The tumor tissues were excised and implanted into 37 normal adult WAG/Rij rats that weighed 225-275 g, as described GSI-IX previously[13]. MRI All rats were initially anesthetized by inhalation of 2% isoflurane and maintained with 0.8% isoflurane for MRI. A clinical 1.5T MRI system (Sonata, Siemens, Erlangen, Germany) was used with a maximum gradient capability of 40 mT/m. The following sequences were acquired in the transverse plane for all rats, with a slice thickness of 2 mm and an inter-slice gap of 0.2 mm: (1) Fat saturated T2-weighted fast spin echo MRI (T2W-MRI) with a repetition/echo time (TR/TE) of 3860/106 ms, a turbo factor of 19, a field of view (FOV) of 140 mm �� 70 mm, and an acquisition matrix of 256 �� 256 (in-plane resolution: 0.5 mm �� 0.3 mm).
Three signals were acquired, in a scan time of 1 min 25 s; (2) Contrast-enhanced fat saturated T1-weighted fast spin echo MRI (CE-T1W-MRI) immediately after an iv bolus of 0.3 mmol/kg gadoterate meglumine (Dotarem?, Guerbet, France), with the following parameters: a TR/TE of 535/9.2 ms, a turbo factor of seven, a FOV of 140 mm �� 70 mm, and an acquisition matrix of 256 �� 256 (in-plane resolution: 0.5 mm �� 0.3 mm). Four signals were acquired, in a scan time of 1 min 24 s; (3) DW-MRI with a 2-dimensional (2D), spin echo, echo-planar imaging sequence. We used a TR/TE of 1700/83 ms, a FOV of 140 mm �� 82 mm, and an acquisition matrix of 192 �� 91 (in-plane resolution: 0.7 mm �� 0.9 mm). For the DW-MRI, six signals were acquired, including repeated measurements for 10 different b values (0, 50, 100, 150, 200, 250, 300, 500, 750, and 1000 s/mm2) in 3 directions (x, y, and z) and averaged for the calculation of the isotropic ADC value.
A parallel imaging technique was applied to reduce susceptibility artifacts and examination times. The total examination time was 4 min 51 s. Tissue processing and histology All rats were euthanized for tissue processing and histology at the end of the experiment. First, animals were over-anesthetized by an intraperitoneal injection of pentobarbital (50 mg/kg) (Nembutal, Sanofi Sante Animale, Brussels, Belgium). Then, the livers were collected, fixed with formalin, embedded in paraffin, and sliced into transverse sections. The sections were 2 mm thick, and were positioned on the same planes used for the MRI scans, based on a grid (Agar Scientific, England).
The tumor slices (5 ��m thick) were stained with hematoxylin and eosin (HE). MRI analysis An off-line LINUX workstation with dedicated software (Biomap, Novartis, Basel, Switzerland) was used for image analyses. Two experienced radiologists delineated the entire tumor with operator-defined regions of Entinostat interest (ROI) in consensus to obtain robust measurements and to facilitate comparisons between different treatment approaches. All ROIs were larger than 10 pixels in size.