High Throughput 3D Cell Assay for Metastatic Prostate Cancer

描述（由申请人提供）：前列腺癌是美国男性中最常见的非皮肤癌。尽管最近取得了进展，前列腺癌的死亡率仍然很高，因为出现了治疗耐药的癌细胞转移。目前缺乏针对转移性癌症的有效治疗方法，至少部分原因是缺乏针对转移性前列腺癌细胞及其微环境的独特性质的药物筛选平台。最近，我们的合作者发现前列腺癌（PCa）细胞像分子寄生虫一样转移到骨骼，并从造血干细胞（HSC）生态位环境中获取资源，在那里它们保持休眠状态并对常规抗癌药物产生抗性。如果能够在体外重建寄生于HSC生态位的休眠转移性PCa细胞，它们将成为筛选特异性抗转移性PCa药物的理想平台。我们最近的研究表明，3D共培养球体可以模拟PCa细胞寄生的HSC生态位，从而使PCa细胞处于生理上更安静的状态。为了利用转移性前列腺癌微组织工程能力，我们已经获得了药物筛选应用，需要开发这些类型的共培养球体的高通量格式。为此，第一阶段STTR提案将：目标1：验证384阵列悬挂降板系统，用于制备共培养球体。这是3-D biommatrix， LLC的主要关注点。我们已经修改了我们发布的平台（Tung et al. 2011），以增强液滴稳定性，促进移液管尖端插入，减轻蒸发，并在光学分析期间保持无菌（图1A）。我们将对荧光、透射光和生物发光实验（Z因子）进行高通量性能验证，并根据需要进一步完善设计。目的2：建立静止状态下骨转移癌的微工程三维组织阵列。这将是密歇根大学高山实验室的研究重点。开发利用384阵列悬滴板形成PCa细胞、骨髓基质细胞（MSCs）和人骨髓内皮细胞（HBMECs）共培养球体的方法，类似于以前在低通量微流控装置中制备的方法。我们还将证实这些共培养球体中的PCa细胞的静止性。激发这项研究的生物学假设是：我们可以通过开发专门针对寄生在HSC生态位的休眠转移性PCa细胞的药物来更好地治疗转移性PCa。PHS 398 （Rev. 11/07）第1页

DESCRIPTION (provided by applicant): Prostate cancer is the most common non-skin cancer in United States men. Despite recent advances, prostate cancer mortality still remains high due to the emergence of therapy-resistant cancer cells that metastasize. This lack of effective therapies against metastatic cancer exists, at least in part, because of lack of drug screening platforms that address the unique nature of metastastatic prostate cancer cells and their microenvironment. Recently, our collaborators have found that prostate cancer (PCa) cells act as molecular parasites as they metastasize to bone and harvest resources from the hematopoietic stem cell (HSC) niche environment where they stay dormant and resistant to conventional anti-cancer drugs. If the dormant metastasized PCa cells that parasitize the HSC niche could be recreated in vitro, they would serve as ideal platforms to screen specifically for anti-metastatic PCa drugs. We have recently shown that 3D co-culture spheroids can mimic the PCa cell parasitized HSC niche thereby maintaining PCa cells in a physiological, more quiescent state. What is required to take advantage of the metastatic PCa microtissue engineering capability we have acquired for drug screening applications is to develop a high throughput format of these types of co-culture spheroids. Towards this end, this Phase I STTR proposal will: Aim 1: Validate a 384 array hanging drop plate system for preparation of the co-culture spheroids. This is a major focus of 3-D Biomatrix, LLC. We have already modified our published platform (Tung et al. 2011) to: enhance droplet stability, facilitate pipette tip insertin, mitigate evaporation, and preserve sterility during optical analysis (Figure 1A). We will perform high throughput performance validations for fluorescence, transmitted light, and bioluminescence assays (Z'- factor), and further refine the design as necessary. Aim 2: Create arrays of microengineered 3D tissues of bone metastasized PCa in the quiescent state. This will be the focus of the Takayama lab at the University of Michigan. To develop procedures to utilize the 384 array hanging drop plate to form co-culture spheroids of PCa cells, marrow stromal cells (MSCs), and human bone marrow endothelial cells (HBMECs) similar to those prepared previously in low throughput microfluidic devices. We will also confirm quiescence of the PCa cells in these co-culture spheroids. The biological hypothesis that motivates this study is: We can treat metastatic PCa better by developing drugs that specifically target dormant metastatic PCa cells that are parasitizing the HSC niche. PHS 398 (Rev. 11/07) Page 1