Contrast-Enhanced Ultrasound For Treatment Assessment In Models Of Triple Negative Breast Cancer

Part ? Establishment of Three Negative Breast Cancer Chemotherapy Model in Nude MiceObject: To explore the experimental basis for the establishment of triple negative breast cancer(TNBC)chemotherapy model in nude mice.Methods: The effect of adriamycin,cetuximab and the combination of the two drugs on the viability of MDA-MB-231 cells was detected by CCK-8 method.Nude mice subcutaneously transplanted tumor model were established in vivo.The experimental group was given chemotherapy and control group was treated with normal saline.To confirm the inhibitory effect of adriamycin and cetuximab on TNBC in nude mice,the change of tumor size was measured by ultrasonography and the volume of tumor was calculated.Results: The results of CCK-8 showed that the combination of adriamycin and cetuximab significantly reduced the viability of the cells.At 48 hours of the action duration,the cell viability of the combination group was significantly lower than that of the adriamycin alone group(P <0.05)and the cetuximab alone group(P <0.05).In the experimental group,there were 16 nude mice and 9 rats in the control group.The observation period was set to 7 days,and the rats were treated on the 1st,3rd and 5th day respectively.On the 1st,3rd,5th and 7th day,the length and width of the tumor was measured and the tumor volume was calculated.There was no significant difference in tumor volume between the two groups(P = 0.590)on the 1st day.On the 7th day,the tumor volume of the experimental group was not significantly increased compared with the first day(P = 0.205),while that of the control group was significantly increased(P = 0.003).Conclusions: The combined use of adriamycin and cetuximab can be successfully simulated TNBC chemotherapy model in nude mice.Part ? 2D Contrast-enhanced Ultrasound for Treatment Assessment in Models of Triple-negative Breast CancerObject: To evaluate the efficacy of 2D contrast-enhanced ultrasonography(2D CEUS)for treatment assessment of three-negative breast cancer(TNBC)in nude mice.Methods: According to the first part,TNBC chemotherapy model was established.The mice with tumor were randomly divided into experimental group A and control group A,experimental group B and control group B.The ultrasound equipment and contrast agent of group A were Esaote Mylab 90 and Sono Vue,respectively,which of B group were TOSHIBA Aplio 500 and Prime.2D CEUS was performed before the administration on day 1,3 and 5,and before sacrifice on day 7.All CEUS videos were quantitatively analyzed.The quantitative parameters were obtained by method A: 1)Peak intensity(Peak),2)Time to peak(TTP),3)Sharpness,4)Area under the curve(AUC).The quantitative parameters were obtained by method B: 1)Peak intensity(Peak),2)Time to peak(TTP),3)Mean transit time(m TT),4)Slope,5)Area under the curve(Area),6)Area wash in.The difference of the above parameters between experimental group and control group was analysis.All mice were sacrificed on the 7th day after CEUS.The semi-quantitative analysis of CD31 and VEGF immunohistochemistry was performed.Semi-quantitative parameters were obtained: 1)Area-p,2)Integrated optical density(IOD).The correlation analysis was made between quantitative parameters and immunohistochemistry.Results: In the experimental group A,there were 16 nude mice and 5 rats in the control group A.In the experimental group B,there were 8 nude mice and 5 rats in the control group B.In both method A and method B,the peak value of the experimental group on the 7th day was significantly decreased,and there was significant difference with the control group(P <0.05).Compared with control group B,the difference between the peak value at 3,5 and 7 days and the peak value before administration on day 1 was statistically significant(P <0.05)in experimental group B.There was no difference between experimental group A and control group A.In the semi-quantitative analysis of immunohistochemistry,the expression of MVD and VEGF in the experimental group was significantly lower than that in the control group(P <0.05).In the correlation analysis,the peak value of day 7 in method B had the highest correlation with immunohistochemistry(r> 0.600,P <0.05).Conclusions: The peak value of 2D CEUS can predict the efficacy of chemotherapy in nude mouse model of TNBC.Moreover,the peak value of method B has the highest correlation with immunohistochemistry,and it is more meaningful to predict the curative effect of chemotherapy.Part ? 3D Contrast-enhanced Ultrasound and 3D Ultrasound Molecular Imaging for Treatment Assessment in Models of Triple-negative Breast CancerObject: On the basis of the second part,the efficacy of 3D contrast-enhanced ultrasonography(3D CEUS)and 3D ultrasound molecular imaging(3D USMI)for treatment assessment of triple-negative breast cancer(TNBC)in nude mice was studied to find a more appropriate assessment method for the chemotherapy evaluation.Methods: According to the first part,TNBC chemotherapy model was established.The mice with tumor were randomly divided into experimental group and control group.Assessment time and dosing cycle were same with the second part.3D CEUS was performed using TOSHIBA Aplio 500 ultrasound equipment and Prime contrast agent.VEGFR-2 targeting Labeler contrast agent was used for 3D USMI destruction-reperfusion evaluation.All CEUS videos were quantitatively analyzed.The quantitative parameters were obtained by 3D CEUS: 1)Peak intensity(Peak),2)Time to peak(TTP),3)Mean transit time(m TT),4)Slope,5)Area under the curve(Area),6)Area wash in.The quantitative analysis parameters of 3D USMI were signal intensity(SI),which was the signal intensity of the VEGFR-2 target microbubbles actually combined with the target.The difference of the above parameters between experimental group and control group was analysis.All mice were sacrificed on the 7th day after CEUS.The semi-quantitative analysis of CD31 and VEGF immunohistochemistry was performed.Semi-quantitative parameters were obtained: 1)Area-p,2)Integrated optical density(IOD).The correlation analysis was made between quantitative parameters and immunohistochemistry.Results: There were 8 nude mice in the experimental group and 5 in the control group.All of them were evaluated by 3D CEUS.6 nude mice in the experimental group were subjected to 3D USMI after 3D CEUS.The control group was all subjected to 3D USMI after 3D CEUS.In the analysis of 3D CEUS,the peak value of the experimental group showed a decreasing trend,while the peak value of the control group showed an increasing trend,and there was a significant difference between the two groups(P <0.05)from 5th day.Other parameters such as TTP,Slope,Area,Area wash in,showed significant differences between the two groups on the 7th day(P <0.05).In the analysis of SI,there was no significant difference in SI value between the experimental group and the control group on the first day.From the third day,the SI value between the two groups showed a significant difference,and the SI value of the experimental group showed a decreasing trend,while the control group SI value showed an increasing trend.In the correlation analysis,quantitative analysis parameters and immunohistochemistry showed a certain correlation,especially of SI value on the 5th and 7th day and ?SID3-D1,?SID5-D1,?SID7-D1(R> 0.600,P <0.05).Conclusions: 3D CEUS was more sensitive and comprehensive for chemotherapy assessment.3D USMI not only reflected the expression of VEGF molecules in the tumor directly,but also could reflect the expression changes of microvascular molecular markers.