Exploring The Mechanism Of Peritumoral Spines Regulating Tumor Microenvironment And Promoting Vascular Normalization Based On DIA Proteomic

Background:The occurrence and development of tumor depended on tumor angiogenesis.Different from normal blood vessels,tumor blood vessels were characterized by high disorder,tortuosity,expansion,uneven thickness,excessive branches,etc.,which may easily lead to blood flow disorder,hypoxia and acid accumulation.The process of vascular normalization referred to promoting the transformation of abnormal blood vessels in tumor to normal blood vessels,making the vascular structure tend to be regular,the vascular basement membrane tend to be complete,the perivascular cells proliferate,improving the perfusion of tumor tissue,increasing oxygen saturation,correcting the acidic microenvironment,enhancing the ability of vascular transportation and supply,more effectively transporting drugs to tumor cells,so as to improve the anti-tumor effect.The mechanism of vascular normalization mainly focused on the regulation of vascular endothelial cells and the protection of vascular barrier function.But as a part of tumor ecosystem,tumor blood vessels were regulated by the whole tumor microenvironment.Tumor microenvironment was a complex ecosystem,which was characterized by hypoxia,low pH,chronic inflammatory cell infiltration,ECM remodeling.Based on the overall view of tumor ecosystem,the viewpoint of improving tumor microenvironment to promote vascular normalization has become the focus of attention.Peritumoral acupuncture was a technique of acupuncture treatment along the edge of the tumor with the tumor as the center.It could not only target and adjust the microenvironment of tumor,but also connect the local meridians,collaterals,superficial collaterals and the skin.It could regulate Qi and activate blood circulation,remove blood stasis and dredge meridians,and theoretically promote the normalization of tumor blood vessels.The previous research found that peritumoral acupuncture could effectively increase the local drug concentration of the tumor,and effectively promote the recruitment and coverage of perivascular cells and the stability of vascular basement membrane two hours after the intervention.However,whether peritumoral acupuncture could promote vascular normalization,whether it has the corresponding time window and the mechanism of promoting vascular normalization still need to be further studied.This research used animal experimental methods to clear the function of peritumoral acupuncture in promoting vascular normalization and the relevant time window.The mechanism of peritumoral acupuncture promoting vascular normalization was explored by using the method of DIA proteomics from the perspective of tumor microenvironment.It could provide modern research basis for promoting vascular normalization by peritumoral acupuncture.There were two parts in this study:From the perspective of modern science,the molecular mechanism of tumor vascularization was summarized;From the perspective of clinical practice of traditional Chinese medicine,this research introduced the clinical application and curative effect of surrounding acupuncture.The experimental part of this study:Purpose:To study the effect of peritumoral acupuncture on the normalization of tumor blood vessels.To clear the time window of vascular normalization.From the perspective of regulating tumor microenvironment,the mechanism of promoting vascular normalization was explained and the related candidate target proteins were screened out.Methods:In this study,the 4T1 breast cancer model was established by using animal experimental method.After the intervention of peritumoral electro-acupuncture,the time window of tumor vascular normalization was determined by the observation of photoacoustic imaging in vivo and detection of micro vascular parameters in vitro.Based on the quantitative proteomics technology of DIA,in the time window,the differential proteins expression in the peritumoral electro-acupuncture group,bevacizumab group and model group were analyzed.Through the analysis of bioinformatics network,the molecular mechanism of peritumoral acupuncture promoting the normalization of tumor blood vessels in 4T1 breast cancer mice was explored.The clinical correlation analysis of the hub differential proteins was conducted by the Oncomine database and GEPIA tools.Result:1.The effect of peritumoral acupuncture on tumor microvascular photoacoustic imaging signal in 4T1 breast cancer mice.Peritumoral acupuncture could significantly reduce the microvascular density in the T2-5 time range(PT2=0.0032,PT3<0.0001,PT4<0.0001,PT5<0.0001).The trend of micro vascular density began to decline from T2,and reached the maximum effect at T3,and gradually recovered from T4-T5.2.The effect of peritumoral acupuncture on tumor promoting vascular normalization in 4T1 breast cancer mice.In the aspect of CD31 expression,compared with the tumor bearing model group,the expression of T2 in peritumoral acupuncture group began to decrease(P=0.0064),the expression of T3 reached the lowest(P<0.0001),the expression of T4 began to rise slightly,but it was still significantly lower than that in the tumor bearing model group(P<0.0001),the expression of T5 further increased(P=0.0037),and there was no statistical difference in the expression of T6-7.In the aspect of α-SMA expression,compared with the tumor bearing model group,the expression of peritumoral acupuncture group increased significantly from T1 time point to T7 time point,and the effect was the highest at T3 time point(P<0.0001).Compared with bevacizumab group,the expression was significantly increased at T1-T3 time point(PT1=0.0160,PT2=0.0418,PT3=0.0007).In terms of COL Ⅳ expression,compared with tumor bearing model group,the expression of COL Ⅳ in peritumoral acupuncture group increased significantly from T1 to T7(PT1=0.0189,PT2=0.0236,PT3=0.0062,PT4=0.0340,PT5=0.0352,PT6=0.0461,PT7=0.0018).Compared with bevacizumab group at T1-T2 and T7 time points,the expression was also significantly increased(PT1=0.0032,PT2=0.0044,PT3=0.0006).In the aspect of HIF1A expression,compared with the tumor bearing model group,the expression of peritumoral acupuncture group decreased significantly from T2 time point to T7 time point(PT3=0.0001,PT4=0.0012,PT5<0.0001,PT6<0.0001,PT7=0.0017).Compared with bevacizumab group,the expression was significantly decreased at T5-T7 time point(PT5<0.0001,PT6=0.0176,PT7=0.0305).3.The difference of protein expression of tumor tissue in 4T1 breast cancer mice by peritumoral acupuncture.There were 8425 proteins and 86242 peptides in the DIA Library.According to the general 50%principle,the protein with quantitative value results in more than 50%of samples was used for subsequent bioinformatics and statistical analysis.A total of 5063 proteins were identified in this study,of which 4167 were quantitative proteins in more than 50%of the samples.The differential expression protein was screened by the standard of expression Fold Change(FC)greater than 1.5 times and P-value less than 0.05.Compared with TG group,there were 274 differentially expressed proteins in EATG group,90 of which were up-regulated and 183 of which were down-regulated.According to P-value<0.05 as the significant difference standard,70 items were screened out in the relevant biological processes,mainly involved oxidative stress,energy and material metabolism,angiogenesis,hypoxia and apoptosis,immune response,and regulation of protein synthesis and processing,etc.27 entries were screened out.It mainly included protein synthesis and processing,material and energy metabolism,and oxidative stress.Compared with BTG group,there were 154 differentially expressed proteins in EATG group,79 of which were up-regulated and 75 of which were down-regulated.According to P-value<0.05 as the significant difference standard,159 items were screened out in the relevant biological processes,mainly involved immune regulation,protein synthesis and processing,mitochondrial energy metabolism,vascular and embryonic development,cell cycle and other aspects.17 entries were screened out.It mainly involved lipid metabolism and immune response related pathways.Compared with TG group,there were 203 differentially expressed proteins in BTG group,of which 78 were up-regulated and 125 were down-regulated.According to P-value<0.05 as the significant difference standard,139 items were screened out in the relevant biological processes,mainly included embryonic development,lipid metabolism,protein synthesis and processing,muscle tissue movement and apoptosis.27 entries were screened out.It mainly included protein synthesis and processing,myocardial movement,energy and material metabolism.4.Validation of clinical public sample dataAccording to Oncome database,GLO1,SOAT1,PIK3AP1 and SLC25A5 were all highly expressed in clinical breast cancer samples(PGLO1=1.96E-28,PSOAT1=0.003,PPIK3AP1=1.77E-4,PSLC25A5=0.0 1 6).Through the analysis of GEPIA online analysis platform,the expression of GLO1,SOAT1,PIK3AP1 and SLC25A5 was positively correlated with VEGFA expression in breast cancer(PGLO1=4.6E-09,PSOAT1=2.9E-10,PPIK3AP1=0.044,PSLC25A5=1E-07).The expression of GLO1,SOAT1,PIK3AP1 and SLC25A5 was also positively correlated with HIF1A expression in breast cancer(PGLO1=6E-09,PSOAT1=7E-61,PPIK3AP1=3.3E-49,PSLC25A5=6.8E-27).The expression of GLO1,SOAT1,PIK3AP1 and SLC25A5 did not show significant correlation in the whole clinical stage of breast cancer(PGLO1=0.142,PSOAT1=0.71,PPIK3AP1=0.457,PSLC25A5=0.334).GLO1,SO AT1 and PIK3AP1 showed no significant correlation with the overall survival of breast cancer patients(PGLO1=0.13,PSOAT1=0.39,PPIK3AP1=0.93).The expression level of SLC25A5 has a significant effect on the overall survival of patients with invasive breast cancer(PSLC25A5=0.0019).The lower the expression level,the better the survival prognosis.Conclusion:1.Peritumoral acupuncture could promote vascular normalization in 4T1 breast cancer mice transplanted tumor,which was manifested in the decrease of photoacoustic signal,the decrease of microvascular density reflected by CD31,the increase of pericyte recruitment and coverage reflected by α-SMA,the enhancement of vascular basement membrane integrity reflected by COL Ⅳ,and the improvement of hypoxic microenvironment reflected by HIF1A.2.There was a specific time window for the effect of peritumoral acupuncture on vascular normalization in 4T1 breast cancer mice.The range was from the second day to the fifth day after the intervention,and the third day was the best time point.3.Peritumoral acupuncture may play a role in promoting vascular normalization by regulating oxidative stress,improving glucose metabolism and lipid metabolism,and regulating immune microenvironment.4.The target candidate proteins GLO1,SOAT1,PIK3AP1 and SLC25A5 were all highly expressed in clinical breast cancer samples and were positively correlated with VEGFA and HIF1A expression in breast cancer,but not clearly correlated with the stage of breast cancer patients.The low expression of SLC25A5 had a significant positive effect on the overall survival prognosis of breast cancer patients.