Preliminary Study Of IVIM-DWI In Monitoring Of The Anti-tumor Immune Response Induced By Cyclophosphamide In Glioma Xenograft Model

Objective:To explore the value of intravoxel incoherent motion diffusion-weighted imaging(IVIM-DWI)in monitoring the anti-tumor immune response induced by medium dose intermittent cyclophosphamide(CPA)in glioma xenograft model.Methods:Three kinds of mouse xenograft glioma models,including GL261(n=22,CPA group n=14,control group n=8),CT-2A(n=18,CPA group n=10,control group n=8)and KR158(n=18,CPA group n=10,control group n=8),were established in C57BL/6 male mice.Recorded the length and width of the transplanted tumors with vernier calipers twice a week after tumor formation and the tumor volume was calculated according to a specific formula.When the average xenograft tumor volume reached 400 mm3,the rats of each glioma model was randomly divided into two groups:the CPA and control groups.The rats in CPA group received the MEDIC regimen(CPA 140 mg/kg,intraperitoneal injection every 6 days for a total of 2 times),whereas the controls were intraperitoneally injected with the equivalent saline at the same time.All glioma-bearing mice underwent MRI scan,including T1-weight imaging(T1WI),T2-weight imaging(T2WI)and IVIM DWI sequences before,6 and 12 days after first injection.After MRI scan,the rats were harvested at each time point for pathological examination.In addition,reverse transcription-quantitative polymerase chain reaction(RT-qPCR)was performed to analyze the expression of immune-related genes in tumor cells,including(Ifnb1,CD8a,Mx1 and Cxc110).The tumor volume was compared between the two groups and the tumor-time growth curve was finally drawn.The IVIM-DWI parameters of the glioma,including D,D*f and derived parameter fD*,were measured for all rats and differences in these parameters were examined between the two groups for each model.Spearman rank correlation test was used to analyze the correlation between the IVIM-DWI parameters and pathological results.Results:1.Tumor growthFor both GL261 and CT-2A models,the tumor volume of the CPA group significantly decreased on the 6th day after first CPA administration,while that of the control group continued to increase.There were the significant differences in tumor size between the two groups on the 12th day after first administration in both GL261 and CT-2A models(P<0.001).However,for KR158 model,the tumor volume cannot be accurately measured in vivo due to the tumor infiltration into the abdominal cavity.2.IVIM-DWI measurements of the glioma and comparison between the two groupsGL261 and CT-2A models:There was no statistical difference in IVIM parameters between the CPA and the control groups at baseline(P>0.05).After administration,the D value of the CPA group showed an upward trend,which was higher than that of the control group on the 6th day and 12th day(P<0.05).The D*,f,and fD*of the CPA group significantly decreased on the 6th day and increased on the 12th day after the first injection,while the opposite trend with first rising and then falling was found in the control group.The D*,f and fD*values of the CPA group on the 6th and 12th days were lower than those of the control group(P<0.05).KR158 model:The D value of the CPA group was higher than that of the control group on the 6th day(P<0.05).There were no statistical differences in any other parameters between the CPA and the control groups at each time point.3.Pathological findingsRT-qPCR results showed that the CPA groups in GL261 and CT-2A models had the higher expression of immune-related genes and the results of immunohistochemistry showed obvious infiltration of inflammatory cells within the tumor.The D value on the 12th day was significantly correlated with the genes for immune cell recruitment and expression.However,in KR158 model,the expression of immune-related genes in the CPA group was suppressed and there was no obvious immune cell infiltration.Conclusion:1.The CPA groups of GL261 and CT-2A models had high expression of immune-related genes and obvious infiltration of inflammatory cells within the glioma,suggesting that the immune response was successfully induced by the MEDIC CPA protocol.Instead,the immune response was suppressed in KR158.2.The immune response induced by a metronomic CPA could be effectively evaluated by using IVIM DWI.The D and D*values are more sensitive in monitoring anti-tumor immune response.