Mechanistic Insights and Diagnostic Applications for Hypoxia-Induced Vasorin in Pancreatic Cancer

项目摘要 随着癌症免疫疗法的出现，患者的可获得性急剧增加。 各种恶性肿瘤的治疗选择。然而，这些方法中的许多在治疗固体方面缺乏疗效。 肿瘤，也就是因为存在于这些肿瘤中的一组独特的环境因素。其中一个 肿瘤微环境(TME)中最普遍的特征是缺氧，也称为 缺氧。长期以来，低氧一直与转移增加和患者预后较差有关。作为回应 对于低氧应激，癌细胞激活高度调控的细胞通路和基因程序，从而促进 患者的生存、迁移、免疫豁免和死亡率增加。在所有实体肿瘤中，胰腺 癌症表现出最严重的缺氧表型，这可能是导致高死亡率的原因之一。 这种癌症类型。这些低氧特异的下游效应表明，肿瘤低氧可用于 阐明用于诊断和治疗目的的实体胰腺癌的选择性生物标志物。在这里，我利用 细胞表面蛋白质组学，以确定胰腺癌中一个新的缺氧调节靶点，称为血管蛋白(VASN)， 这是一个以前与胶质母细胞瘤进展有关的靶点。此外，我还展示了VASN的作用 在低氧应激下胰腺癌的生长和存活中起重要作用。最后，我分离出了 表达了10个针对VASN胞外区的抗体克隆。使用这些抗体，我发现 VASN在体外低氧条件下经历了大量的切割事件，这与以前发表的报道是独一无二的 结果。我们推测VASN裂解对于胰腺癌的生存和增殖是必要的。 以及VASN裂解特异性形式在相关肿瘤中表达水平分类 模型将有助于基于抗体的缺氧性胰腺癌诊断的下游发展。至 检验这一假设，我将使用蛋白质组学特征和重组蛋白表达来鉴定 VASN蛋白分解的体外细胞后果。为了分离出朝膜方向保留的克隆 对于切割形式的VASN，我将使用一种既定的基于噬菌体展示的重组抗体策略。精挑细选的 将实施基于抗体的生物素化策略，以寻找新的膜相互作用伙伴- 保留形式的裂解VASN，可能在下游信号转导中发挥重要作用。最后，我会 将我现有和未来的抗VASN抗体克隆功能化，用于基于血清的ELISA检测和 分别对VASN进行基于放射性示踪剂的免疫PET成像，这将验证蛋白水解物的存在 VASN在人血清和肿瘤以及体内nu/nu小鼠胰腺癌模型中的表达。这个 拟议的研究将提供对VASN蛋白降解在体内的功能后果的机械性见解 并为发展非侵入性、早期检测诊断提供证据 一种高度转移性和致命性疾病的替代品。

Project Summary With the advent of cancer immunotherapies, patients have seen dramatic increases in the availability of treatment options for various malignancies. However, many of these modalities lack efficacy in treating solid tumors, namely because of the unique set of environmental factors that exist within these tumors. One of the most ubiquitous features within the tumor microenvironment (TME) is oxygen deprivation, also known as hypoxia. Hypoxia has long been linked to increased metastasis and poorer prognoses for patients. In response to hypoxic stress, cancer cells activate highly regulated cellular pathways and gene programs that promote survival, migration, immune privilege, and increased mortality for patients. Across all solid tumors, pancreatic cancer exhibits the most severe hypoxic phenotype, which may contribute to the high mortality rate associated with this cancer type. These hypoxia-specific downstream effects suggest that tumor hypoxia can be used to elucidate selective biomarkers of solid pancreatic cancer for diagnostic and therapeutic purposes. Here, I utilize cell surface proteomics to identify a novel hypoxia-regulated target in pancreatic cancer called vasorin (VASN), a target previously implicated in the progression of glioblastoma. Furthermore, I have shown that VASN plays an important role in the growth and survival of pancreatic cancer under hypoxic stress. Finally, I have isolated and expressed ten unique antibody clones against the ectodomain of VASN. Using these antibodies, I found that VASN undergoes numerous cleavage events under hypoxia in vitro, which are unique from previously published results. We hypothesize that VASN cleavage is necessary for the survival and proliferation of pancreatic cancer under hypoxia, and that classification of expression levels for cleavage-specific forms of VASN in relevant tumor models will aid the downstream development of antibody-based diagnostics for hypoxic pancreatic cancer. To test this hypothesis, I will employ proteomic characterization and recombinant protein expression to identify the in vitro cellular consequences of VASN proteolysis. In order to isolate clones towards the membrane-retained form of cleaved VASN, I will use an established phage display-based recombinant antibody strategy. A selective antibody-based biotinylation strategy will be implemented to identify novel interacting partners of the membrane- retained form of cleaved VASN that may play an important role in downstream signaling. Finally, I will functionalize my existing and future anti-VASN antibody clones for serum-based ELISA detection and radiotracer-based immunoPET imaging of VASN, respectively, which will validate the presence of proteolyzed VASN in human serum and tumors, as well as in an in vivo nu/nu mouse model of pancreatic cancer. The proposed studies will provide mechanistic insights into the functional consequences of VASN proteolysis in pancreatic cancer, as well as providing evidence for the development of non-invasive, early-detection diagnostic alternatives for a highly metastatic and deadly disease.