【作者】 孙涛；

【导师】 孙保存； 赵秀兰；

【作者基本信息】 天津医科大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 第一部分肿瘤细胞与骨髓间充质干细胞相互作用促进内皮细胞表型呈现[目的]利用体外细胞共培养系统研究小鼠骨髓间充质干细胞(BMSCs)与黑色素瘤细胞的相互作用,观察肿瘤细胞诱导BMSCs向血管内皮细胞分化过程中的表型改变,同时观察BMSCs对肿瘤细胞表达内皮标志分子的诱导作用。为进一步了解在与肿瘤细胞接触的微环境中BMSCs向血管内皮细胞方向分化的程度和特点及其对肿瘤细胞表达内皮标志分子的影响,为肿瘤血管生成中血管内皮细胞的来源研究奠定基础。[方法]1)分离C57BL小鼠股骨骨髓细胞进行原代培养,差速贴壁纯化后进行流式细胞术分析确认BMSCs表型；2)建立Transwell非接触式共培养模型,利用免疫荧光观察共培养过程中BMSCs和黑色素瘤细胞出现内皮细胞的表型变化,利用透射电镜观察诱导后BMSCs细胞内是否出现W-P小体；定时收集共培养细胞和培养基,ELISA检测培养基中VEGF-a水平变化,Western Blot检测细胞裂解液中VEGFR1、VEGFR2和FactorⅧ的时序性水平变化；3)利用免疫荧光染色比较非接触式共培养和接触式共培养细胞表型和形态变化。[结果]1)由C57BL小鼠股骨分离获得的细胞,经纯化培养后表型鉴定为CD44+/CD73+/CD90+/CD105+/CD166+/CD34-/CD45-/CD133-,符合BMSCs表型；2) mBMSCs与B16细胞共培养72h后,可观察到mBMSCs随着培养时间的推移逐渐呈现并上调血管内皮标志分子CD34、CD45、FactorⅧ、VEGFR-1和VEGFR-2,而原有的CD44和CD105则表达下调；流式细胞术分析显示诱导前FactorⅧ阳性细胞率为1.09%,诱导后为30.17%。超微结构分析发现,经72h诱导后的mBMSCs可在透射电镜下观察到内皮细胞胞浆所特有的Weibe-Palade小体出现。3)非接触式共培养条件BMSCs和B16细胞的增殖率均明显增高,mBMSCs在共培养组倍增时间均值为25.02h、对照组为28.15h,B16细胞在共培养组倍增时间均值为26.30h、对照组为30.76h,差别具有统计学意义；4)非接触共培养体系中,mBMSCs可以被B16细胞诱导出现内皮细胞表型,同样B16细胞也被观察到相关内皮细胞标志分子VEGFR1、VEGFR2和FactorⅧ的表达上调。B16细胞和BMSCs直接混合共培养(接触式共培养)72h,发现细胞群体的VEGFR1、VEGFR2和FactorⅧ的阳性细胞率显著降低,可见到B16细胞包绕mBMSCs生长的现象；5)共培养体系中培养液VEGF-a水平随培养时间延长而显著增高。[小结]1)在与肿瘤细胞相互作用的微环境中,BMSCs可以被肿瘤细胞诱导出现表型转变并分化为血管内皮细胞；2)肿瘤细胞亦可与BMSCs相互作用,出现内皮细胞标志分子的上调；3) BMSCs和肿瘤细胞可相互作用,彼此促进增殖；4)培养基中VEGF-a水平的时序性升高与BMSCs和肿瘤细胞出现内皮细胞表型密切相关。第二部分临床病理分析—EMT相关蛋白在人肝癌血管生成拟态中的作用[目的]本部分研究通过对97例随访资料完全的人类肝细胞肝癌临床标本进行分析,观察血管生成拟态(VM)的存在与否及其与临床预后的关系；初步分析上皮-间充质转变(EMT)相关蛋白(表征蛋白E-cadherin、VE-cadherin和调控蛋白Twist1、Twist2、Snail和Slug)和VM相关蛋白(CD31/PAS、MMPs)之间的关系,及其对临床预后的影响,筛选与VM关系密切的EMT相关蛋白,为肿瘤细胞向血管内皮方向塑形研究提供依据。[方法]收集天津医科大学总医院和附属肿瘤医院2001.2-2005.12年经手术切除标本随访资料完整并经病理医师诊断为肝细胞肝癌的病例97例,对临床病历资料和随访资料进行分析；利用HE染色和CD31/PAS双染法观察97例HCC组织中是否存在VM,并计数PAS/VM数量；以免疫组织化学方法检测EMT相关蛋白和VM相关蛋白,分析二者之间的相关性及其与VM、临床预后之间的关系,进行统计学分析。[结果]1)97例HCC临床病例样本,发现18例(18／97,19%)具有典型的VM结构；2) Twist1可弥漫表达于HCC细胞胞浆内,在部分病例表达于细胞核或核浆共表达,局部可出现强阳性灶性表达,Twist1核表达在VM+组和VM-组间差别具有统计学意义,Pearson检验结果显示Twist1-cyto和Twist1-nu均与VM-PAS阳性环计数具有相关性,Twist2、Snail、Slug均与VM关联性不强；3)EMT表征蛋白VE-cadherin与VM具有相关性,与Twist1-cyto、Twistl-nu、Slug均存在相关性,而与Twist2和Snail无相关性；MMP9与VM、Twist1表达存在相关性,MMP2在本研究中与VM、Twist1无关；4)生存分析结果表明Twist1-cyto、Twist1-nu、Snail、VE-cadherin、MMP9表达阳性的病人生存时间较短,E-cadherin阳性病人生存时间较长,Slug和MMP2的表达与否则与生存时间的关系无统计学意义；5)Cox回归分析结果表明Twist1核表达对生存预后影响最大。[小结]1) VM结构主要存在于分化差、恶性度高的实体型及低分化型HCC中,提示VM与HCC恶性生物学行为相关；2)EMT调控蛋白Twist1核表达与VM关联性最为密切,影响病人生存预后；3) Twist1与VE-cadherin、MMP9表达具有相关性,推测肿瘤细胞可能通过Twist1的调节表达VE-cadherin,转变为具有内皮细胞特征的肿瘤相关内皮细胞(TAEs),进而形成VM；4)EMT调控蛋白Twist2、Snail和Slug与VM形成无明确关联性。第三部分Twist1增强肿瘤细胞迁移、侵袭和成血管塑形能力[目的]利用HCC细胞系对Twist1与细胞迁移、侵袭和成血管塑形能力的影响进行体外研究,以进一步证明Twist1和肿瘤细胞塑形、VM的关系,分析Twist1对VE-cadherin表达的调控作用和对MMPs活性的调节。从分子层面研究肿瘤细胞向内皮细胞方向塑形的分子机制。[方法]筛选HCC细胞系,构建Twist1表达质粒和shRNA质粒,转染细胞获得Twist1上调和下调模型,Western Blot和RT-PCR鉴定转染效果；利用细胞伤口愈合实验、Transwell侵袭实验评价Twist1对细胞迁移、侵袭能力的影响,进一步利用Matrigel三维培养模型分析Twist1对细胞管道化塑形能力的影响；利用染色质免疫共沉淀、荧光素酶报告基因检测分析Twist1对VE-cadherin转录的调控作用,利用明胶酶谱法分析Twist1对MMP2和MMP9活性的影响。[结果]1)在HepG2、PLC、SMMC7221、Be17402和Huh-7几种细胞系中,PLC、HepG2、Huh-7等细胞系在mRNA和蛋白水平均呈现为Twistl低表达,Be17402在mRNA和蛋白水平均高表达；2) pcDNA3-Twistl可以明显上调HepG2的Twistl表达,pGP-Twist1-shRNA可有效下调细胞内Twist1表达；伴随Twist1表达改变,细胞表型出现相应变化；3)上调Twist1的HepG2和下调Twist1的Be17402对比分析表明：Twist1可以促进HCC细胞的迁移和侵袭能力；三维培养条件下,上调Twist1可促进HepG2细胞形成管腔样结构,下调Twist1可抑制Be17402细胞形成管腔样结构；4)三维培养条件下,上调Twist1可有助于HepG2细胞表达VE-cadherin,下调Twist1可抑制Be17402表达VE-cadherin;利用染色质免疫共沉淀和荧光素酶报告基因分析发现Twist1可能在特定培养微环境下与VE-cadherin启动子区结合并增强其转录活性；5) Twist1的表达上调对HepG2和Be17402细胞MMP2和MMP9的活性均有增强作用。[小结]1) Twist1表达可促进HCC细胞的迁移和侵袭作用；2) Twist1表达可通过上调VE-cadherin促进肿瘤细胞在三维基质中出现管道化塑形；3)在三维培养条件下,Twist1方可与VE-cadherin的启动子区域结合并促进VE-cadherin的转录；4) Twist1表达可促进HCC细胞MMP2与MMP9的活性增强；5) Twist1蛋白是肿瘤细胞经EMT形成VM的重要调节分子。第四部分抗凋亡蛋白Bcl-2与EMT调控蛋白Twist1相互作用[目的]课题组前期工作已观察到缺氧与VM形成密切相关,缺氧可使肿瘤细胞形成“线形程序性坏死”(LPPCN),进而引流血液形成VM,而LPPCN形成又与抗凋亡蛋白Bcl-2家族密切相关。为研究启动Twist1表达和使之行使功能的上游机制,探讨其是否与抗凋亡蛋白有关,本部分研究将模拟缺氧微环境以评价肿瘤细胞抗凋亡蛋白Bcl-2和Twist1的时相表达。利用酵母双杂交、真核过表达Twist1免疫共沉淀和内源蛋白免疫共沉淀方法分析Twist1与抗凋亡蛋白Bcl-2的直接相互作用,以期进一步明确缺氧微环境诱导肿瘤细胞经EMT机制向血管内皮细胞方向塑形最终形成VM的分子机制,丰富血管生成理论。[方法]利用缺氧袋法制造肿瘤细胞缺氧微环境,以B16细胞系为模型研究缺氧条件下抗凋亡蛋白Bcl-2和Twist1表达的时相性变化；以酵母双杂交系统筛选与Twist1相互作用的抗凋亡蛋白；对肝癌细胞HepG2过表达Twist1后进行免疫共沉淀,分析Twist1与Bcl-2的直接相互作用；在缺氧条件下,进行内源蛋白的免疫共沉淀,分析内源Twist1与Bcl-2的直接相互作用。[结果]1)缺氧处理黑色素瘤细胞B16,经过15h和30h缺氧诱导后B16细胞表达Twist1,可见30h的缺氧显著上调Twist1表达；同时Bcl-2出现时序性表达增强,其中24-36h为表达高峰,随后下降。在30h缓解缺氧可检测到Twistl、VEGFR2、VE-cadherin的高表达；2)酵母双杂交筛选与Twistl相互作用蛋白,结果显示阳性克隆中含有BCL-2基因：3)对HepG2细胞转染pcDNA3-Twistl后48小时,可以检测到Bcl-2上调。利用Co-IP分别以Twist1和Bcl-2抗体钓取,均可在产物中检测到相应的Twistl和Bcl-2；4)在缺氧30h缓解缺氧1d后可以检测到内源Bcl-2和Twistl的相互作用。[小结]1)缺氧可致肿瘤细胞出现Bcl-2、Twistl的响应性表达上调,缺氧-缓解缺氧对Twistl的上调程度高于单纯缺氧；2)酵母双杂交实验表明Twistl蛋白可以与Bcl-2蛋白发生直接相互作用；3)肿瘤细胞过表达Twist1模型可观察到Twist1蛋白与Bcl-2蛋白发生相互作用；缺氧条件下可检测到肿瘤细胞内源Twist1蛋白和Bcl-2蛋白发生相互作用。4)缺氧及抗凋亡机制可能诱导肿瘤细胞发生EMT,抗凋亡蛋白Bcl-2上调可能是EMT的启动因素之一。结论本文通过对BMSCs和肿瘤细胞相互作用向内皮细胞分化、EMT相关蛋白和VM的关系、以及缺氧抗凋亡机制与TAEs形成的关系等多个角度,证明肿瘤细胞向血管内皮细胞方向转变的"LPPCN-EMT-TAEs, LET"理论和TAEs存在的可能及分子机制。其中包括所涉及的Bcl-2与Twist1相互作用、肿瘤相关内皮细胞标志物呈现的时序关系以及相应的组织、细胞在分子和功能学多角度的评价。“LET”理论可以更好的解释肿瘤细胞在特定微环境下的生物学行为,进一步完善肿瘤血管生成理论和EMT理论。同时,其还为肿瘤细胞分化的基础研究提供了新的思路和切入点,对肿瘤血管生成的转化医学研究提供了新的药物靶点和治疗信息,对深入理解肿瘤的生物学行为和临床诊治具有一定得价值和意义。更多还原

【Abstract】 Part 1 Tumor cells and bone marrow mesenchymal stem cells coordinate with each other in acquiring endothelial cell phenotype[Purpose]A co-culture system was used to investigate the interaction between mouse bone marrow mesenchymal stem cells (BMSCs) and B16 melanoma cells, observe phenotypic changes of tumor cells in the process of inducing BMSCs to differentiate into cells with endothelial phenotypic features as well as the role of BMSCs in inducing tumor cells to express endothelial markers. This is important for further understanding the degree and characteristics of differentiation of BMSCs into endothelial cells and its effect on the expression of endothelial markers in tumor cells in the contact co-culture microenvironment, and lays a foundation for identifying the origin of vascular endothelial cells.[Methods]1) Limb bone marrow from C57 mice was separated for primary culture. After purification by differential centrifugation, BMSCs were identified with stem cell phenotype with flow cytometry.2) A transwell co-culture model was established and immunofluorescence was used to observe the phenotypic changes of the BMSCs and melanoma cells in the process of co-culture. Transmission electron microscopy was used to confirm whether there were W-P corpuscles after BMSCs were induced. Cells and mediums were collected at various time regularly. ELISA was used to detect the change of the level of VEGF-a in medium and Western Blot was used to detect the time-dependent change of the level of VEGFR1, VEGFR2 and FactorⅧin cell lysate.3) Immunofluorescence staining was performed to compare the change of cell phenotype and morphology between cells of non-contact co-culture and that of contact co-culture.[Results]1) Phenotype of cells separated from limb bone marrow from C57 mice after purified culture was identified as:CD44+/CD73+/CD90+/CD105+/CD166+/CD34-/ CD45-/CD133-, consistent with the phenotype of BMSCs.2) After co-culture with B16 melanoma cells for 72h, the expression of endothelial phenotype CD34,CD45,FactorⅧ,VEGFR and VEGFR-2 in mBMSCs was observed to be up-regulated gradually in a time-dependent manner, while the expression of CD4 and CD 105 was down-regulated. Flow cytometry showed that the percent of Factor VIII positive cells was 1.09% before induction and 30.17% after induction. Ultrastructure analysis found that Weibe-Palade bodies, which were specific for endothelial cells, could be observed under transmission electron microscope after co-culture for 72h.3) Proliferation rates of BMSCs and B16 cells from co-culture system increased significantly. The doubling time of mBMSCs in co-culture group was 25.02h, while that was 28.15h in control group. The doubling time of B16 cells in co-culture group was 26.30h, while that was 30.76h in control group.4) In non-contact co-culture system, mBMSCs can be induced by B16 cells to acquire endothelial cell phenotype. At the same time, B16 melanoma cells was observed to up-regulate the expression of VEGF-a, VEGFR-1, VEGFR-2 and Factor VIII. After directly co-culture (contact co-culture) of B16 cells and BMSCs for 72h, the percent of VEGFR1, VEGFR2, and FactorⅧpositive cells decreased significantly. B16 cells surrounding mBMSCs was observed.5) The level of VEGF-a in culture medium increased significantly with the extension of incubation time in co-culture system.[Conclusions]1) In the microenvironment of co-culture with the tumor cells, BMSCs can be induced by tumor cells to differentiate into endothelial cells and showed phenotype changes;2) Tumor cells can interact with BMSCs and showed up-regulation of endothelial cell markers;3) BMSCs and tumor cells can interact with each other to promote proliferation;4) The time-dependent increase of VEGF-a in culture medium was associated with appearance of endothelial markers of BMSCs after co-culture with tumor cells.Part 2 Clinicopathologic analysis----role of Twistl in vascular mimicry in human hepatocellular carcinoma[Purpose]Clinical specimens of human hepatocellular carcinoma of 97 patients with complete follow-up data were studied in this part to observe the existence of VM and its relationship with clinical prognosis and to analyze preliminarily the relationship between EMT-associated protein (characterizing protein E-cadherin and regulating protein Twistl, Twist2, Snail and Slug) and VM-related protein (CD31/PAS, VM-cadherin, MMPs) and its effect on clinical prognosis. EMT-related proteins that closely associated with VM were screened.[Methods]Resected specimens of 97 patients identified as hepatocellular carcinoma by pathologists with complete follow-up data were obtained from General Hospital and Cancer Institute and Hospital of Tianjin Medical University between February 2001 and December 2005 to analyze the medical records and clinical follow-up data; HE staining and CD31/PAS double staining were used to identify the existence of VM and the number of PAS/VM was counted; immunohistochemistry was used to detect EMT-related protein and VM-related protein and analyze their correlations between each other and the association with VM and clinical diagnosis with Statistical methods.[Results] 1) In 97 cases of HCC patients,18 cases (18/97,19%) had typical VM structures;2) Twistl expressed diffusely in the cytoplasm of HCC cells. In some cases, it expressed in the nucleus, or in the nucleus and cytoplasm simultaneously. There may be strong positive foci-like expression locally. The difference of nuclear expression of Twistl between two groups was significant. Results of Pearson test showed that there was a correlation between twist1-cyto,Twistl-nu and the number of VM-PAS-positive loop. The association between Twist2, Snail, Slug and VM was not strong;3) EMT-related characterizing protein VE-cadherin was correlated with VM as well as Twistl-cyto, Twistl-nu and Slug, but it was not correlated with Twist2 and Snail. MMP9 was correlated with VM and Twistl. MMP2 had no correlation with VM and Twistl in this study;4) Results of survival analysis showed that patients positive for Twistl-cyto, Twistl-nu, Snail, VE-cadherin, and MMP9 had a shorter survival and patients positive for E-cadherin had a longer survival. The expression of Slug and MMP2 had no significant correlation with the survival time.5) Results of Cox regression analysis showed that the expression of Twist1-Nu did the greatest contribution to the survival of patients.[Conclusions]1) VM was mainly existed in the poorly differentiated and high-malignant HCCs who belonged to solid and poorly differentiated type, suggesting that VM was associated with malignant biological behaviors of HCC;2) The nuclear expression of EMT regulatory protein Twistl had the most close association with VM and it affected the prognosis.3) The expression of Twistl was correlated with the expression of VE-cadherin and MMP9. Tumor cells may regulate the expression of VE-cadherin through the regulation of Twistl, thereby form tumor associated endothelial cells (TAEs) with characteristics of endothelial cells;4) Correlation between EMT regulatory proteins Twist2, Snail, Slug and VM formation were not observed.Part 3 Role of Twistl in enhancing tumor cell migration, motility and angiogenic remodeling[Purpose]HCC cell lines were used to study the effect of Twistl on migration, invasion and angiogenic remodeling of tumor cells in vitro to further identify the association between Twistl and remodeling of tumor cells and VM, and analyze the regulation of Twistl on the expression of VE-cadherin and the activity of MMPs.[Methods]HCC cell lines were selected. Twistl expression plasmid and shRNA plasmid were constructed. Cells were transfected to acquire up-regulation model and down-regulation model. Western Blot and RT-PCR were used to identify the transfection efficiency. Wound healing assay and transwell invasion assay were performed to evaluate the effect of Twistl on cell motility and invasion; three-dimensional matrigel culture was used to further analyze the effect of Twistl on angiogenic remodeling of tumor cells; chromatin immunoprecipitation assay and reporter gene assays were performed to investigate the regulation of Twistl on VE-cadherin transcription; zymography assays were performed to detect the effect of Twistl on the activity of MMP2 and MMP9.[Results]1) Expressions of twistl mRNA and protein in PLC, HepG2 and Huh-7 cell lines showed a low level, while those in Be17402 presented a high level;2) pcDNA3-Twistl can significantly up-regulate twistl expression in HepG2 and pGP-Twist1-shRNA can effectively reduce twistl expression in tumor cells. Along with the changes of Twistl expression, cell phenotypes changed correspondingly.3) Comparison between the up-regulated cell model in HepG2 and knockdown cell model in Be17402 suggested that Twist1 can enhance cell migration, motility and invasiveness. In three-dimensional culture, up-regulation of Twistl can promote HepG2 cells to form tubular structure, while down-regulation of Twistl can inhibit Be17402 from forming tubular structure.4) In three-dimensional culture, up-regulation of Twistl induced HepG2 cells to express VE-cadherin while down-regulation of Twistl inhibited expression of VE-cadherin in Be17402. Chromatin immunoprecipitation assay and reporter gene assays suggested that Twistl can combine with the promoter of VE-cadherin in a particular microenvironment and enhance its activity of transcription.5) Up-regulation of Twistl in HepG2 cells and Be17402 cells enhanced the activity of MMP2 and MMP9.[Conclusions]1) Expression of Twist1 can promote cell migration, motility and invasiveness.2) Expression of Twistl can promote tubular remodeling in three-dimensional matrix through up-regulation of VE-cadherin;3) In the three-dimensional culture, Twistl combined with the promoter of VE-cadherin to promote the transcription of VE-cadherin;4) Expression of Twistl can enhance the activity of MMP2 and MMP9 in HCC cells;5) Twistl was the important molecular in the formation of VM by tumor cells via EMT. Part 4 Interaction between anti-apoptotic protein Bcl-2 and EMT regulatory protein Twistl[Purpose]We have reported that the VM was induced by hypoxia. Tumor cells formed LPPCN by hypoxia inducition and then obtain blood. LPPCN formation was associated with the anti-apoptosis protein Bcl-2. To investigate the upstream mechanism that initiates Twist1 expression, hypoxic micro-environment was simulated to evaluate time-dependent expression of anti-apoptotic protein Bcl-2 and Twistl. Yeast two hybrid assay, co-immunoprecipitation assay of overexpressing Twistl in eukaryotic cells and co-immunoprecipitation of endogenous protein were used to analyze direct interactions between Twistl and Bcl-2 to further identify the molecular mechanism of hypoxic microenvironment inducing tumor cells to remodel into vascular endothelial cells via EMT and enrich the theory of angiogenesis.[Methods]GasPak was used to simulate hypoxic tumor microenvironment. B16 cells were used as the model to study the time-dependent changes of the expression of Bcl-2 and Twistl; yeast two-hybrid assay was used to select the anti-apoptosis proteins those interacted with Twistl; co-immunoprecipitation was performed after Twistl was expressed in HepG2 cells to analyze the direct interaction between Twistl and Bcl-2. In hypoxia microenvironment, co-immunoprecipitation of endogenous protein was performed to analyze the direct interaction between Twistl and Bcl-2.[Results]1) After hypoxia for 30h, Twistl was significantly overexpressed in melanoma B16 cells, at the same time, Bcl-2 was overexpressed in a time-dependent manner with a peak expression between 24-36h followed by the decrease. Transcription factor Twistl, VEGFR2 and VE-cadherin were detected to be highly expressed when hypoxia was terminated at 30h.2) Detection of proteins those interacted with Twistl selected by Yeast two-hybrid assay suggested that there was Bcl-2 gene.3) After transfection with pcDNA3-Twistl for 48h, Bcl-2 and Twistl were detected to be overexpressed. Co-IP was used to precipitate Twist land Bcl-2 respectively, Bcl-2 and Twistl can be detected correspondingly.4) After hypoxia for 30h followed by re-oxygen for 24h, co-immunoprecipitation of endondogeneous Bcl-2 and Twistl can be detected.[Conclusions]1) Hypoxia can induce responsive overexpression of Bcl-2 and Twistl.The overexpression induced by hypoxia-reoxygen was greater than that by hypoxia.2) Yeast two-hybrid assay suggested that Twistl can interact with Bcl-2 directly.3) In tumor cells overexpressing Twistl, the direct interaction between Twistl and Bcl-2 can be detected; in hypoxia environment, the direct interaction between endogenous Twistl and Bcl-2 can be detected.4) Hypoxic and anti-apoptosis mechanism may induce tumor cells to undergo EMT and the overexpression of anti-apoptosis protein Bcl-2 may be one of initiators of EMT. Conclusions for whole manuscriptBased on the interaction between MSCs and tumor cells resulting in differentiation into endothelial cells, the relationship between EMT-related proteins and VM and the relationship between Bcl-2voverexpression and the formation of TAE in hypoxia, the possible mechanism of "LET" that could clarify the transformation from tumor cells to vascular endothelial cells was investigated. The contents were as followings:interactions between Bcl-2 and Twistl, time-dependent associations of appearance of tumor-associated endothelial cell markers, and molecular and functional evaluations of the corresponding tissues and cells. "LET" theory can better explain the biological behavior of tumor cells in particular microenvironment and further perfect the theory of tumor angiogenesis and EMT. At the same time, the theory provides a new idea and an access for basic research of tumor cells differentiation, provides new drug targets and information of treatment for transformational medicine research of tumor angiogenesis. It plays an important role in further understanding the biological behavior of tumor and in clinical diagnosis.更多还原