The Role And Mechanism Of Tumor Endothelial Cell Autophagy In Tumor Vascular Normalization

Objective:Abnormal tumor vasculature has a profound influence on tumor microenvironment and malignant progression,and normalization of tumor vasculature can optimize tumor microenvironment,reduce metastasis as well as improve the function of tumor blood flow and enhance the effects of radiotherapy and chemotherapy.At present,the normalization of tumor vasculature in clinic is realized mainly by inhibiting the VEGF/VEGFR pathway,however,the effect is unstable and this method is susceptible to drug resistance.Thus,it is necessary to further study the characteristics and mechanism of tumor blood vessels and develop more effective drugs for tumor blood vessel normalization.Autophagy is an important protective mechanism for eukaryotes to cope with adverse environment.Cells can provide energy for themselves and maintain cell homeostasis through autophagy.At the same time,autophagy also plays important role in resisting cell apoptosis.Previous studies have proposed the potential role of autophagy in tumor endothelial cells,and autophagy may promote tumor vascularization.The autophagy blocker chloroquine was shown to improve the tumor milieu via normalizing tumor vessel structure and function and increasing perfusion.In this study,we will further explore the autophagy of tumor endothelial cells and evaluate the role of various autophagy inhibitors in tumor vascular normalization.In addition,the lack of efficient,cost-effective,and accurate tumor vascular models seriously limits the development of this field.In previous studies,we found that the transgenic zebrafish xenograft tumor model has the potential for tumor vascular research,but the existing zebrafish xenograft tumor models remain to be improved and optimized.To sum up,in this study,we will improve and optimize the existing zebrafish xenograft tumor vascular model,and verify its effectiveness in the field of tumor vascular research.Eventually,it will be used in vivo study model,and the autophagy characteristics of tumor vascular endothelial cells were discussed by combining with various experimental data information obtained from in vitro cell experiments and tumor tissue samples of human and mouse,and the screening and evaluation of the effects of various autophagy inhibitors on tumor vascular normalization were attempted.Materials and Methods:The xenograft vascular model was established by tumor cell microinjection in transgenic zebrafish.In Tg(flk: e GFP)zebrafish,the vascular endothelia were labelled by green fluorescence protein(GFP),and in Tg(flk: e GFP/gata1: ds Red)zebrafish,the vascular endothelia were labelled by GFP and the erythrocytes were labelled by red fluorescence.The structure and function characteristics of tumor vessels were quantitatively observed by laser confocal in real-time.The anti-angiogenesis function and adverse effects of the vascular endothelial growth factor receptor tyrosine kinase inhibitors(VEGFR-TKI)in zebrafish tumor xenograft model were tested.The therapeutic window of VEGFR-TKI was obtained by calculating the half inhibitor dose(ED50)and maximum tolerated dose(MTD).Through compare the results with previous reported experiment results,the practicability of zebrafish model was evaluated.Search was conducted on GEO(a public functional genomics data platform)to obtain the single-cell transcriptome sequencing data of tumor endothelial cells and normal endothelial cells,and the differences in the expression of autophagy-related genes between tumor endothelial cells and normal endothelial cells were detected by algorithm comparative analysis and gene enrichment analysis.The expression of autophagy protein LC3 B in tumor endothelial cells in a various tumor type of clinical specimens and lung metastases of mouse melanoma were detected by immunofluorescence staining,which using the platelet endothelial cell adhesion molecule(PECAM-1/CD31)to label vessels and microtubule binding protein 1 light chain 3B(LC3B)to label autophagy.Immunofluorescence staining was used to compare the differences of autophagy marker LC3 B between normal endothelial cells and tumor endothelial cells in gastric and colon tissues.The difference of autophagy between tumor vessels and normal blood vessels in autophagy specific gene transgenic zebrafish Tg(flk:GFP/flk:LC3-m Cherry)were quantitatively observed by laser confocal real-time analysis.Human umbilical vein endothelial cells(HUVEC)were cultured in hypoxia,sugar-free and serum-free in vitro to simulate the deficiency of oxygen and nutrition in tumor microenvironment.The expression of autophagy protein LC3 B in HUVEC induced under the condition of hypoxia and nutrition deficiency was detected by immunofluorescence staining,and the expression of LC3 B and p62 in HUVEC was detected by Western blotting(WB).The apoptosis of HUVEC induced by the autophagy inhibitors(chloroquine,ULK-101)was detected by flow cytometry and WB.The effects of multiple autophagy inhibitors(3-MA,MHY1485,chloroquine,LY294002 and ULK-101)on tumor blood vessels were tested using transgenic Tg(flk: e GFP)zebrfish xenograft model.After screening the autophagy inhibitors with the optimal effect of tumor vascular normalization,the changes in structure and function of tumor vascular were further evaluated,including the following two aspects:(1).Tg(flk: e GFP/gata1: ds Red)zebrafish xenograft tumor model was used to evaluate the blood flow in zebrafish xenograft tumor after treatment;(2).Dextran fluorescent with different fluorescence were injected into the intersegmental vessels of zebrafish to further evaluate the permeability of tumor vessels.And chloroquine was used as a positive control drug.Results:1.We successfully transplanted CT26 and GL261 cells into the pericardial cavity of transgenic zebrafish Tg(flk:e GFP)and Tg(flk:e GFP / gata1:ds Red).In this model,we observed the complex tumor vascular network with small part of functional tumor vessels and a large number of nonfunctional tumor blood vessels.2.The efficacy and adverse effects of VEGFR-TKIs in zebrafish xenograft tumor model were consistent with other results in other animal models and clinical reports.It was preliminarily confirmed that zebrafish xenograft tumor vessel model had good clinical predictive value.3.Autophagy protein LC3 B was widely expressed both in the tumor endothelial cells of clinical tumor specimens and mouse melanoma lung metastases.Compared with the normal tissue endothelial cells,the expression of autophagy protein LC3 B is higher in tumor tissues.4.In the tumor vascular network of mouse lung metastatic tumor and autophagy specific gene transgenic zebrafish Tg(flk:GFP/flk:LC3-m Cherry),the autophagy was higher expressed in nonfunctional tumor vessels than functional tumor vessels.5.Autophagy of HUVEC could be induced after 24 hours of hypoxia,sugar-free and serum-free in vitro and the apoptosis of HUVEC cells could be induced by the autophagy inhibitors(chloroquine,ULK-101).6.Autophagy inhibitors(3-MA,LY294002)can induce the death of nonfunctional tumor endothelial cells,while autophagy inhibitors(chloroquine,MHY1485 and ULK-101)can normalize tumor blood vessels.7.ULK-101,an autophagy inhibitor,showed the optimal function of tumor vascular normalization in zebrafish tumor model.ULK-101 can selectively inhibit the killing of non-functional tumor vessels,preserve and normalize the functional and tumor vessels.Conclusion:In this study,we have successfully optimized the zebrafish xenograft tumor vascular model.This model can achieve real-time quantitative observation and analysis of tumor vessels.And the model has good clinical predictive value,which can be used as an efficient and accurate model in the study of tumor vessels and tumor vascular normalization.Furthermore,by using this model and other in vitro cell experiments and immunofluorescence staining of tumor specimens,we preliminarily confirmed that tumor endothelial cells have the characteristic of high expression of autophagy compared with normal vascular endothelial cells,and there are differences in autophagy expression among different structures and functions of tumor blood vessels.Autophagy can be used as an important protective mechanism for tumor endothelial cells to resist apoptosis in the tumor microenvironment under tumor hypoxia and nutritional deficiency.Inhibition of autophagy plays an important role in tumor vascular normalization.Autophagy inhibitors targeting ULK have potential application prospects in the field of tumor vascular normalization.More safe and effective ULK inhibitors need to be further developed and verified.