attenuated Herpes simplex virus-1 (HSV-1) decreases neointimal hyperplasia and increases luminal area. This experiment tested the hypothesis that R7020, a more highly attenuated mutant of HSV-1, would modulate the vascular remodeling response of experimental vein grafts chronically exposed to low shear stress. METHODS: The external jugular veins of 31 New Zealand white rabbits were clamped and intraluminally exposed to vehicle (phospate-buffered saline solution, n = 11), R7020 2.5 x 10(8) plaque forming units [PFU]/mL (n = 8), or R7020 2.5 x 10(9) PFU/mL (n = 12) for 10 or 30 minutes at an average pressure of 80 mm Hg. After exposure, an end-to-side distal external jugular-to-common carotid artery anastomosis was created, resulting in a widely patent arteriovenous fistula. The external jugular was suture-ligated just proximal to the thoracic inlet, distal to a small 10- to 50-microm venous tributary, creating a reversed vein "graft" segment immediately and abruptly exposed to arterial pressure (48 +/- 3 mm Hg) and low shear stress (0.12 +/- .02 dyne/cm(2)). In the 29 animals (N = 31) that survived to harvest, 26 grafts were found to be patent and were analyzed further. Nine grafts were harvested within the first week after operation, snap frozen in liquid nitrogen, and assayed for the presence of the Herpes viral immediate-response protein ICP0 by Western blot analysis. The 17 remaining grafts were perfusion-fixed, excised, stained, and analyzed morphometrically by digital planimetry. RESULTS: In patent grafts, the hemodynamic environment of low shear stress was maintained (shear stress at harvest, 0.26 +/- .06 dyne/cm(2)). Western blot analysis revealed the presence of ICP0 in R7020-exposed vein grafts after 2, 3, 7, and 14 days; ICP0 was not detected in unexposed vein grafts or adjacent carotid arteries. After 4 weeks, vein grafts exposed to R7020 exhibited a statistically significantly increased ratio of luminal radius to wall thickness, indicating altered remodeling (vehicle, 6.7 +/- 1.3; R7020 2.5 x 10(8), 9.1 +/- 1.3; R7020 2.5 x 10(9) ratio, 11.3 +/- 1.4; P < .05 for high dose compared with vehicle). CONCLUSION: A brief exposure of the neurovirulence-attenuated HSV-1 strain R7020 results in an increased ratio of luminal radius to wall thickness in experimental vein grafts chronically exposed to low shear stress.