The Application Value Of Multimodal Magnetic Resonance Imaging To Monitor The Early Antitumor Mechanism Of CuS@GOD Nanoparticles In 4t1 Breast Cancer Xenograft Model

Objective:To explore the early antitumor mechanism of CuS@GOD nanoparticles in a female Balb/c mouse 4T1 breast cancer xenograft model by multimodal magnetic resonance imaging(MRI)and evaluate its application valueMethods:Female Balb/c mice 4T1 breast cancer xenograft models were established and randomized into four groups:normal saline(n=25),CuS@GOD(n=25),CuS+laser(n=25)and CuS@GOD+laser(n=25)groups.Both CuS and CuS@GOD nanoparticles were chelated with gadolinium contrast Gd-DTPA.The 0.1ml saline or nanoparticles(0.7mg/ml)were directly injected within the tumor center.About 1 hour and 55 minutes after the intratumoral injection,the near infrared(NIR)laser light was used to irradiate the tumors in the CuS+laser and the CuS@GOD+laser groups for 5 minutes and infrared thermal imaging were subsequently performed by the infrared thermal mapping apparatus to measure the tumoral temperatureTen mice in each group were randomly selected for intravoxel incoherent motion-diffusion weighted imaging(IVIM-DWI)and blood oxygenation level dependent functional magnetic resonance imaging(BOLD-fMRI)scan at baseline and serially at 0,0.5,1,2,4,24 hours after infrared thermal images taken in the CuS+laser and the CuS@GOD+laser groups,or 2 hours after intratumoral administration in the normal saline and CuS@GOD groups.At the end of the 24h timepoint scanning in each group,3 mice were randomly chosen and sacrificed for pathological examinationThree mice in each group were randomly chosen and sacrificed at the same timepoints corresponding to the MR scan after treatment and then the tumors were removed and fixed.In addition,3 model mice were used as controls.After fixation,the tumors were sliced in the central transverse section and the hematoxylin eosin(HE)staining was performed.Then the antibodies against ki67,VEGF,EGLN1 and ?-H2AXser139 were used for tumor immunostaining.Besides,TUNEL assays of tumor tissues were done.The results of MRI and pathology were statistically analyzed and correlatedResults:The D values slightly increased at Oh compaired to the baseline in the CuS+laser and the CuS@GOD+laser groups,then reduced at 0.5h but rebounded till the 24h(P<0.05).The D values in the normal saline and CuS@GOD groups,however,remained almost constant.The D*value in the CuS+laser group increased from baseline to the 1h,followed by the gradual drop to the 24h,which was similar at baseline(P=0.017).The D*value of the other three groups showed almost no change.After the continuous reduction from the baseline,the f values in the CuS+laser and the CuS@GOD+laser groups reached to the minmum at 1h and 2h timepoints,respectively,followed by the sustaining increasing to the maximum at 24h(P<0.05).Nevertheless,the f values decreased at Oh compared to the baseline in the normal saline and CuS@GOD groups and kept constant at the later timepoints(P<0.05).There were a sudden surge of R2*values in the CuS+laser and the CuS@GOD+laser groups at Oh,followed by a continuous signal decay afterwards to the baseline level at 24h(P<0.05).But the decreasing speed of the R2*value in the CuS@GOD+laser group was smaller than that in the CuS+laser group.The average R2*values in the control groups were relatively stable.The mean IOD of ki67 staining in the CuS+laser and CuS@GOD+laser groups continuously reduced after treatment,reaching the minimum at 24h(P<0.05).The mean IOD in the normal saline and the CuS@GOD groups decreased at Oh compared to the baseline,while kept stable at the remaining timepoints.After treatment,the TUNEL positive staining rate(%)in the CuS+laser and CuS@GOD+laser groups continuously increased and reached the maximum at 24h(P<0.05).The positive staining rate(%)in the normal saline and the CuS@GOD groups at Oh were higher than that at the baseline(P<0.05),then remained stable at the subsequent timepoints.The mean IOD of the VEGF staining reduced in the all groups after treatment and reached to the minimum at 1h.Thereafter,the mean IOD maintained at the lowest level in the normal saline and the CuS@GOD groups(P<0.05).The mean IOD in the CuS+laser and CuS@GOD+laser groups started to increase at 2h and reached to the maximum at 24h(P<0.05).The tendency of the mean IOD of the EGLN1 staining in the CuS+laser and the CuS@GOD+laser groups were similar to that of the mean R2*values.The mean IOD increased slightly in the normal saline and CuS@GOD groups at Oh after treatment,and then remained stableThe positive staining rates(%)of ?-H2AXser139 successively increased from the normal saline,CuS@GOD,CuS+laser to CuS@GOD+laser groups,which were 55.9±1.4,58.8±0.4,60.7±0.8 and 64.3±2.3,respectively(P=0.000)The average temperatures(?)of the tumors before and after treatment in the CuS+laser and the CuS@GOD+laser groups were 33.9±3.0 and 56.6±4.7,34.5±2.6 and 55.2±5.1,respectivelyThe D value of IVIM-DWI was negatively correlated with the mean IOD of ki67 staining,and positively correlated with TUNEL and ?-H2AXser139 staining.The f and the R2*values of BOLD-fMRI had a significantly positive linear correlation with VEGF and EGLN1 staining,respectivelyConclusion:The IVIM-DWI and BOLD-fMRI were capable of monitoring the early antitumor mechanism of CuS@GOD nanoparticles in vivo:photothermal effect,photodynamic effect and photocatalytic effect.The D,f and R2*values are the most sensitive biomarks for evaluation.