Contrast-Enhanced Ultrasound In Evaluation Of Angiogenesis And Radiotherapy Effect Of Transplanted Nasopharyngeal Carcinoma In Nude Mice

Part?: Contrast-enhanced Ultrasound in Evaluation of Angiogenesis and Tumor Staging of Nasopharyngeal Carcinoma in Nude MiceObjective: To explore the use of contrast-enhanced ultrasound(CEUS)in evaluating angiogenesis in a xenograft nasopharyngeal carcinoma model in nude mice and the evolution of CEUS parameters according to the growth of nasopharyngeal carcinoma.Methods: Nude mice were divided into three groups according to experiments conducted at various times from tumor implantation(8 mice/group;group A: 4 weeks from implantation;group B: 6 weeks from implantation;group C: 8 weeks from implantation).CNE-2 cells were transplanted in 24 nude mice and CEUS evaluations of the tumors were performed at 4,6 or 8 weeks from implantation.CEUS parametric perfusion images and pathological findings were recorded.R version 3.4.4 software was used to analyze the CEUS parameters and pathological findings.Results: One-way anova analysis indicated statistically significant differences among the three groups with the parameters of peak intensity(PI)(P<0.001),area wash in(AWI)(P<0.001),area wash out(AWO)(P<0.001),tumor volumes(P<0.001)and microvessel density(MVD)(P<0.001).Pearson correlation coefficient analysis indicated that MVD was correlated with tumor volume(r=0.644,P=0.001),PI(r=0.904,P<0.0001),AWI(r=0.547,P=0.008)and AWO(r=0.744,P<0.0001).Tumor volume was correlated with MVD(r=0.644,P=0.001),PI(r=0.625,P=0.002),AWI(r=0.528,P=0.012)and AWO(r=0.784,P<0.001).The percentage of necrosis in histological sections was correlated with the percentage of CEUS unperfused area(r=0.446,P=0.038).Spearman rank correlation coefficient analysis indicated that vascular endothelial growth factor(VEGF)was correlated with PI(r=0.462,P=0.032).Welch t test indicated PI,AWI and AWO parameters were significantly lower than that of kidneys(P<0.001,P=0.009,P=0.005).Conclusions: The CEUS parameters PI,AWI and AWO indirectly reflect the MVD and the tumor volume in our model of subcutaneous transplanted nasopharyngeal carcinoma in nude mice,providing precious information on angiogenesis and tumor growth.Meanwhile,VEGF may play a role in promoting angiogenesis of nasopharyngeal carcinoma.Part?: Contrast-enhanced Ultrasound Evaluation of Radiotherapy Effect on Transplanted Nasopharyngeal Carcinoma in Nude MiceObjective: To study CEUS perfusion characteristics and changes of MVD and VEGF after radiotherapy(RT)for transplanted nasopharyngeal carcinoma in nude mice,analyze the correlation between CEUS quantitative parameters of transplanted tumor and histopathological results,and explore whether CEUS can reflect angiogenesis of transplanted nasopharyngeal carcinoma after RT and whether it is suitable for evaluation of RT effect.Methods: A total of 30 nude mice models of human nasopharyngeal carcinoma cell line(CNE-2)were established and randomly divided into control group and RT group with 15 mice each.CEUS imaging was performed on nude mice of each group when the tumor grew to a diameter of 1-1.5cm.The subcutaneous transplanted tumor on the back of nude mice in RT group was irradiated with 15 Gy single dose with Co-60 GWXJ80.The volume changes of transplanted nasopharyngeal carcinoma after RT were measured.Six nude mice were randomly selected from the control group and RT group respectively for CEUS examination 7 days after RT.The rest of the nude mice in the control group and RT group were given CEUS examination14 days after RT.CEUS perfusion characteristics of nasopharyngeal carcinoma were recorded.ROI was selected for TIC analysis to study the differences of quantitative parameters PI,AWI and AWO among the groups.The nude mice were killed immediately after CEUS,and nasopharyngeal carcinoma tissues were retained for HE staining,CD34 and VEGF staining.The differences of MVD and VEGF and the correlation between quantitative parameters and MVD and VEGF were analyzed.R version 3.4.4 software was used for statistical analysis.Results: There was no significant difference in CEUS parameters PI,AWI and AWO between control group and RT group before RT when the ROI was the whole tumor region or the high enhancement region.On the 7th and 14 th days after RT,the tumor volume in RT group was significantly smaller than that in control group.On the 7th day after RT,the mean values of CEUS parameters PI and AWO of nasopharyngeal carcinoma transplanted tumor in RT group were lower than those in control group.The expression of MVD and VEGF in tumor tissues in vitro in RT group was lower than that in control group.On the 14 th day after RT,the CEUS parameters PI,AWI and AWO of nasopharyngeal carcinoma transplanted tumor in RT group were lower than those in control group.The expression of MVD and VEGF in tumor tissues in vitro in RT group was lower than that in control group.CEUS quantitative parameters PI,AWI,AWO and VEGF expression of nasopharyngeal carcinoma transplanted tumor in RT group after RT were positively correlated with MVD.Tumor volume is positively correlated with CEUS quantitative parameters PI,AWI,AWO and MVD respectively.CEUS parameters PI,AWI and AWO of nasopharyngeal carcinoma transplanted tumor in RT group and control group were positively correlated with tumor volume,MVD and VEGF expression,respectively.Aditional,VEGF expression in tumor tissues was positively correlated with MVD.Conclusion: Radiation can reduce MVD and VEGF expression in nasopharyngeal carcinoma transplanted tumor tissue established by CNE-2 cell line,indicating that radiation can inhibit tumor by inhibiting nasopharyngeal carcinoma angiogenesis.There is a positive correlation between tumor volume and MVD and PI after RT,which indicates that the reduction of tumor volume after RT not only by killing tumor cells through direct action of radiation,but also may inhibit tumor growth through reduction of tumor microvessel density.The average value of CEUS quantitative parameters PI and AWO of subcutaneous nasopharyngeal carcinoma transplanted tumor in RT group is lower than that of the control group after RT,which is similar to the changes of MVD and VEGF expression.Meanwhile,the quantitative parameters PI,AWI,AWO and VEGF expression are positively correlated with MVD,which indicates that RT leads to the reduction of blood volume of nasopharyngeal carcinoma transplanted tumor,and CEUS can reflect the microvascular perfusion change of nasopharyngeal carcinoma transplanted tumor after RT,so CEUS imaging can be used to realize the blood supply of nasopharyngeal carcinoma after RT and evaluate the RT effect.Part?: Bioinformatics Analysis of Differentially Expressed Genes Related to Nasopharyngeal CarcinomaObjective: Gene expression data which were related to nasopharyngeal carcinoma will be obtained from the Gene Expression Omnibus.Bioinformatics analysis will be used to integrate and analyze data to obtain differentially expressed genes and related signal pathways related to the occurrence and development of nasopharyngeal carcinoma.To verify the relationship between VEGF and nasopharyngeal carcinoma,and to explore other tumor-related genes and signal pathways that may be involved in the occurrence and development of nasopharyngeal carcinoma.Methods: RStudio-1.1.456 software was used to download the gene expression profiles GSE53819 data set from Gene Expression Omnibus.The gene expression profiles GSE53819 was m RNA gene expression profiles which containing from 18 nasopharyngeal carcinoma tissues from nasopharyngeal carcinoma patients and 18 non-cancerous nasopharyngeal tissue from non-cancerous nasopharyngeal chronic inflammation patients.Extract the expression matrix of the gene expression profiles dataset and extract clinical information and gene annotation.Identified differentially expressed genes using limma function package,meanwhile,|log FC | > 1 and P value < 0.01 were considered to be significant statistical differences.Volcano map and heat map of the differentially expressed genes were presented.The function and pathway enrichment of differentially expressed genes were analyzed,and the protein-protein interaction network was carried out using STRING online analysis software.The top 20 central genes related to nasopharyngeal carcinoma were identified using Cytoscape 3.4.0 software and Cyto Hubba App plug-in.Results: A total of 658 differentially expressed genes were obtained under the| log FC | > 1 and P value < 0.01 conditions.267 genes were up-regulated and391 genes were down-regulated from the differentially expressed genes.Functional enrichment analysis and pathway enrichment analysis of differentially expressed genes showed that up-regulated genes differentially expressed in nasopharyngeal carcinoma mainly accumulated in extracellular matrix and basement membrane,participated in biological processes such as secretion and regulation of cytokines and regulation of cell-matrix adhesion.Kyoto encyclopedia of genes and genomes analysis showed that the differentially expressed genes relating nasopharyngeal carcinoma were enriched in extracellular matrix-receptor interaction,micro RNAs in cancer,cytokine-cytokine receptor interaction,p53 signal pathway,cancer pathway and other pathways.Down-regulated genes differentially expressed in nasopharyngeal carcinoma were mainly enriched in motor cilia,axons,dynein complexes,external power arms.They were mainly involved in biological processes such as activation,proliferation and regulation of B cells,cilia movement and so on.Kyoto Encyclopedia of Genes and Genomes analysis showed that they were significantly enriched in B cell receptor signaling pathways,calcium signaling pathways and so on.Meanwhile,20 hub genes including VEGFA,FN1,CCND1,PTGS2 were screened out through protein-protein interaction network analysis.Cytoscape software analysis suggested that the hub genes might play an important role in the occurrence and development of nasopharyngeal carcinoma.Conclusion: This study has screened out differentially expressed genes and hub genes that are statistically significantly related to nasopharyngeal carcinoma with bioinformatics analysis.In this study,gene ontology analysis and kyoto encyclopedia of genes and genomes analysis have revealed the relevant genes and pathways enrichment with biological significance in the molecular mechanism of nasopharyngeal carcinoma.VEGF has been proven to be closely related to the occurrence and development of nasopharyngeal carcinoma.At the same time,other relevant genes with biological significance in the molecular mechanism of nasopharyngeal carcinoma and their pathways of action have also been found,providing potential biological targets and directions for the research on the occurrence and development of nasopharyngeal carcinoma.