Designing technologies to visualize protease activity in cancer models

项目总结 癌症是一组由细胞不受控制的增殖引起的疾病。这是第二个 美国主要死因约40%的美国人将被确诊 在他们生命中的某个时刻患上癌症。蛋白水解酶是一种裂解肽键的酶，起着至关重要的作用。 在癌症进展的许多步骤中，包括迁移和转移。以蛋白酶活性为靶点的药物 都进入了临床试验，但都没有成功。新的抗蛋白酶疗法正在研发中 开发了更好的酶专一性，以减少不想要的副作用。从而增加了我们的 需要了解肿瘤微环境中的蛋白酶活性，才能使这些有希望的药物 诊所。 这个项目的目标是开发生物分子偶联物，它可以直观地显示 建立组织模型，以了解癌细胞如何调节其他局部细胞的活动。这些 连接物将核酸和多肽结合在一起，从而使切割多肽的蛋白酶激活核酸。 用于装订的酸。这将被用来量化单个细胞类型周围的蛋白酶活性。由于核酸 杂交是序列特异性的，可以在单个水凝胶中同时研究多种蛋白酶。 这些偶联物将通过“点击”化学作用偶联到水凝胶上。这使得分子能够被添加 在细胞培养过程中，以研究特定时间点的蛋白酶活性。这些共轭化合物一直是 设计使它们可以在自动合成器上制造，并可以很容易地整合到大多数 生物材料平台。 这项研究计划包括两个特定的目标，旨在开发对蛋白酶反应的偶联物， 将它们加入到水凝胶中，并可视化模型组织中的时空蛋白酶活性。第一个目标 将开发能够与荧光团标记的互补链结合的核酸-多肽结合物 只有在它们被蛋白水解酶“激活”之后。第二个目标是将这些偶联物加入水凝胶中。 用于同时时空显示水凝胶中同时含有癌性和非癌性的蛋白酶 以更好地了解转移过程。我们假设癌细胞 与非癌症细胞一起培养时，蛋白酶活性会发生变化。这种方法很强大，因为它可以 易于被其他实验室改编，可用于多种酶，并可用于大多数生物材料 系统。由于蛋白水解酶催化多肽键的裂解，所以它们特别适用于 刺激反应疗法。因此，这项研究可以帮助跨学科的研究人员更多地发展 有效的生物医学干预。

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.