胰腺癌血管内皮细胞基底微绒毛的形态功能鉴定和标志物筛选及参与肿瘤生物学行为的机制研究

Pancreatic ductal adenocarcinoma (PDAC), among the most intractable of human malignancies, is a highly aggressive and nearly lethal neoplasm. It is more often than not hypovascularized and replete with stromal-rich ‘‘deserts’’ that are largely avascular and indeed may even be actively antiangiogenic which confers inefficient drug delivery to the tumorous milieu. Clinically, the neoplastic cells have robust glucose uptake, the paradox inherent suggest another pariticluar way in vascular trafficking and cargo delivery for cellular uptake persist. Performing an adapted thick human tumor sections immunostainingandemployingconfocal and electronmicroscopywith 3-dimensionalconstruction imaging method, we previously identified an undiscovered distinctive feature of the mature and angiostaticmicrovasculature in PDAC, which had long and intensive, hairy-like, actin-cytoskeleton projections on the surface of the basal membrane of microvessels that we refer to as “basal microvilli”. However, the clinical implications and underlying molecular mechanism of basal microvilli’s sprouts is still unclear. In this study, we plan to (1) analyze the relationship between basal microvilli and malignant bebehavior of PDAC. (2) To determine whether or not these basal endocilia contain endocytic and exocytic properties, as well as glucose transporter positivity, suggesting they typecast as glucose trafficker and cargo deliverer to facilitate cellular uptake within the tumor microenvironment. (3) To clarify mutational spectra characteristic of basal microvilli, whole genome sequencing of genomic DNA extracted from laser microdissection-enriched basal microvilli was utilized, meanwhile, high output RNA-sequencing was also performed. (4) Prospectively to screen the molecules that regulate and sustain growth of basal microvilli in human and animal model. In summary, the mechanisms of basal microvilli’s sprout in tumor metabolism and its clinical implications in tumor behavior are expectable from this project, which will definitely explode our understading of estimates of pancreatic carcinogenesis and contribute to the clinical screening of tageted therapy, indicating that a long window of opportunity may exist for threatment of PDAC patients by removal of basal microvilli.

胰腺癌细胞如何在纤维间质丰富和血管贫乏的环境中获取高水平代谢所需能量，同时有效清理肿瘤微环境代谢废物的机制尚未阐明。申请人首次证实胰腺癌中微血管内皮细胞基底面延伸出致密的绒毛状突起，将此具有肌动蛋白骨架的病理结构命名为血管内皮细胞基底微绒毛（basal microvilli），并揭示其形态特征。本研究将在人胰腺癌组织和基因工程胰腺癌小鼠模型上进一步探索胰腺癌血管内皮细胞基底微绒毛采取高效运输方式的相关机制，揭示胰腺癌血管内皮细胞基底微绒毛发生的基因组、转录组和蛋白组表达谱型，研究其发生的分子机制，并筛选参与调控胰腺癌血管内皮细胞基底微绒毛生长的关键分子。该研究对于阐明胰腺癌的肿瘤代谢机制以及恶性生物学行为具有重要意义，为有效治疗靶点的研发提供理论支持。通过清除胰腺癌血管内皮细胞基底微绒毛，饥饿肿瘤细胞可能成为胰腺癌治疗新策略。

该项目原计划将在人胰腺癌组织和基因工程胰腺癌小鼠模型上进一步探索胰腺癌血管内皮细胞基底微绒毛采取高效运输方式的相关机制，揭示胰腺癌血管内皮细胞基底微绒毛发生的分子机制，并筛选参与调控其生长的关键分子。获得该项目资助后，我们研究顺利，在人胰腺癌组织和基因工程胰腺癌小鼠模型上明确了胰腺癌微血管内皮细胞基底微绒毛表达CD34和具有肌动蛋白的骨架以及血管新生抑制特征的病理形态结构。阐明了胰腺癌血管内皮细胞基底微绒毛增强了微血管与肿瘤细胞之间的葡萄糖转运，采取高效的囊泡运输方式在肿瘤细胞间进行能量物质的摄取和投送。于2015在J Pathol上发表了包含后续补充实验的论文，提前圆满完成了预定目标。此后，我们及时调整战略，把目标对准了血管内皮细胞基底微绒毛这一病理形态结构是否在其它恶性实体肿瘤中存在，进行其广泛性和特异性的初步探讨。同时探索胰腺癌血管内皮细胞基底微绒毛新生的相关分子事件，以及评价胰腺癌血管内皮细胞基底微绒毛与患者临床病理学特征的关系和肿瘤恶性生物学行为的相关性，探索胰腺癌血管内皮细胞基底微绒毛潜在的临床意义和预后指导价值，为胰腺癌的治疗和判断预后提供理论依据和临床指导。.我们研究发现，血管内皮细胞基底微绒毛结构在人胆管癌、转移性胰腺神经内分泌肿瘤和乳腺癌中均存在，但在人肝细胞癌、胶质母细胞瘤、肾透明细胞癌、口腔鳞状细胞癌中不存在。对于胰腺癌血管内皮细胞基底微绒毛，Pathway分析显示VEGF/Jak1/Stat3信号通路明显激活。VEGFA和GLUT-1可能是与其生长密切相关的基因，其生长不依赖传统血管新生的VEGFR2通路。血管内皮细胞基底微绒毛的参数与胰腺癌患者临床病理学特征紧密相关，是判断预后的独立危险因素。胰腺癌血管内皮细胞基底微绒毛具有潜在的临床意义和预后指导价值，能够为胰腺癌的治疗和判断预后提供理论依据和临床指导。该研究结果部分发表在（2016 J Mol Biomark Diagn, 2017 Oncotarget），后续还有论文发表，并进行专利申报。项目执行期间资助2名博士研究生完成其课题。