Designing technologies to visualize protease activity in cancer models

项目摘要 癌症是由细胞不受控制的增殖引起的疾病的集合。这是第二 美国的主要死因，大约40%的美国人将被诊断出 患上了癌症蛋白酶是切割肽键的酶， 在癌症进展的许多步骤中，包括迁移和转移。靶向蛋白酶活性的药物 已经进入临床试验，但都没有成功。新的抗蛋白酶疗法 开发了改进的酶特异性，以减少不必要的副作用。从而增加我们的 需要了解肿瘤微环境中的蛋白酶活性，以使这些有前途的药物， 诊所 本项目的目标是开发生物分子缀合物，其可以可视化蛋白酶活性， 为了了解癌细胞如何调节其他局部细胞的活性，这些 缀合物联合收割机将核酸和肽结合，使得切割肽的蛋白酶激活核酸 酸结合。这将用于量化单个细胞类型周围的蛋白酶活性。因为核酸 杂交是序列特异性，可以在单个水凝胶内同时研究多种蛋白酶。 使用“点击”化学将缀合物偶联至水凝胶。这使得分子能够被添加到 在细胞培养期间，以研究在特定时间点的蛋白酶活性。这些共轭物已经被 设计，使他们可以在自动合成器，并可以很容易地纳入大多数 生物材料平台。 这项研究计划包括两个具体的目标，旨在开发蛋白酶反应性缀合物， 将它们结合到水凝胶中，并可视化模型组织中的时空蛋白酶活性。第一个目标 将开发能够结合荧光团标记的互补链的核酸-肽缀合物 只有在它们被蛋白酶“激活”之后。第二个目标是将这些共轭物纳入水凝胶 为了同时时空可视化含有癌性和癌性蛋白酶的水凝胶中的蛋白酶， 为了更好地了解转移过程。我们假设癌细胞 与非癌细胞一起培养的细胞将具有改变的蛋白酶活性。这种方法很强大，因为它可以 易于被其他实验室采用，可用于许多蛋白酶，并可用于大多数生物材料 系统.由于蛋白酶催化肽键的裂解，因此它们尤其可用于制备 刺激反应疗法因此，这项研究可以帮助跨学科的研究人员开发更多 有效的生物医学干预措施。

PROJECT SUMMARY Cancer is a collection of diseases that are caused by the uncontrolled proliferation of cells. It is the second leading cause of death in the United States of America and approximately 40% of Americans will get diagnosed with cancer at some point in their life. Proteases, which are enzymes that cleave peptide bonds, play vital roles in many steps in cancer progression, including migration and metastasis. Drugs that target protease activity have entered clinical trials, however they were all unsuccessful. New anti-protease therapies are being developed with improved enzyme specificity to reduce unwanted side-effects. Thus increasing our understanding of protease activity in the tumor microenvironment is needed to bring these promising drugs to the clinic. The objective of this project is to develop biomolecular conjugates which can visualize protease activity within model tissues in order to understand how cancerous cells modulate the activity of other local cells. These conjugates combine nucleic acids and peptide such that a protease cleaving the peptide activates the nucleic acid for binding. This will be used quantify protease activity around individual cell types. Since nucleic acid hybridization is sequence-specific, multiple protease can be studied simultaneously within a single hydrogel. The conjugates will be coupled to the hydrogel using “click” chemistry. This enables the molecules to be added during cell culture in order to study protease activity at specified time points. These conjugates have been designed so that they can be made on automated synthesizers, and can be easily incorporated into most biomaterials platforms. This research plan includes two specific aims that were designed to develop protease-responsive conjugates, incorporate them into hydrogels, and visualize spatiotemporal protease activity in a model tissue. The first aim will develop nucleic acid-peptide conjugates which are able to bind fluorophore-labelled complementary strand only after they been “activated” by proteases. The second aim will incorporate these conjugates into hydrogels for simultaneous spatiotemporal visualization proteases in a hydrogels containing both cancerous and noncancerous cells in order to better understand metastatic processes. We hypothesize that cancer cells cultured with non-cancerous cells will have altered protease activity. This approach is powerful because it can be easily adapted by other labs, can be used for many proteases and incorporated into most biomaterial systems. Since proteases catalyze the cleavage of a peptide bond, they are especially useful for making stimuli-responsive therapies. Thus this research can help researchers across disciplines develop more effective biomedical interventions.