Experimental Design
Two weeks after VX2 tumor implantation, the tumor-bearing rabbits were randomly divided into the ALPPS group (with the left branch of portal vein being ligated and the liver parenchyma being split between the left medial lobe and right lobe) and the Sham group (with the hepatic artery and portal vein being dissected without ligated). Three rabbits were selected from each group to have a DCE-MRI scan and a PET/CT scan with 18F-FDG, 18F-FCH, and 18F-FLT on days 0, 1, 3, 7 and 14 after surgery. After imagings, the rabbits were sacrificed for future pathological studies.
The ALPPS model in rabbits with liver VX2 tumor
This study was approved by the Animal Care Committee of Harbin Medical University. The rabbit liver VX2 tumor (Guang Zhou Jennio Biotech Co., Ltd) model was used in this study. Adult male New Zealand White rabbits, weighing 2.0-2.5 kg (provided by Harbin Medical University) were anesthetized with an intramuscular injection of 50 mg/kg zolazepam (Zoletil; Virbac, Carros, France). After a midline subxyphoid abdominal incision, and crushing of VX2 tumor into pieces, approximately 1 mm3 of tumor was directly implanted into the left lobe of liver. The VX2 tumor was allowed to grow for two weeks. The largest tumor, which measured in average 1.5 cm in diameter by baseline T2-weighted axial MRI, was selected to establish the ALPPS model. The tumor-bearing rabbits were anesthetized by intramuscular injection of 50mg/kg zolazepam and maintained on a mixture of 1-3% isoflurane (RWD Life Science Co, Ltd) and 50% oxygen and air (0.8-1.5 L/min). The animals were fixed in the Trendelenburg position on the operating table. After a midline laparotomy, the left branch of portal vein was dissected, marked, and ligated with 4-0 silk (Ethicon, Somerville, NJ). The ischemic line between the left medial lobe (LML) and the right liver (RL) then became prominent, and the liver parenchyma was then split along the ischemic line [16]. All operations were performed under an operating microscope (Binocular Operation Microscope; Type XTS-4A; Jiangsu Zhenjiangzhongtian Optical Instruments Co, Ltd. China) (Fig.2A).
The ALPPS model in rabbits with liver VX2 tumor
This study was approved by the Animal Care Committee of Harbin Medical University. The rabbit liver VX2 tumor (Guang Zhou Jennio Biotech Co., Ltd) model was used in this study. Adult male New Zealand White rabbits, weighing 2.0-2.5 kg (provided by Harbin Medical University) were anesthetized with an intramuscular injection of 50 mg/kg zolazepam (Zoletil; Virbac, Carros, France). After a midline subxyphoid abdominal incision, and crushing of VX2 tumor into pieces, approximately 1 mm3 of tumor was directly implanted into the left lobe of liver. The VX2 tumor was allowed to grow for two weeks. The largest tumor, which measured in average 1.5 cm in diameter by baseline T2-weighted axial MRI, was selected to establish the ALPPS model. The tumor-bearing rabbits were anesthetized by intramuscular injection of 50mg/kg zolazepam and maintained on a mixture of 1-3% isoflurane (RWD Life Science Co, Ltd) and 50% oxygen and air (0.8-1.5 L/min). The animals were fixed in the Trendelenburg position on the operating table. After a midline laparotomy, the left branch of portal vein was dissected, marked, and ligated with 4-0 silk (Ethicon, Somerville, NJ). The ischemic line between the left medial lobe (LML) and the right liver (RL) then became prominent, and the liver parenchyma was then split along the ischemic line [16]. All operations were performed under an operating microscope (Binocular Operation Microscope; Type XTS-4A; Jiangsu Zhenjiangzhongtian Optical Instruments Co, Ltd. China).
Synthesis of 18F-FDG, 18F-FLT and 18F-FCH
18F-FDG was acquired from daily productions at the TOF-PET/CT/MR center of the Fourth Hospital of Harbin Medical University. 18F-FLT and 18F-FCH were synthesized using the commercially available TRACERlab FXFN synthesis module (GE Healthcare). 18F-fluorothymidine was synthesized following published methods[17]. In brief, 3-N-Boc-1-[5-O-(4,4’dimethoxytrityl)-3-O-nosyl-2-deoxy-β-D-lyxof-uranosyl]thymine( Jiangsu huayi technology Co., Ltd) was used as a precursor and subjected to radiofluorination.18F-FCH was synthesized using the method reported by Melissa E.et al. Ditosylmethane( jiangsu huayi technology Co., Ltd) was fluorinated with fluorine-18 and the fluorotosylme-thane was alkylated intermediate with dimethylethanolamine( jiangsu huayi technology Co., Ltd) [18]. The final labeled products of both 18F-FLT and 18F-FCH had > 95% radiochemical purity as assessed by high-performance liquid chromatography.
PET/CT and MR imaging protocol
PET/CT imaging was performed on the tumor-bearing rabbits on days 0, 1, 3, 7, and 14 after surgery. All animals were fasted for at least 6 h before 18F-FDG PET/CT imaging. 18F-FDG, 18F-FLT or 18F-FCH (37 MBq/kg) was injected into each rabbit through the marginal ear vein at one separate day individually; PET/CT (GE Discovery PET/CT Elite, American) was performed 50–60 min after tracer injection. The rabbits were fixed in the supine position after anesthesia with intramuscular injection of zolazepam. The CT was performed before the PET. The CT parameters were: 120 kV, 10 mA, and a 3.33 mm pitch. Emission data were acquired at 3 min per bed position. The images were reconstructed using an aniterative reconstruction algorithm to obtain CT, PET, and PET/CT fusion images.
MRI examinations were performed on a 3.0T MR system (GE Discovery MR750W American). After anesthetized, the rabbits were placed in a prone position within the eight channel knee coil (3T HD T/R Knee Array, GE Healthcare). DCE-MRI was performed using a three-dimensional T1-weighted fat-suppressed fast spoiled gradient-echo (3D-FS-FSPGR): TR/TE of 7.7/2.1 ms; FOV of 170×170 mm; imaging acquisition matrix of 272×160, flip angle of 20°, Slice thickness of 2.5mm,gap of 1.0mm. In total, 60 phases were acquired. An intravenous bolus of gadodiamide (GE Healthcare AS) at a dose of 0.1 mmol/kg was administered by manual injection through the marginal ear vein at 4 ml/s and was immediately followed by a 5 ml saline flush after the first 12 phases. For T1 mapping, four pre-contrast images were acquired with the same imaging parameters by using different flip angles (5°, 10°, 20°, and 30°). It took approximately 10–11min to complete a DCE-MRI sequence with 60 phases, with each phase taking 9s. The total acquisition time of each examination was approximately 15 min.
DCE-MRI data were translated into a quantitative software (Omni-Kinetics, GE Healthcare). The results in the liver were calculated based on dual-input extended Tofts mode[19]. The input function was drawn on the abdominal aorta and the portal vein. The ROI was placed manually in the largest slice of FLR, which outlined the liver shape, excluding the major vessel. A single-input Tofts model was used to fit the tumor[20]. The ROIs were used to delineate the entire tumor and avoid the necrosis area. The enhanced-tumor on all tumor-containing image slices were selected. The Ktrans (volume transfer constant) measures the efflux of the contrast agent from the intravascular space to the extravascular extracellular space (EES). Based these kinetic models, the Ktrans of tumor and FLR were calculated.
Liver volume and volume increase rate measured by MR imaging
FLR volume (FLRVMRI) was calculated on a workstation (AW5.0; GE Medical Systems). On T2WI, the FLR area was manually delineated with a freehand ROI on all FLR containing images. The total FLRVMRI was calculated using the following equation:
FLRVMRI =ΣFLR area on each FLR-containing slice×(Slice thickness + Gap)
The percentage FLR volume increase after ALPPS was based on the FLRVMRI data and calculated using the formula.
PET/CT data analysis
For the result of 18F-FDG, 18F-FLT and the 18F-FCH, the region of interest (ROI) was drawn on the tumor and the FLR area; The maximum standardized uptake value (SUVmax) was calculated by measuring the maximal concentration of radioactivity in a ROI and correcting it for body weight and the injected dose. The SUVmean was then calculated as the average of SUV values in all voxels within the threshold-defined tumor volume. The tumor was automatically delineated on the 18F-FDG PET/CT fusion images on workstation (AW5.0; GE Medical Systems) by an outline extraction method in three-dimensional mode (with a threshold of 42% of the SUVmax) and then the metabolic tumor volume (MTV) was calculated. The FLR functional volume (FLRVFCH) was manually outlined on the three-dimensional reconstruction of 18F-FCH PET images on a workstation (AW5.0; GE Medical Systems) to exclude adjacent non-liver structures and a threshold of 42% of the SUVmax within the volumetric region of interest was used to calculate the FLRVFCH.
Valid volumetric function
The functional liver is discernible as the region of apparent 18F-FCH uptake. To represent the functional liver region (FLR), a Valid Volumetric Function (VVF) was defined as follows:
Valid Volumetric Function (VVF) = FLR SUVmean × FLRV FCH
The rate of VVF increase after ALPPS was calculated using the formula:
Liver volume measurement by water displacement method
Three rabbits were selected to be sacrificed. The liver was fully dissected and placed into a bottle filled with water. Liver volume (FLRVwater) was equal to the final water volume (with liver) subtracted by the baseline water volume (without liver).
Histopathologic analysis
After euthanasia, the liver and tumor tissues were fixed in 10% buffered formalin, embedded, and sectioned for future histopathological analysis. Hepatocyte and tumor cell proliferation was measured by immunohistochemistry with Ki67 antibody (ab15580, Abcam, UK). The sections were also stained with the CD31 antibody (ab199012, Abcam, UK) to measure the angiogenesis and stained with a-SMA (ab7817, Abcam, UK) to access vessel maturation. The microvessel density(MVD)marked with CD31 was quantified by using the Weidner method as follows: three areas of highest MVD were selected for scoring under high magnification (×200) within each slide, and then the average value of these three areas was recorded as the MVD[21]. Any brown-staining endothelial cell or endothelial cell cluster was considered as a single countable microvessel. The percentage of a-SMA positive stained area was determined by analysing three randomly selected fields at high magnification (×200) from each tumor and FLR section using ImageJ (NIH, Bethesda, MD, USA). The glucose transporter 1 (ab128033, Abcam, UK) was stained to reflect the glucose metabolism. The Ki67-positive rate was quantified with Image J software (National Institutes of Health, Bethesda, MD). The scores of GLUT1 were estimated according to staining intensity and number of stained cells, as previously described[22]. We also performed hematoxylin and eosin (H&E) stainings to observe the histopathological.
Statistical analysis
Data were expressed as mean ± standard error of the mean (SEM). Differences among each time point and differences between groups were analyzed by two-way analysis of variance. Further data comparison between the two groups at each time point was performed using the variance homogeneity test. The independent or paired t-test was used for equal variance, and the Pearson or Spearman correlation test was used to evaluate correlations between parameters of imaging and histopathologic analyses. All statistical tests were performed with two-tailed distribution and a P<0.05 was considered statistically significant. Statistical analyses were performed with GraphPad Prism8.0 (GraphPad Software, San Diego, CA).