Tissue Factor splicing and pancreatic tumor progression: pilot studies

描述（由申请人提供）：我们将确定交替剪接组织因子（asTF）的（过）表达是否促进胰腺肿瘤的发生，以及asTF表达的降低是否抑制胰腺肿瘤的发生。高TF活性在胰腺导管腺癌（PDAC）中已被长期观察到。直到最近，在PDAC和其他形式的实体癌中，高TF表达的功能意义被认为与全长TF的蛋白水解功能密不可分。全长TF是一种完整的膜蛋白，作为丝氨酸蛋白酶VIIa的辅助因子。我们发现，分泌的选择性剪接TF变体asTF通过整合素的连接非蛋白性地激活癌症相关的信号通路。1， PDAC中原发性肿瘤生长和转移扩散的主要因素。6 ?1在PDAC细胞上的表达与转移率相关；因此,asTF - ? 6 ?相互作用可能以不依赖血管生成、细胞自主的方式增强转移性扩散。我们的最新数据表明，asTF蛋白在PDAC病变中丰富，但在正常导管中并不丰富，并通过上调内皮细胞上的细胞粘附分子来促进单核细胞募集。选择性靶向asTF的化合物可能对癌症进展有重大影响，而对止血的影响最小。我们将进行体外研究，以及在胸腺裸/SCID小鼠中进行WT/低水平内源性TF和表达asTF的人PDAC细胞系的原位模型。为了评估asTF表达升高的影响，我们将生成响应多西环素的PDAC细胞亚克隆（过）表达asTF。为了评估降低asTF水平的效果，我们将用表达高效特异性抗asTF shRNA的慢病毒转导PDAC细胞。定期对癌细胞和肿瘤血管进行体内成像；处死动物，采集组织标本，评估血管化程度/微血管密度/单核细胞浸润程度及增殖、凋亡指标。分析的中心目标：1。服用强力霉素后，i)我们是否观察到癌症进展加快和/或生存率降低？ii)原发肿瘤生长是否增强？iii)转移的发生率是否增加？2. asTF水平的降低是否抑制肿瘤生长和/或扩散？为了探索肿瘤asTF水平与疾病进展相关的可能性，将利用切除的PDAC组织进行临床病理研究。我们的研究结果将包括对asTF对胰腺癌病理生物学的贡献的定性理解。为了产生asTF-（过）表达的PDAC细胞，我们将使用我们实验室开发的一种创新的迷你基因。体内研究将使用先进的实时成像方法-标记SapC(H2)- dops囊泡。sapc偶联囊泡与其他方法相比具有优势，因为它们可以选择性地靶向肿瘤血管和富含磷脂酰丝氨酸的肿瘤细胞，并且可以快速进行体内评估，同时与传统的免疫组织化学技术完全兼容。

DESCRIPTION (provided by applicant): We will ascertain whether (over)expression of alternatively spliced Tissue Factor (asTF) promotes pancreatic tumorigenesis, and whether a decrease in asTF expression suppresses pancreatic tumorigenesis. High TF activity has been long observed in pancreatic ductal adenocarcinoma (PDAC). Until recently, the functional significance of high TF expression in PDAC and other forms of solid cancer was thought to be inseparable from the proteolytic function of full-length TF, an integral membrane protein that serves as a co-factor for the serine protease VIIa. We found that asTF - the secreted alternatively spliced TF variant - non-proteolytically activates cancer-associated signaling pathways via ligation of integrin ?6?1, the major contributor to primary tumor growth and metastatic spread in PDAC. ?6?1 expression on PDAC cells correlates with the rate of metastases; thus, asTF-?6?1interactions may enhance metastatic spread in an angiogenesis-independent, cell-autonomous fashion. Our newest data indicate that asTF protein is abundant in PDAC lesions, but not normal ducts, and promotes monocyte recruitment via upregulation of cell adhesion molecules on endothelial cells. Compounds selectively targeting asTF may have a major impact on cancer progression while having minimal impact on hemostasis. We will perform in vitro studies, as well as the orthotopic model in athymic nude/SCID mice with WT/low levels on endogenous TF and human PDAC cell lines expressing asTF. To evaluate the effects of elevated asTF expression, we will generate subclones of PDAC cells (over)expressing asTF in response to doxycycline. To evaluate the effects of lowering asTF levels, we will transduce PDAC cells with a lentivirus expressing a highly effective and specific anti-asTF shRNA. In vivo imaging of cancer cells and tumor vessels will be carried out at regular intervals; the animals wil be sacrificed, tissue specimens collected, and the degree of vascularization / microvessel density / monocyte infiltration evaluated along with the proliferation and apoptosis indices. The central objectives of the analysis: 1. Following doxycycline administration, i) do we observe enhanced cancer progression and/or decreased survival? ii) Is the primary tumor growth augmented? iii) Is there an increased incidence of metastases? 2. Does a decrease in asTF levels inhibit tumor growth and/or spread? To explore the possibility that the levels of tumor asTF correlate with disease progression, clinicopathological studies utilizing resected PDAC tissue will be performed. Our findings will comprise a qualitative gain in the understanding of asTF's contribution to the pathobiology of pancreatic cancer. To generate the asTF-(over)expressing PDAC cells, we will employ an innovative mini-gene developed in our laboratory. In vivo studies will be carried out using cutting-edge real-time imaging methodology - labeled SapC(H2)-DOPS vesicles. SapC-conjugated vesicles present advantage over other methodologies as they selectively target tumor vasculature and phosphatidylserine-enriched tumor cells, and allow rapid in vivo evaluation while being fully compatible with conventional immunohistochemical techniques.