Notch Signaling Modulates Tumor Vessel Stability And Tumor Progression Via Phenotypic Transition Of Vascular Mural Cells

The tumor microenvironment contains a variety of cellular components,such as endothelial cells,pericytes/smooth muscle cells,immune cells,and fibroblasts,etc.Studies have shown that these cells are not"bystanders"of tumor malignancy but are involved in tumorogenesis in many ways.Among them,vessels are not only necessary for tumor growth,but also participate in the progressive disease such as local invasion and distant metastasis.Tumor blood vessels are significantly abnormal,with uncontrolled angiogenesis and immature structure,which exhibit the lack of clear classification,the disconnected endothelial cells,the discontinued basement membrane,incomplete or even lack of mural cell coverage,leading to the failure of perfusion,increased interstitial pressure in the tumor,hypoxia,tumor metastasis,and decreased drug delivery and therapy resistance.Vascular mural cells compromise vascular smooth muscle cells（v SMCs）and pericytes,which are one of the main cellular components of blood vessels and play a multidimensional role in both physiological and pathological angiogenesis.Under physiological conditions,mural cells are essential for maintaining the stability of vascular structures and the regulation of vasoconstriction/relaxation.Besides,mural cells are also believed to be sources of cancer-associated fibroblasts（CAFs）.Elucidating the precise function and regulatory mechanism of tumor vascular mural cells will provide new strategies for effective tumor treatment.The Notch signaling is an evolutionarily conserved signaling pathway,which plays a key role in cell fate determination,vascular development and homeostasis maintenance.Notch signaling is known to modutate the development,differentiation and functional regulation of mural cells.Mutations or disorders of Notch signaling members have been reported to be involved in many human vascular genetic diseases and cardiovascular diseases related to mural cells.The understanding of the abnormal phenotype of vascular mural cells and the underlying mechanisms is still limited.The exact role of Notch signaling in tumor mural cells is not clear either.Aims:This study aims to elucidate the significance of mural cells in tumor vascular system and tumor growth,to explore the effect of Notch signaling on phenotype regulation of tumor vascular mural cells,as well as on tumor vascular structure and function,tumor growth and metastasis,and to preliminarily investigate the molecular mechanism of Notch signaling in the regulation of tumor vascular mural cell phenotype.Methods:1.Effect of mural cells ablation on tumor blood vessels and tumor growthSM22α-Cre ER^T2 was mated with Rosa-Stopfloxed-DTA mice to construct mice DTA^SM22 which expressed diphtheria toxin specifically in the mural cells,and tamoxifen was administred to induce specific mural cell ablation.Subcutaneous LLC xenograft was established.H&E and Pimo anoxic probe staining were utilized to identify the anoxia and necrosis of the tumor,and immunofluorescence was used to observe the tumor vascular structure.The volume and weight of the tumor at 21 days post-injection were monitored to compare the effect of mural cell ablation on tumor growth.2.Notch signaling of vascular mural cells in TMEFirstly,the tissue microarray of human non-small cell lung cancer and cancer-adjacent lung tissue were used to compare the differences in the expression of Notch1 protein in SM22αpositive cells in lung cancer and normal tissues by immunofluorescence staining.Secondly,we constructed SM22α-driven td Tomato-labling mice and establish LLC subcutaneous xenografts.Tumor tissues were taken at 21 days post-injection.td Tomato positive cells in LLC tumors and lungs were havested through FACS and the expression of Notch downstream target gene of td Tomato positive cells was detected by real-time quantitative PCR.Lasty,v SMCs-DA was cultured in vitro in LLC and B16 conditioned-medium.The expression of Notch signaling constituents was detected by real-time quantitative PCR and Western blot.3.The effect of Notch signaling activation or blockade on tumor growth and metastasisWe constructed the transgenic mice in which the intracellular domain of Notch1,NICD,was pecifically overexpressed in vascular mural cells.Similarly,vascular mural cell specific RBP-J knockout transgenic mice were generated.LLC and B16 subcutaneous xenografts were established.H&E staining was used to detect necrosis,and PIMO hypoxia probe and GLUT1 staining were used to detect hypoxia in tumor sections.To evaluate tumor metastasis,GFP and luciferase were stably transfected in LLC cells.The number of GFP positive cells entering peripheral blood to form circulating tumor cells was counted using fluorescence microscope.The number of lung metastasis was detected by luciferin-based imaging.The volume and weight of the tumor at 21 days for LLC and 14 days for B16post-injection were evaluateed to compare the effect of mural cell ablation on tumor growth.4.The effect of Notch signaling activation or blocade on tumor vascular structure and functionBefore tumor tissue havest,fluorescent labeled dextran was injected through tail vein to evaluate the perfusion function of tumor blood vessels.The tumor tissue was made into frozen sections,and CD31 was stained withα-SMA,SM22α,NG2 respectively to observe the coverage of vascular mural cells.5.The effect of Notch signal activation or blockade on the phenotype of vascular mural cellsThe expression of contraction-related proteins such asα-SMA and SM22αaround endothelial cells was counted in immunoflurence staining images.v SMCs-DA were infected with N1ICD adenovirus vector to activate the Notch signaling,or treated with the DAPT,aγ-endocrine enzyme inhibitor,to inhibit the Notch signaling.The transcriptome of v SMCs-DA infected by ad NICD and ad Ctrl was sequenced to detect the changes in contraction and secretion phenotype profile.The results of bioinformatics analysis were further verified by real-time quantitative PCR and Western blot in NICD overexpressed or DAPT treated v SMCs-DA.6.Downstream molecular mechanism of Notch signaling activation or blocking in vascular mural cellsRNA-seq results were analyzed by Gene Ontology（GO）and gene enrichment analysis（GSEA）to find out the changes of signaling pathway related to phenotype transition after Notch signaling activation and verified by real-time quantitative PCR and Western blot in NICD overexpressed or DAPT-treated v SMCs-DA.Results:1.The depletion of vascular mural cells leads to the disordered tumor vasculature.Mural cell absence led to dramatically anoxic and necrotic tumor,and the tumor growth is significantly limited.2.Compared with mural cells in normal lung tissues,the expression of Hey1 and Hey2m RNA,two Notch target genes,in tumor vascular mural cells was significantly downregulated;the expression of Notch receptor,ligand and downstream gene m RNA in v SMCs-DA was decreased after treatment with tumor-conditioned medium,and the expression of Jagged1 and Hes1 was also decreased.3.The activation of Notch signaling in the mural cells could deter tumor growth,reduce hypoxia and necrosis within the tumor,and inhibit tumor cell proliferation.On the other hand,inhibition of the Notch signaling in mural cells could accelerate tumor growth,aggravate hypoxia and necrosis inside the tumor,promote tumor cell proliferation,increase the number of circulating tumor cells in the peripheral blood,and increase metastasis.4.The activation of Notch signaling in the mural cells improved the perfusion of tumor internal blood vessels,increased the expression ofα-SMA and SM22αaround the blood vessels.The blockage of Notch signaling in the mural cells weakened the perfusion function of tumor internal blood vessels,decreased the expression ofα-SMA and SM22αaround the blood vessels,and the positive signal of SM22αwas far away from CD31 positive endothelial cells and distributed to tumor matrix.5.The expression ofα-SMA and SM22αwas significantly increased in v SMCs-DA upon N1ICD overexpression.While proliferation and migration of v SMCs-DA was decreased.Besides,conditioned medium of N1ICD-expressed v SMCs-DA inhibited the invasion of LLC cells.On the contrary,the expression contraction-related proteins,α-SMA and SM22αin v SMCs-DA was decreased after DAPT treatment.Proliferation and migration of v SMCs-DA was enhanced with DAPT treatment and the invasion ability of LLC cells was promoted by conditioned medium derived from DAPT treated v SMCs-DA.6.RNA-seq data following N1ICD overexpression,Jagged1 knockout and DAPT treatment indicated that activated Notch signaling prompted v SMCs-DA contraction phenotype while repressed the sectatory phenotype.The expression of Toll-like receptor（TLRs）was generally reduced,and NF-κB signal pathway was inhibited after Notch activation.The expression of TLRs was highly upregulated in Notch inhibited cells and NF-κB signaling pathway was significantly activated after Notch blockade.Conclusions:The results of the present study showed that vascular mural cells are the key regulatory factors in maintaining the structural and functional homeostasis of tumor vascularature and are neccessary for tumor growth.Compared with normal tissues,the Notch signaling in vascular mural cells within tumor microenviroment is significantly repressed.Decreased Notch signaling in mural cells results in the disordered and malfunctional tumor vessles,increased hypoxia within the tumor,and ultimately increased tumor growth and metastasis.On the contrary,activation of Notch signaling in mural cells markedly improves tumor vascular structure and function and inhibits tumor growth.In vitro,Notch signaling activation enhances the mural cell contractile phenotype and inhibits its secretory phenotype,which is manifested by reduced proliferation ability,weakened secretion profile,and enhanced adhesion to endothelial cells,which can further inhibit tumor cell proliferation and invasion.In contrast,blocking Notch signaling can promote the enhancement of mural cell secretion phenotype,which is manifested by amplified proliferation and secretion ability,weakened adhesion to endothelial cells,and ultimately promote tumor cell proliferation and invasion.Mechanistically,Notch signaling may inhibit TLRs-NF-κB signaling activation and thereby inhibit the secretory ability.This study systematically investigated the modulatory role of Notch signaling in vascular mural cells,and the effect of this regulation on tumor vascular structure and functional homeostasis and tumor progression.Besides,the study demonstrated that the activation of Notch signaling on mural cells improves tumor vessel funciton and inhibits tumor progression,providing a new target for tumor microenvironment-based anti-cancer treatment.