Experimental Study On Reconstruction Of Tumor Vessels And Microenvironment By Magnetic Resonance/Photoacoustic Bimodal Imaging

Background:Malignant tumor is an important disease that threatens human health,and the mechanism of the interaction between tumor cells and blood vessels has not been fully elucidated.Different from normal tissues,the disorder of vascular structure in tumor shows uneven distribution,tortuous dilatation,insufficient blood flow,high permeability and other functional abnormalities.Tumor neovascularization network determines tumor growth,invasion,metastasis and response to treatment.Recent studies have confirmed that appropriate dose of radiotherapy can change the structure and function of tumor microvessels,and the use of drugs such as immune checkpoint inhibitors can change the state of "immunosuppression" such as hypoxia in tumor microenvironment.in order to achieve the therapeutic effect.Therefore,it is very important to explore an effective monitoring method to observe the changes of tumor vessels noninvasively in vivo.Objective:MR/PA bimodal imaging technique was used to dynamically monitor the changes of tumor vessels induced by radiotherapy in a mouse model,and to observe the changes of immune-related tumor microenvironment combined with biological techniques,so as to further clarify the mechanism of vascular remodeling on inhibiting tumor growth and optimize the combined therapy.Methods:This study was approved by the Ethics Committee of the third affiliated Hospital of Kunming Medical University,and all animal experimental procedures are in accordance with the institutional guidelines on animal care and use.72 BALB/c female mice were purchased from the Experimental Animal Center of Kunming Medical University to establish the model of breast cancer-bearing mice.The mice were randomly divided into tumor control group（n=12）,anti-PD-L1 drug group（n=12）,high-dose radiotherapy group（high-dose group）and low-dose radiotherapy group（low-dose group）.High dose radiotherapy plus PD-L1 group（n=12）,low dose radiotherapy fractionation + PD-L1 group（low dose combined therapy group）.The radiotherapy regimen is as follows: low-dose radiotherapy received 2Gy irradiation for 14 times,high-dose radiotherapy fractionation received 8Gy radiation for 3 times.The PD-L1 antibody was diluted with normal saline into a PD-L1 monoclonal antibody solution with concentration of 1mg/ml,and then slowly injected into the abdominal cavity of 0.01mg/g,and the treatment period was 14 days.The changes of tumor blood vessels and microenvironment after radiotherapy were monitored by magnetic resonance/ photoacoustic bimodal imaging.The changes of tumor vascular density,expression of immune cells and expression of apoptotic proteins were quantitatively analyzed by immunofluorescence,flow cytometry and Western blotting.In the process of tumor combined therapy,the survival rate of mice in each experimental group was observed and recorded,and the effects of radiotherapy and combined therapy were analyzed and compared.All the data obtained are expressed as mean ±standard mean error（SEM）.Magnetic resonance / photoacoustic image data,gray value of target protein in Westernblot,cell expression data in flow cytometry and immunofluorescence data were analyzed by one-way analysis of variance（one-way ANOVA）,and mouse survival data were analyzed by Carmelan-Mel single factor survival analysis（Kaplan-Meiertest）.If the data does not meet the requirements of normal distribution,nonparametric test analysis is used.SPSS22.0 software,Sigmaplot14.0 and Origin18.0 software were used to analyze the experimental data and make images.Results:1.Establishment of breast cancer model and radiotherapy 4T1 breast cancer-bearing mice received radiotherapyThere was no significant difference in tumor volume among the tumor control group,low dose group and high dose group before radiotherapy in 4T1 breast cancer bearing mice,but the tumor volume growth rate in the radiotherapy group decreased with the increase of treatment days after different radiotherapy fractionation.Compared with the tumor control group,radiotherapy significantly inhibited tumor progression（P < 0.001）,and the tumor growth in the high dose group was lower than that in the low dose group（P < 0.001）.2.Magnetic resonance / photoacoustic bimodal imaging shows tumor blood vessels and microenvironment changes（a）Manganese ion enhanced magnetic resonance images showed that after radiotherapy,the tumor volume of the tumor control group increased significantly（P < 0.01）,and the image showed no significant difference in the degree of enhancement（P > 0.05）.The tumor volume of low dose group and high dose group increased significantly after radiotherapy（P < 0.01）.The image showed a wide range of low enhancement areas,which was significantly different from that before radiotherapy（P < 0.05）.（b）Photoacoustic parameters showed that the levels of Hb T and Hb R in high dose group and low dose group were significantly lower than those in control group,and the levels of Hb O₂ and SO₂ in high dose group and low dose group were higher than those in tumor control group,and there was significant difference between high dose group and tumor control group.3.Results of pathology and Western blot detection in each group（a）Compared with the tumor control group,the number of Ki-67 positive proliferative cells decreased and the number of TUNEL positive cells increased in low dose group and high dose group after radiotherapy.In addition,the number of Ki-67 positive cells decreased significantly and the number of TUNEL positive cells increased significantly in high dose group.（b）After radiotherapy,compared with the tumor control group,the tumor vascular density in the low dose group decreased significantly to48.93%（P < 0.05）.In addition,the vascular maturity increased significantly to 55.13% in the low dose group after radiotherapy（P <0.05）.（c）Compared with the tumor control group,the expression of PD-L1 in the high dose group increased significantly after radiotherapy.（d）After radiotherapy,the expression of cleaved caspase-3 protein increased significantly in both high-dose group and low-dose group.4.Changes of tumor immune microenvironment after radiotherapyAfter different dose fractionation radiotherapy,compared with the tumor control group,CD4+ in the high dose group increased by 73.96%（P =0.006）,CD8+ cells increased by 74.91%（P = 0.015）,NK cells increased by 29.25%（P = 0.001）,and the low dose CD4+ group increased by67.46%,3.89% and 15.98%（P < 0.001）.5.Effect of different combined treatment regimens for tumorsCompared with anti-PD-L1 alone and tumor control group,the tumor progression of radiotherapy combined with anti-PD-L1 treatment group was significantly slower and the survival rate tended to increase（P <0.01）.In addition,the survival rate of low dose irradiation + anti-PD-L1 antibody treatment group was significantly increased（P < 0.035）,and the survival rate of high dose radiation + anti-PD-L1 antibody treatment group was the most significant（P < 0.002）.Conclusions:1.Radiotherapy with high and low dose fractionation regimens can inhibit tumor progression in mice to varying degrees,including the proliferation of tumor parenchyma cells,the changes of tumor vascular structure and the dynamic changes of tumor immune microenvironment;2.MR/PA bimodal imaging can non-invasively monitor the changes of tumor blood vessels and microenvironment,which provides a new method for evaluating tumor treatment;3.High dose radiotherapy for tumor may promote the effect of immune drugs and improve the response rate and survival cycle.