| Objective: 1) Establish the rabbit VX2 limb soft tissue tumor model with ultrasound guidance for further intervention study and imaging evaluation; 2) Using neoadjuvant radiotherapy on tumor models, and increase the dose of radiation with volumetric modulated arc therapy and simultaneous integrated boost(VMAT-SIB) technique in the tumor area adjacent to the bone. Then prepare a “necrotic zone” defined as surgical margin with local radiation injury, for further evaluation of feasibility and effectiveness of the surgical margin; 3) To evaluated its safety of implementation of hypofraction radiotherapy to prepare surgical margin on the tumor model. Methods: 1) 30 male New Zealand white rabbits were used and randomly divided into control group(n=6)and model group(n=24), injecting with 0.5ml saline and 0.5ml tumor tissue suspension,respectively. Magnetic resonance diffusion weighted imaging(MRDWI) and three-dimensional ultrasound were used in two groups to analyze tumor morphology,signal characteristics and volume changes. Histological changes were collected from sacrificed animals after capturing image in the control group. Eighteen animals were randomly sacrificed at 14 d, 21 d and 28 d after histological changes were collected in the model group. The remaining six animals were not killed, to observe survival time with tumor; 2) 30 tumor model rabbits were selected and randomly divided into three groups,control group(n=10), intensity modulated radiation therapy(IMRT) group(n=10) and VMAT-SIB group(n=10). The MRDWI and three-dimensional power Doppler angiography(3D-PDA) of the 3 groups were collected on the 7 day after radiotherapy.The animals were sacrificed later. Histological and immunohistochemical examination were used to evaluate proliferation, apoptosis, vascular density of tumor tissue; 3) 40 tumor model rabbits were selected and randomly divided into four groups, control group(n=10), VMAT-SIB 3Gy Group(n=10), VMAT-SIB 4Gy Group(n=10), and VMAT-SIB5 Gy group(n=10). General situation of the skin was observed after radiotherapy in four groups, referring to CTCAE grading. HE staining was used to observe morphological changes of the skin and bone tissue. TEM was performed to observe ultrastructural changes of bone cells. FISH was used to assay the expression of PDGF-B and EGF in skin tissue. Real-time quantitative PCR was used to assay gene expression of VEGF,BFGF in skin tissue. Results: 1) Three-dimensional ultrasound and MRDWI confirmed that 24 animals growing tumorigenic rate was 100%, and 24 model rabbits were all survived. Three-dimensional ultrasound findings showed that there was no abnormal echo at injection site in the control group. The volume of tumor increased gradually as time goes in the model group, and the statistically was significant compared with the control group(P<0.05). MRDWI showed that no abnormal signal was observed in the control groups. Tumor imaging was captured in the left hind limb, and high signal was detected in tumor tissues with DWI maps in model group, while low signal was observed with ADC maps. Histology result: with HE staining, normal striated muscle and bronchoalveolar lavage tissue was shown in the control group; in the model group with HE staining, vigorous growing tumor cells were detected in an irregular dense arrangement and striated muscle was invased at the 14 d; the boundary of tumor and patchy necrosis zone were unclear, cells were much more dense in the tumor area at the21 d; nucleus of tumor cells were partially condensed and broken at the 28 d; tumor cells were observed in the border zone of tumor, and necrotic cells can be observed with inflammatory cell infiltration and fibrosis. Gross anatomy showed diffuse metastatic nodules on the lungs, and small nodules partially merged, and bronchi and alveolar were invased by tumor cells at the 60 d; 2) Two tumor model rabbits were died in the IMRT group, due to anesthesia overdose one rabbit dead, and the other one was given euthanasia because of tumor ulceration to the skin during radiotherapy. ROI(tumor tissues adjacent to bone) among three groups were significantly different in the DWI and ADC maps. It was shown a relatively high signal in DWI maps, and significant low signal in ADC maps in the control group. The signal was unevenly low signal in DWI maps and high in ADC maps in IMRT group. The signal was low signal in DWI maps and high in ADC maps in VMAT group. 3D-PDA results: VI, FI and VFI among the three groups were significantly different. IMRT and VMAT group were significantly lower than the control group. Histology result: compare with the control group, striated muscle was invased with tumor cells and normal cell structures were injured with HE staining;IMRT group, diffuse necrotic cells were observed in the tumor parenchyma and boundarytissue, accompanied by inflammatory cell infiltration and fibrosis with HE staining;VMAT group, a necrotic zone was formed in tumor tissue adjacent to bone, and obvious cell necrosis and fibrosis with inflammatory cell infiltration was observed with microscope. Proliferation activity and vascular density were decreased and apoptosis increased in IMRT and VMAT group after radiotherapy; 3) There was no abnormal of local skin in control group, 3Gy group and 4Gy group during and after the radiotherapy. 8animals were presented with mild erythema and dry desquamation on local skin during radiotherapy in 5Gy group. Structures of each layer in epidermis were intact and normal skin tissue can be observed in control group, 3Gy group and 4Gy group. A lot of keratosis was presented under epidermis, and inflammatory cell infiltration was presented under the epithelium in 5Gy group. Normal bone structures were seen under microscope in the control group, 3Gy group and 4Gy group. Bone cells were loose arranged and obvious atrophy was observed in the surface of cells with microscope in 5Gy group. TEM results showed that bone morphology was normal in the control group, 3Gy group and 4Gy group. Rough endoplasmic reticulum expansion and vesicle-like change were observed in5 Gy group. FISH: The difference was statistically significant of EGF mRNA and PDGF-B mRNA among control group, 3Gy group, 4Gy group and 5Gy group(P<0.05).Real-time quantitative PCR: the difference was statistically significant of VEGF m RNA and bFGF mRNA among control group, 3Gy group, and 4Gy group(P<0.05), but there was no difference comparing with 5Gy group(P>0.05). Conclusion: 1) Inject tumor tissue suspension according to ultrasound guidance with a high tumor formation rate and limited tumor growth range. Radiographic and histological results suggested that late intervention study should be conducted around 14 days of tumor implantation, in order to reduce the impact of liquefaction necrosis within the tumor; 2) It is feasible to applied VMAT-SIB technique to perform neoadjuvant radiotherapy on tumor model. With MRDWI, 3D-PDA, and histology, surgical margin can be effectively defined by necrosis zone in tumors adjacent to the bone; 3) Implementation of hypofraction radiotherapy on tumor models had shown that animals of VMAT-SIB 3Gy group and VMAT-SIB 4Gy group can tolerate early local toxicity, and it is a relatively safe dose fraction protocol. |
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