High Throughput 3D Cell Assay for Metastatic Prostate Cancer

描述(申请人提供)：前列腺癌是美国男性最常见的非皮肤癌。尽管最近取得了进展，但由于出现了转移的耐药癌细胞，前列腺癌的死亡率仍然很高。这种缺乏针对转移性癌症的有效治疗方法的存在，至少在一定程度上是因为缺乏解决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)细胞在转移到骨骼和从造血干细胞(HSC)利基环境中获取资源时扮演着分子寄生虫的角色，在那里它们处于休眠状态，对传统抗癌药物具有耐药性。如果寄生于HSC利基的潜伏转移的PCa细胞能够在体外重建，它们将成为专门筛选抗转移的PCa药物的理想平台。我们最近发现，3D共培养的球体可以模拟PCa细胞寄生的HSC生态位，从而使PCa细胞保持在生理的、更静止的状态。为了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，需要开发一种高通量形式的这些类型的共培养球体。为此，这项第一阶段STTR提案将：目标1：验证用于制备共培养球体的384阵列悬挂滴板系统。这是3D Biomatrix，LLC的一个主要关注点。我们已经修改了我们发布的平台(董等人)。提高液滴稳定性，促进吸管尖端插入，减少蒸发，并在光学分析过程中保持无菌(图1A)。我们将对荧光、透射光和生物发光分析(Z‘因子)进行高通量性能验证，并根据需要进一步改进设计。目的2：建立静止骨转移性前列腺癌的三维微工程组织阵列。这将是密歇根大学高山实验室的重点。开发利用384阵列悬挂板形成与先前在低通量微流控设备中制备的球体类似的方法，以形成PCa细胞、骨髓基质细胞(MSCs)和人骨髓内皮细胞(HBMECs)的共培养球体。我们还将证实在这些共培养的球体中的PCA细胞是静止的。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利基的潜伏的转移性PCa细胞的药物来更好地治疗转移性PCa。小灵通398(11/07版)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