The Experimental Study Of ⁹⁹Tc^m-3PRGD₂ Imaging In The Diagnosis And Evaluation Of Treatment In Bone Transplanted Tumor

Objective: Research ⁹⁹Tc^m-3PRGD₂ SPECT imaging on diagnosis and evaluation of treatment after radiotherapy in bone transplanted tumor,and discuss the significance of ⁹⁹Tc^m-3PRGD₂ SPECT imaging in the evaluation of treatment after radiotherapy for bone transplanted tumor.Methods:1 Establishment of rat model of bone transplanted tumorThe Walker256 rat brest cancer cells which stored in liquid nitrogen tanks were recovered and injected into the abdominal cavity of female Wistar rats which wight 140g¹60g.After 7¹0 days,the rat abdominal bulge.The ascites of Walker 256 rat breast cancer cells was extracted,centrifugation,and the cell concentration was adjusted to 108/ml.80 female Wistar rats with body weight of 220g²50g were selected and were anesthetized with chloral hydrate,then drilled in the left hind leg 1.5cm from the tibial articular surface.70 of 80 female Wistar rats were selected,5uL ascites mixture（about 5×105 tumor cells）were injected in marrow cavity.The ascites mixture boiled 30 min was inactivated,and injected into the other 10 rats（the same position,volume and quantity）The rats tibia were scanned with CT to determine the tumor formation on 10 th,20th and 30 th day.Tumor formation criteria: reduced density area appeared in bone marrow cavity and cortical bone wsa defect.There were 40 rats eventually format tumor in the 70 rats,the other 10 rats injected with inactivated tumor cells are not tumorigenic.2 The measurement of volume on bone transplanted tumorThe rats with tumor were scanned by CT,the long diameter and short diameter of the tumor were calculated by AW4.5,and the volume was calculated（V=Long diameter×Short diamete2/2）.3 Synthesis and radiochemical purity test of ⁹⁹Tc^m-3PRGD₂Fresh and aseptic Na99TcmO4 leacheate was add into 3PRGD2 freeze-dried product.Put them into 100℃ water bath pot boiling 20 min after fully shaking and then cooled to room temperature.The imaging agent was mixed with the rat serum and placed in a water bath at 37℃.Determine of the radiochemical purity of ⁹⁹Tc^m-3PRGD₂ at 1h,2h,4h and 6h by thin layer paper chromatography.¹⁸F-FDG provided by NanJing JiangYuan Andike Pharmaceutical Group Company,the radiochemical purity ≥90%.4 Experimental groupingThe 40 rats which eventually format tumor were randomly divided into 4 groups according to random number table method,each group was divided into 10 groups.They were diagnostic tumor bearing group,sham radiotherapy group,5Gy radiotherapy group and 25 Gy radiotherapy group.The other 10 rats which were injected with inactivated tumor cells was the diagnostic control group.5 External radiation therapyIn the sham radiotherapy group,5Gy radiotherapy group and 25 Gy radiotherapy group,the single dose of 0Gy,5Gy and 25 Gy 6MeV linear accelerator were used to treat the bone transplanted tumor at the end of the first SPECT imaging,respectively.6 ⁹⁹Tc^m-3PRGD₂ SPECT imaging and ¹⁸F-FDG PET imaging⁹⁹Tc^m-3PRGD₂（74MBq/0.2ml）was injected into caudal vein on the 30 th day after operation in diagnostic tumor bearing group and diagnostic control group,SPECT imaging was performed 1h after injection.The ROI（Region of interest）technique was utilized to measure the radioactivity count ratio of the bone transplanted tumor site and the same size site of the corresponding region,that is the T/NT ratio,ROI was about 20mm²,each rat was repeated 5 times and the mean value was taken as the result.¹⁸F-FDG PET imaging（37MBq/0.2ml）was performed in sham radiotherapy group,5Gy radiotherapy group and 25 Gy radiotherapy group on the 30 th day after operation and 10 th days after radiotherapy.AW4.5 workstation automatically ploted the volume of interest（Volume of interest,VOI）and calculated the SUVmax.⁹⁹Tc^m-3PRGD₂ SPECT imaging were performed one day intervals and calculated the ratio of T/NT.7 HE staining and immunohistochemical staining of αⅤβ3 receptor and Ki67 antigen in bone transplanted tumorThe rats in each group were sacrificed at the end of the imaging.The bone transplanted tumor was fixed,decalcified,embedded,sectioned and stained with HE.Immunohistochemical staining was performed on tumor sections in each group,and the proportion of αⅤβ3 positive cells and proliferating cell nuclear antigen Ki67 positive cells were calculated,respectively.8 Statistical analysisSPSS21.0 statistical software was used for statistical analysis and the measured datas consistent with normal distribution were expressed as mean±standard deviation（`x±s）.Two sample T-test or Wilcoxon rank sum test was used for the comparison of diagnostic tumor bearing group and diagnostic control group.One-way ANOVA or Kruskal-Wallis H rank sum test was used for the comparison among sham radiotherapy group,5Gy radiotherapy group and 25 Gy radiotherapy group,and compared between every two groups with SNK test.T/NT ratio and SUVmax were correlated with αⅤβ3 receptor and Ki67 antigen expression by linear correlation analysis or Spearman rank correlation.Result:1 Establishment of bone transplanted tumor model40 rats were finally diagnosed as tumor successfully by CT in 70 rats,and the rate of tumor formation was 57.1%.10 rats in diagnostic control group were not found bone destruction.2 Radiochemical purity of ⁹⁹Tc^m-3PRGD₂The solution of ⁹⁹Tc^m-3PRGD₂ was colorless and transparent,and the radiochemical purity of 1h,2h,4h and 6h were 97.6%,97.2%,96.1%,and 95.4% respectively at 37℃.3 Volume change of bone transplanted tumor before and after treatmentThere was no significant difference among the sham radiotherapy group,5Gy radiotherapy group and 25 Gy radiotherapy group in volume of pretherapy bone transplanted tumor（20.98±2.90mm3,21.29±3.26mm3,22.11±3.19 mm3,F=0.343,P=0.700）;There was significant difference among the three groups in volume of post-therapy bone transplanted tumor（26.75±2.85 mm3,23.44±3.17 mm3,22.29±3.03 mm3,F=6.016,P=0.007）.The volume of bone transplanted tumor in 5Gy radiotherapy group and 25 Gy radiotherapy group were less than that of sham radiotherapy group（P = 0.021,0.002）,but there was no statistical difference between 5Gy radiotherapy group and 25 Gy radiotherapy group（P=0.379）.4 ⁹⁹Tc^m-3PRGD₂ SPECT imaging and ¹⁸F-FDG PET imagingIn diagnostic tumor bearing group,the bone transplanted tumor in left tibia was significantly concentrated by SPECT imaging,but there was no significant concentration of radioactive nuclide or only mild enhancement in the surgery area of diagnostic control group by SPECT imaging.There was difference between the two groups of T/NT ratio（3.48±0.44,1.10±0.84,t=16.66,P=0.000）.There was obvious radioactive concentration in the bone transplanted tumor in sham radiotherapy group,5Gy radiotherapy group and 25 Gy radiotherapy group before radiation therapy.T/NT ratio were（3.70±0.30,3.87±0.24,3.96±0.26）respectively,and there was no statistical difference among the three groups（F =2.516,P=0.100）.The radioactive concentration of the left tibia was still visible on the 10 th day after radiation therapy,the T/NT ratio were（4.03±0.33,3.51±0.31,2.00±0.28）respectively,and there was significant difference among the three groups（F=116.535,P=0.000）.The T/NT ratio of 5Gy radiotherapy group and 25 Gy radiotherapy group were less than that of sham radiotherapy group（P<0.05）and the T/NT ratio of 25 Gy radiotherapy group was less than that of 5Gy radiotherapy group（P=0.000）.There were obvious radioactive concentration in bone transplanted tumor by PET scan before radiotherapy in sham radiotherapy group,5Gy radiotherapy group and 25 Gy radiotherapy group.There was no statistical difference among the three groups in SUVmax（12.83±1.04,13.58±1.56,13.00±1.01,F=1.050,P=0.364）.But there was significant difference among the three groups（14.34±2.16,9.06±1.76,8.43±2.34,F=23.876,P=0.000）in SUVmax after radiotherapy.The SUVmax of 5Gy radiotherapy group,25 Gy radiotherapy group were less than sham radiotherapy group（P<0.05）,but there was no significant difference in SUVmax value between 5Gy radiotherapy group and 25 Gy radiotherapy group.5 Bone transplanted tumor HE stainingThe bone matrix structure is not complete in diagnostic tumor bearing group,sham radiotherapy group,5Gy radiotherapy group,25 Gy radiotherapy group,and a large number of tumor cells were seen in bone marrow cavity.There were a large number of necrotic tumor cells in 5Gy radiotherapy group and 25 Gy radiotherapy group,especially in 25 Gy radiotherapy group.In the diagnostic control group,the bone cortex of the tibia was complete and clear.6 Immunohistochemical expression of integrin αⅤβ3 receptor and proliferating cell nuclear antigen Ki67The immunohistochemistry result of diagnostic tumor bearing group show that integrin αⅤβ3 receptor were in the cell membrane and cytoplasmic of tumor cells and vascular endothelial cells.While the expression of αⅤβ3 receptor was not detected in diagnostic control group.The proportion of positive cells in sham radiotherapy group,5Gy radiotherapy group and 25 Gy radiotherapy group were（67.12%±5.86%,42.96%±7.07%,32.41%±4.17%）respectively,and there was significant difference among the three groups（F=93.394,P=0.000）.The proportion of integrin αⅤβ3 receptor positive cells in sham radiotherapy group was significantly higher than that of 5Gy radiotherapy group and 25 Gy radiotherapy group（P=0.000,0.000）,and 5Gy radiotherapy group was higher than 25 Gy radiotherapy group（P=0.000）.The nuclei were stained with brown yellow as Ki67 positive cells in sham radiotherapy group,5Gy radiotherapy group and 25 Gy radiotherapy group.The proportion of positive cells were（66.20%±14.94%,36.20%±13.01%,32.62%±13.63%）respectively,and there was significant difference among the three groups（F=16.489,P<0.05）.The proportion of positive cells in 5Gy radiotherapy group and 25 Gy radiotherapy group were less than sham radiotherapy group（P=0.000,0.000）,but there was no statistical difference between the 5Gy radiotherapy group and 25 Gy radiotherapy group（P=0.565）.7 Correlation analysis between T/NT ratio and SUVmax with Ki67 and αⅤβ3 receptor expressionThe T/NT ratio of SPECT imaging and SUVmax of PET imaging were correlated with the expression level of αⅤβ3 receptor（r1= 0.847,r2=0.679,P= 0.000,0.000）and Ki67 antigen（r1= 0.584,r2=0.806,P= 0.001,0.000）.Conclusion1 ⁹⁹Tc^m-3PRGD₂ is easy to label,high radiochemical purity and well stability.2 ⁹⁹Tc^m-3PRGD₂ SPECT imaging can diagnosis and evaluate the radiotherapy efficacy of bone transplanted tumor.3 ⁹⁹Tc^m-3PRGD₂ SPECT imaging and ¹⁸F-FDG PET imaging can both be used to evaluate the radiotherapy efficacy of bone transplanted tumor.When the ¹⁸F-FDG PET imaging can not be performed,⁹⁹Tc^m-3PRGD₂ SPECT imaging can be used to replace PET imaging.