Sumeet Virmani

Comparison of Hypoxia-inducible Factor-1α Expression before and after Transcatheter Arterial Embolization in Rabbit VX2 Liver Tumors

PURPOSE To test the hypothesis that transcatheter arterial embolization (TAE) induces expression of hypoxia-inducible factor-1alpha (HIF-1alpha) within the same rabbit VX2 liver tumor. MATERIALS AND METHODS Seven VX2 tumors were grown in the livers of five New Zealand white rabbits. Ultrasonography-guided biopsy was performed before and 10 minutes after TAE in all tumors. Pre- and post-TAE tumor biopsy specimens along with post-TAE whole liver tumor sections were stained with HIF-1alpha antibody and analyzed for percentage of HIF-1alpha-positive nuclei by using a spectral unmixing system mounted on a high-powered microscope. Statistical data comparisons were performed with the Wilcoxon signed-rank test (alpha = 0.05). RESULTS TAE of liver tumors resulted in a statistically significant increase in the mean percentage of HIF-1alpha expression. The mean percentage of HIF-1alpha-positive stained nuclei increased from 23% +/- 3.5 in pre-TAE biopsy specimens to 41% +/- 8.7 in post-TAE biopsy specimens (P < .02). The increase was even more significant when the mean percentage of HIF-1alpha-positive stained nuclei from the same pre-TAE biopsy specimens was compared with sections from post-TAE whole tumor specimens (60% +/- 8.9, P < .02). CONCLUSIONS The results of this study revealed that hypoxia caused by TAE of VX2 liver tumors activates HIF-1alpha, a transcription factor that in turn regulates other pro-angiogenic factors.

Comparison of Two Different Methods for Inoculating VX2 Tumors in Rabbit Livers and Hind Limbs

PURPOSE To compare two methods to (a) propagate VX2 cell strain in rabbit hind limbs and (b) inoculate liver parenchymal tumors in rabbits. MATERIALS AND METHODS One hundred forty-two New Zealand white rabbits were used for this study (60 with hind limb tumor [donors] and 82 with liver tumors [recipients]). In the donor group, nine rabbits received frozen VX2 cell suspension and 51 were injected with freshly prepared VX2 cell suspension. In the recipient group, 32 rabbits were injected with VX2 tumor cells and 50 were implanted with a small tumor fragment in the liver parenchyma. Success rates in terms of tumor growth were compared by using chi(2) or Fisher exact tests, with alpha = .05. RESULTS Hind limb and liver tumors were successfully grown in 48 of the 60 rabbits in the donor group (80%) and 57 of the 82 rabbits in the recipient group (70%). The success rate of growing hind limb tumors increased from 33% (three of nine rabbits) to 88% (45 of 51 rabbits) when fresh VX2 cells instead of frozen were injected percutaneously (P < .0011). Similarly, the success rate for VX2 liver tumors almost doubled from 47% (15 of 32 rabbits) to 84% (42 of 50 rabbits) when a tumor fragment instead of VX2 cell suspension was used (P < .00036). This also significantly reduced the frequency of metastasis (P < .005). CONCLUSIONS The authors recommend (a) the use of fresh VX2 cell suspension for percutaneous injection in the hind limbs of rabbits to maintain the VX2 cell strain and (b) the surgical implantation of freshly harvested VX2 tumor fragment into the liver parenchyma to establish liver tumors.

MR Tracking of Iron-labeled Glass Radioembolization Microspheres during Transcatheter Delivery to Rabbit VX2 Liver Tumors: Feasibility Study

PURPOSE To prospectively test the hypothesis that iron labeling of radioembolization microspheres permits their visualization by using magnetic resonance (MR) imaging for in vivo tracking during transcatheter delivery to liver tumors. MATERIALS AND METHODS All experiments were approved by the Institutional Animal Care and Use Committee. Phantom studies were performed to quantify microsphere relaxivity and volume susceptibility properties and compare image contrast patterns resulting from aggregate deposition of unlabeled and iron-labeled microspheres. In seven rabbits in which nine VX2 liver tumors were implanted, T2*-weighted gradient-echo (GRE) MR images with negative image contrast (NC), white-marker (WM) GRE images with positive image contrast (PC), and on-resonance water-suppression turbo spin-echo (SE) images with PC were obtained before and after catheter-directed administration of microspheres into the hepatic artery. During each injection, serial GRE acquisitions were performed for real-time visualization of microsphere delivery. Contrast-to-noise ratios (CNRs) were measured between regions of microsphere accumulation and regions of normal liver parenchyma that demonstrated no apparent microsphere accumulation. Pre- and postinjection CNR measurements at identical spatial positions were compared by using paired t test (alpha = .05). RESULTS Conventional microspheres did not produce detectable image contrast in phantoms. Iron-labeled microspheres produced susceptibility-induced dipole patterns with spatial extent of image contrast increasing with increasing microsphere dose. Real-time image series depicted both preferential delivery to tumor tissues and nontargeted delivery to adjacent organs. T2*-weighted GRE, WM GRE, and on-resonance water-suppression turbo SE each permitted in vivo visualization of the microsphere deposition, with postinjection CNR values (mean, 14.29 +/- 3.98 [standard deviation], 1.87 +/- 0.93, and 19.30 +/- 8.72, respectively) significantly greater than corresponding preinjection CNR values (mean, 2.02 +/- 4.65, 0.02 +/- 0.27, 0.85 +/- 2.65, respectively) (P < .05). CONCLUSION Microsphere tracking during radioembolization may permit real-time verification of delivery and detection of extrahepatic shunting.

Negative Regulation of EGFR-Vav2 Signaling Axis by Cbl Ubiquitin Ligase Controls EGF Receptor-mediated Epithelial Cell Adherens Junction Dynamics and Cell Migration

The E3 ubiquitin ligase Casitas B lymphoma protein (Cbl) controls the ubiquitin-dependent degradation of EGF receptor (EGFR), but its role in regulating downstream signaling elements with which it associates and its impact on biological outcomes of EGFR signaling are less clear. Here, we demonstrate that stimulation of EGFR on human mammary epithelial cells disrupts adherens junctions (AJs) through Vav2 and Rac1/Cdc42 activation. In EGF-stimulated cells, Cbl regulates the levels of phosphorylated Vav2 thereby attenuating Rac1/Cdc42 activity. Knockdown of Cbl and Cbl-b enhanced the EGF-induced disruption of AJs and cell motility. Overexpression of constitutively active Vav2 activated Rac1/Cdc42 and reorganized junctional actin cytoskeleton; these effects were suppressed by WT Cbl and enhanced by a ubiquitin ligase-deficient Cbl mutant. Cbl forms a complex with phospho-EGFR and phospho-Vav2 and facilitates phospho-Vav2 ubiquitinylation. Cbl can also interact with Vav2 directly in a Cbl Tyr-700-dependent manner. A ubiquitin ligase-deficient Cbl mutant enhanced the morphological transformation of mammary epithelial cells induced by constitutively active Vav2; this effect requires an intact Cbl Tyr-700. These results indicate that Cbl ubiquitin ligase plays a critical role in the maintenance of AJs and suppression of cell migration through down-regulation of EGFR-Vav2 signaling.

Diffusion-Weighted PROPELLER MRI for Quantitative Assessment of Liver Tumor Necrotic Fraction and Viable Tumor Volume in VX2 Rabbits

To test the hypothesis that diffusion‐weighted (DW)‐PROPELLER (periodically rotated overlapping parallel lines with enhanced reconstruction) MRI provides more accurate liver tumor necrotic fraction (NF) and viable tumor volume (VTV) measurements than conventional DW‐SE‐EPI (spin echo echo‐planar imaging) methods.

Distinct roles for Rho versus Rac/Cdc42 GTPases downstream of Vav2 in regulating mammary epithelial acinar architecture.

Non-malignant mammary epithelial cells (MECs) undergo acinar morphogenesis in three-dimensional Matrigel culture, a trait that is lost upon oncogenic transformation. Rho GTPases are thought to play important roles in regulating epithelial cell-cell junctions, but their contributions to acinar morphogenesis remain unclear. Here we report that the activity of Rho GTPases is down-regulated in non-malignant MECs in three-dimensional culture with particular suppression of Rac1 and Cdc42. Inducible expression of a constitutively active form of Vav2, a Rho GTPase guanine nucleotide exchange factor activated by receptor tyrosine kinases, in three-dimensional MEC culture activated Rac1 and Cdc42; Vav2 induction from early stages of culture impaired acinar morphogenesis, and induction in preformed acini disrupted the pre-established acinar architecture and led to cellular outgrowths. Knockdown studies demonstrated that Rac1 and Cdc42 mediate the constitutively active Vav2 phenotype, whereas in contrast, RhoA knockdown intensified the Vav2-induced disruption of acini, leading to more aggressive cell outgrowth and branching morphogenesis. These results indicate that RhoA plays an antagonistic role to Rac1/Cdc42 in the control of mammary epithelial acinar morphogenesis.

Four-dimensional transcatheter intraarterial perfusion (TRIP)-MRI for monitoring liver tumor embolization in VX2 rabbits.

Transcatheter intraarterial perfusion (TRIP)‐MRI is an intraprocedural technique to iteratively monitor liver tumor perfusion changes during transcatheter arterial embolization (TAE) and chemoembolization (TACE). However, previous TRIP‐MRI approaches using two‐dimensional (2D) T1‐weighted saturation‐recovery gradient‐recalled echo (GRE) sequences provided only limited spatial coverage and limited capacity for accurate perfusion quantification. In this preclinical study, a quantitative 4D TRIP‐MRI technique (serial iterative 3D volumetric perfusion imaging) with rigorous radiofrequency (RF) B1 field calibration and dynamic tissue longitudinal relaxation rate R1 measurement is presented for monitoring intraprocedural liver tumor perfusion during TAE. 4D TRIP‐MRI and TAE were performed in five rabbits with eight VX2 liver tumors (N = 8). After B1 calibrated baseline and dynamic R1 quantification, subsequent tissue contrast agent concentration time curves were derived. A single‐input flow‐limited pharmacokinetic model and peak gradient method were applied for perfusion analysis. The perfusion Fρ reduced significantly from pre‐TAE 0.477 (95% confidence interval [CI]: 0.384–0.570) to post‐TAE 0.131 (95% CI: 0.080–0.183 ml/min/ml, P < 0.001). Magn Reson Med 60:970–975, 2008.

Development of a VX2 Pancreatic Cancer Model in Rabbits: A Pilot Study

PURPOSE An animal model of pancreatic cancer that is large enough to permit imaging and catheterization would be desirable for interventional radiologists to develop novel therapies for pancreatic cancer. The purpose of this study was to test the hypothesis that the VX2 rabbit model of pancreatic cancer could be developed as a suitable platform to test future interventional therapies. MATERIALS AND METHODS The authors implanted and grew three pancreatic VX2 tumors per rabbit in six rabbits. Magnetic resonance (MR) imaging was performed at 2 weeks to confirm tumor growth. At 3 weeks, the authors selectively catheterized the gastroduodenal artery under guidance of x-ray digital subtraction angiography (DSA). T2-weighted anatomic imaging, diffusion-weighted MR imaging, and transcatheter intraarterial perfusion (TRIP) MR imaging were then performed. After imaging, tumors were confirmed at necropsy and histopathologically. Tumor sizes at 2 and 3 weeks were compared with a paired t test (P = .05). RESULTS VX2 pancreatic tumors were grown in all six rabbits. The difference between tumor sizes at 2 and 3 weeks (1.29 cm +/- 0.39 vs 1.91 cm +/- 0.50, respectively) was significant (P < .001). All tumors were confirmed to be located within pancreatic tissue via histopathologic analysis. DSA and TRIP MR imaging were successful in five rabbits. Diffusion-weighted and anatomic MR imaging were successful in all six rabbits. CONCLUSIONS The VX2 rabbit model of pancreatic cancer is feasible, as verified by imaging and pathologic correlation, and may be a suitable platform to test future interventional therapies.

Intraprocedural Diffusion-Weighted PROPELLER MRI to Guide Percutaneous Biopsy Needle Placement within Rabbit VX2 Liver Tumors

To test the hypothesis that diffusion‐weighted (DW)‐PROPELLER (periodically rotated overlapping parallel lines with enhanced reconstruction) magnetic resonance imaging (MRI) can be used to guide biopsy needle placement during percutaneous interventional procedures to selectively target viable and necrotic tissues within VX2 rabbit liver tumors.