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。尽管最近的进展，前列腺癌死亡率仍然很高，这是由于出现了转移的耐药癌细胞。这种针对转移性癌症的有效疗法的缺乏至少部分是因为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）细胞作为分子寄生虫，因为它们转移到骨和收获资源，从造血干细胞（HSC）利基环境中，他们保持休眠和耐药的常规抗癌药物。如果可以在体外重建寄生在HSC小生境中的休眠的转移的PCa细胞，它们将作为理想的平台来专门筛选抗转移的PCa药物。我们最近已经表明，3D共培养球体可以模仿PCa细胞寄生HSC龛，从而保持PCa细胞在生理上，更静止的状态。利用我们已经获得的用于药物筛选应用的转移性PCa微组织工程能力所需的是开发这些类型的共培养球状体的高通量形式。为此，该I期STTR提案将：目标1：用于制备共培养球状体的384阵列悬滴平板系统。这是3-D Biomatrix，LLC的主要重点。我们已经修改了我们发表的平台（Tung等人，2011），以增强液滴稳定性，促进移液器吸头插入，减轻蒸发，并在光学分析期间保持无菌性（图1A）。我们将对荧光、透射光和生物发光测定（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：在静止状态下创建骨转移PCa的微工程3D组织阵列。这将是密歇根大学高山实验室的重点。开发利用384阵列悬滴板形成PCa细胞、骨髓基质细胞（MSC）和人骨髓内皮细胞（HBMEC）的共培养球体的程序，类似于先前在低通量微流体装置中制备的那些。我们还将证实PCa细胞在这些共培养球体中的静止。促使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小生境中的休眠转移性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