Experimental Study On The Correlation Between T2* Mapping,IVIM,DKI,HIF-1ɑ And Pimonidazole Expression In The Nude Mouse Fibrosarcoma Model Based On Imaging-Pathology Level

Purpose:To study the value of T2*mapping,IVIM,and DKI parameters(including R2*,ADC,D,D*,f,MD,and MK values)in fibrosarcoma nude mouse models for assessing the expression of HIF-1αand pimonidazole,pathological indicators in the central region and the peripheral region of the tumor,using a image-pathology controlled approach.Materials and methods:1.Experimental materialsHT-1080 cell line;30 female Balb/c nude mice,MEM medium,culture dishes,HIF-1αantibody,pimonidazole injection,pimonidazole antibody,ultra clean bench,cell incubator,microscope,surgical instruments,GE750W magnetic resonance scanner,etc.2.Experimental methods2.1 HT-1080 cells culture2.2 Construction of fibrosarcoma murine modelHT-1080 cells were collected and injected into the right thigh muscle of nude mice.The models were considered successful when a significant mass was observed at the seeded tumor site.2.3 MR imaging of fibrosarcoma murine modelThe models were examined on a GE 750W 3.0T MRI scanner,and the scan sequences included:T1WI,T2WI,T2*mapping,IVIM,and DKI.Post-processing was performed to generate gray-scale maps and pseudo-color maps of R2*,ADC,D,D*,f,MD,and MK values.The area inside the tumor less than 1 mm from the edge was classified as the peripheral area of the tumor,otherwise it was classified as the central area of the tumor.The ROIs were outlined using the small sample outline method:five ROIs were outlined in each of the solid parts of central region and the peripheral region in R2*,ADC,D,D*,f,MD,and MK maps with reference to FSE T2-weighted imaging,excluding the areas of hemorrhage,calcification,and cystic lesions.The area of each ROI was 1 mm~2,and the average value of the 5 ROIs was finally taken as the measurement value.2.4 Imaging-pathological control method for nude mouse fibrosarcoma modelA marker line perpendicular to the tumor site of the right hind limb of nude mice with fibrosarcoma was made.A fragrant oil ring was fixed along the marker line,and the MRI scan was made so that the fragrant oil ring was completely visualized in the figure.After the tumor was removed,a surgical suture was tied at each of the three points of the sticky oil ring,and another line was tied at the tumor site near the head side to determine the tumor direction.After formalin fixation for 8h,the tumor was sectioned along the four points marked on the tumor and subsequently stained.2.5 HE staining and observation of the central area and the peripheral area of the tumor of the fibrosarcoma specimen2.6 Immunohistochemical staining and observation of the central region and the peripheral region of the tumor of the fibrosarcoma specimen3.Observational indicators4.Statistical methodsStatistical analysis was performed using SPSS 24.Normal distribution of data was performed using Kolmogorov-Smirnov test.Interclass correlation coefficient(ICC)was assessed for interobserver agreement.Correlation analysis between T2*mapping,IVIM,DKI parameters and HIF-1αand pimonidazole expression was performed using Pearson or Spearman correlation tests.Differences in HIF-1αexpression,pimonidazole expression,T2*mapping,IVIM,and DKI parameters in the central and peripheral regions of the tumor were compared by paired-samples t-test or Wilcoxon signed-rank test.Differences in T2*mapping,IVIM,and DKI parameters at sites with low and high expression of HIF-1αand pimonidazole were compared by independent sample t-test.The ability of T2*mapping,IVIM,and DKI to distinguish between the high and low expression groups of HIF-1αand pimonidazole was determined by ROC analysis,and the area under the curve(AUC),critical value,sensitivity,and specificity were calculated.P<0.05 was considered statistically significant.Results:1.Tumorigenic performance of nude rat fibrosarcoma modelA total of 30 intramuscular nude rat fibrosarcoma sarcoma models were constructed.The tumor volume ranged from 781-1416 mm~3.2.MRI performance of the central region and the peripheral region of the tumor in the nude mouse fibrosarcoma modelThe tumors of 30 nude mice with fibrosarcoma models were oval or irregularly lobulated,with equal or slightly high signal on T1WI.No difference in signal intensity between the central and peripheral regions of the tumors was observed.On the T2*mapping image,it showed inhomogeneous high signal,and the signal in the central region was higher than that in the peripheral region.The MK and R2*values in the post-processing parameters were greater in the peripheral region than in the central region of the tumor,and the f value in the peripheral region was smaller than that in the central region.3.The pathological manifestations of the central region and the peripheral region of the tumor in nude mice fibrosarcoma model3.1 Conventional pathological manifestations of nude mice fibrosarcoma modelThe tumor was oval or irregularly lobulated with soft texture.The surface of the tumor was grayish white or red.Microscopically,the tumor cells showed consistent morphology of spindle-shaped fibroblast-like cells,and the classical bundle-like arrangement was seen.3.2 Immunohistochemical staining of HIF-1αand pimonidazole in the central and peripheral regions of the tumor in nude mice with fibrosarcoma modelIn 30 nude mice with fibrosarcoma model,HIF-1αand pimonidazole positivity was manifested by yellowish,brownish or tan nuclei/stroma.The positive expression rate of HIF-1αand pimonidazole in the peripheral area was greater than that in the central area of the tumor.4.T2*mapping,IVIM and DKI used to evaluate the expression of HIF-1αand pimonidazole in the central and peripheral regions of the tumor in nude mice with fibrosarcoma model4.1 Correlation of T2*mapping,IVIM and DKI with their HIF-1αexpressionR2*and MK values showed moderate positive correlation with HIF-1α(r=0.552,0.550)D value and f value showed low negative correlation with HIF-1α(r=-0.363,-0.362).4.2 Correlation of T2*mapping,IVIM,DKI and their pimonidazole expressionR2*and MK values were strongly correlated with pimonidazole(r=0.714,0.779)D values were moderately negatively correlated with pimonidazole(r=-0.498);ADC,f and MD values were weakly negatively correlated with pimonidazole(r=-0.327,-0.348,-0.369).4.3 Differences in imaging parameters of T2*mapping,IVIM,DKI and their HIF-1αhigh and low expression sitesThe R2*and MK values of HIF-1αhigh expression sites were higher than those of low expression sites;the D and f values of HIF-1αhigh expression sites were lower than those of low expression sites.4.4 Differences in imaging parameters of T2*mapping,IVIM,DKI and their pimonidazole high and low expression sitesThe R2*and MK values of pimonidazole high expression sites were higher than those of low expression sites;the ADC,D,and f values of pimonidazole high expression sites were lower than those of low expression sites.4.5 Diagnostic efficacy of T2*mapping,IVIM,and DKI for assessing their HIF-1αexpressionWhen the area under the curve of R2*,D,f,and MK values were 0.747,0.717,0.714,and 0.713,respectively,the diagnostic thresholds for high and low expression of HIF-1αwere 113.26,0.331,44.8,and 1.87,respectively,with sensitivities of 81%,66.7%,71.8%,and 57.1%,and specificities of 61.5%,82.1%,61.9%,and 82.1%,respectively.4.6 Diagnostic efficacy of T2*mapping,IVIM,and DKI for assessing their pimonidazole expressionWhen the area under the curve of R2*,ADC,D,f,and MK values were 0.804,0.661,0.782,0.705,and 0.920,respectively,the diagnostic thresholds for the ability of pimonidazole expression were 114.39,0.347,0.331,61.43,and 1.86,respectively,and the sensitivities were 89.5%,63.2%,68.4%,48.8%,and 84.2%,and the specificity was65.9%,75.6%,80.5%,94.7%,and 90.2%,respectively.Conclusions:In this study,we successfully constructed 30 nude mice fibrosarcoma models,and carried out experimental studies of T2*mapping,IVIM,and DKI in nude mice fibrosarcoma models with their HIF-1ɑand pimonidazole expression correlation by means of body surface line-imaging surface-pathological surface precision control.We came to the following conclusions:1.Parameters of T2*mapping,IVIM and DKI in nude mouse fibrosarcoma model:R2*value correlated with its HIF-1αexpression and showed moderate positive correlation,D value and f value correlated with its HIF-1αexpression and showed low negative correlation,MK value correlated with its HIF-1αexpression and showed moderate positive correlation,among which R2*value correlated more strongly;i.e.T2*mapping,IVIM and DKI of nude mouse fibrosarcoma model can predict the expression of HIF-1α,among which R2*value is better.2.The parameters of T2*mapping,IVIM and DKI in nude mouse fibrosarcoma model:R2*value was correlated with its pimonidazole expression and showed a high positive correlation,D value was correlated with its pimonidazole expression and showed a moderate negative correlation,ADC value and f value were correlated with its pimonidazole expression and showed a weak negative correlation,MK value was correlated with its pimonidazole expression and showed a high positive correlation,and MD value was correlated with its pimonidazole expression and showed a high positive correlation.The correlation between MD value and pimonidazole expression was high and weak,and the correlation between MD value and pimonidazole expression was weak and negative,among which MK value was stronger;i.e.,pimonidazole expression could be predicted by T2*mapping,IVIM and DKI in nude mouse fibrosarcoma model,among which MK value was better.