| Purpose: The current study employed combined positron emissiontomography/computed tomography (PET/CT) imaging analysis on rabbit VX2 tumormodel, to explore optimal timing for PET/CT imaging, and to understand the initiation,development and metastasis of transplanted tumor. PET/CT imaging evaluation andpathologic examinations were also conducted after Argon-Helium cryoablation treatment,to establish protocols for evaluating clinical effectiveness and outcome.Materials and Methods: In order to establish VX2 transplanted tumor model in rabbitsand observe tumor growth, VX2 carcinoma cell suspension was injected into the rabbitleg muscle or tumor blocks were inoculated into the rabbit liver. 18F-FDG PET/CT scanswere performed at different stages after tumor inoculation to acquire PET/CT images.Standardized uptake values (SUVave) were measured at the lesion sites, along withpathological analysis and immunohistochemical staining, in order to pinpoint optimaltiming for PET/CT imaging. PET/CT imaging was performed on rabbits bearing VX2carcinoma at this selected time point and then at different time points after argon-heliumcryoablation treatment and these images compared. The images were also comparedwith the results of pathologic examination, to demonstrate the changes afterargon-helium cryoablation and to evaluate the role of PET/CT imaging in monitoring theefficacy of argon-helium cryoablation.1. Establishment of rabbit VX2 transplanted tumor modelVX2 carcinoma-bearing rabbits were anesthetized and subcutaneous tumor tissue was dissected free of surrounding fatty tissue. The tumor tissue was subsequently cut intosmall pieces by a pair of scissors in 0.9％normal saline, to make tumor cell suspension tobe used in transplantation. Healthy Japanese white rabbits were anesthetized, ventralabdomen shaved and disinfected, 1ml syringe with 50ml syringe needle was used toinject 0.1ml each of tumor cell suspension into the thigh muscle on both sides and the leftforeleg. Tumor growth was closely observed after inoculation.2. Imaging study on the rabbit VX2 transplanted tumorJapanese white rabbits were randomly divided into groups and inject 18F-FDG atdifferent time points after tumor inoculation to perform PET/CT scanning. 2mL normalsaline solution was injected via marginal ear vein of the rabbits to confirm venous access.Afterwards, 18F-FDG was injected at 0.75mCi/kg body weight, followed by another 2mLof normal saline solution to wash the catheter, and then withdraw the needle and appliedpressure to stop bleeding. CT scanning was performed, followed by PET scanning, tomeasure tumor size. Images were processed and reconstructed using iterativereconstruction algorism to obtain CT, PET and their fused images. Xeleris image fusionworkstation was used to view PET/CT cross-sectional images and obtain cross-sectionaldata. CT images were used to select maximal cross-sectional area of the tumor. Thetumor border was defined to acquire SUVave and SUVmax of tumor tissue; SUVave andSUVmax of normal tissue were calculated from region of interest 2cm away from tumortissue, for comparison.3. Pathologic studyEuthanize rabbits after PET/CT scanning. Tumor tissue was dissected and fixedwith 4％formadelhyde, followed by paraffin sectioning, H&E staining andmorphologically analysis under the light microscope. Vessels were labeled bymonoclonal antibody against Thy and VEGF. Intratumoral microvessel density wasmeasured by a method established by Weidner et al: find the area of high vessel densityunder low-magnification lenses, then switch to high magnification lenses (200x) tomeasure the number of microvessels in 5 fields and use the average number as a readout of tumor microvessel density.Results1. Results from establishing rabbit VX2 tumor modelVX2 tumor cell line grows easily in rabbit skeletal muscle and local tumor growthcan be observed or palpable after tumor cell transplantation. The present study indicatesthat VX2 tumor cell suspension inoculates easily and induces rapid tumor growth.Maximum tumor growth occurs at 2～8 weeks after inoculation and slows down after 8weeks, when swollen lymph nodes were found. Transplanted tumor mass varies in sizeand growth rate. In general, transplanted tumors in the foreleg grow at slower rate andare smaller; whereas tumors in the hind leg and liver grow faster, are bigger in size, andwith more necrosis. The overall survival rate was 80％.2. PET/CT imaging resultsTumor tissue has good uptake and retention of 18F-FDG. 40～100 minutes afterinjection of 18F-FDG, tumor tissue was clearly visualized. Smaller tumors exhibitedpunctuate nodular shadows and bigger tumors exhibited circular, high-density shadows.No staining was observed in necrotic areas, which contrasted well with surroundingtissue. SUVave measurements at 60～80 min after 18F-FDG injection differedsignificantly from SUVave at 20, 40, 100 and 120 minutes (P＜0.05). Tumor SUVave at2, 6 and 8 weeks after VX2 cells inoculation differed significantly from SUVave ofnormal tissue 2cm away from tumors (P=0.006; 0.001, 0.000; 0.007).PET/CT images were compared before and after argon-helium cryoablation. Oneday after the treatment, radioisotope density was significantly lower with lower SUVavemeasurement and with apparent radioactive defect in the center. One week aftertreatment, patchy lesions were observed. One month after the treatment, there was noradioisotope uptake at the treated region. SUVave measurement was significant lowerimmediately after the treatment, followed by a rebound and then a slow decline.3. Pathological findings The increase in tumor size, the number of pathological karyokinesis and averagemicrovessel density were closely related to SUVavg (P=0.009; 0.025, 0.034 respectively;r=0.091, 0.975, 0.966 respectively). Pathological changes before and after argon-heliumcryoablation: 3 days after the treatment, the vast majority of tumor cells had died andexhibited as anucleated, cytoplasmic red staining cells, with undefined cell margins. Inthe treated region, a few infiltrating inflammatory cells can be observed, along withfragmented nucleus, red blood cells and white blood cells. A clear demarcation wasobserved between the treated region and adjacent normal tissue. Necrotic tissue wasimmediately adjacent to normal muscle cells, without transitional morphology. One weekafter the treatment, an inflammatory zone was formed surrounding the necrotic tissue anda large number of infiltrated inflammatory cells were observed. One month after thetreatment, fibroblasts were observed surrounding vessels and cryoablated regionexhibited disorganized structure, with many active lymphocytes and macrophages.Increased angiogenesis was also observed. Necrotic tissues disappeared two months afterthe treatment and fibrous nodules were formed at the previous lesion site, with a fewresidual lymphocytes. The pathological changes fully demonstrated the cellular eventsafter cryoablation treatment: irreversible necrosis of tumor cells, inflammatory cellinfiltration and fibrosis.ConclusionsRabbit VX2 transplanted tumor model was established in this study and PET/CTimaging analysis was conducted on the tumor model. A comparative study betweenPET/CT imaging with pathological findings, before and after argon-helium cryoablation,was also performed. It was found that rabbit VX2 tumor model provided the bestclinical tumor model 4 weeks after tumor transplantation. PET/CT scanning should beconducted 40～100 min after 18F-FDG injection and the image resolution was optimal at60～80 min. SUVave is one of the semiquantative PET parameters to be used in malignanttumor diagnosis and a number lower than 2.5 is considered the threshold for diagnosing malignancy in animal experiments. PET/CT imaging is a reliable and valuable tool intumor diagnosis and can be used to reveal tumor anatomical site, morphology, size andtumor cell metabolism, therefore useful in monitoring metastasis in early phase.PET/CT imaging can faithfully demonstrate the changes after argon-helium cryoablationand can be used to evaluate the effectiveness of cryoablation treatment.
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