Microfluidics-Based Platform for Screening Combinatorial Drug Treatments

描述(申请人提供)：肿瘤性疾病向高度致瘤性、转移性和放化疗耐药表型的转变是一系列基因变化累积的结果。联合疗法已经成为“单一药物疗法”的有力替代品，因为它们通过克服癌细胞中的多重耐药机制来增强细胞死亡。肿瘤坏死因子-1相关的凋亡诱导配体(TRAIL)和抗死亡受体(DR)4和5的激动型抗体因能选择性地诱导转化(恶性)细胞的凋亡而在正常细胞中表现出较低的细胞毒性而受到人们的广泛关注。尽管有这样的前景，但许多肿瘤细胞对TRAIL具有耐药性，因此，放射和化疗基因毒素已被用于增强癌细胞对死亡受体介导的细胞凋亡的敏感性。虽然这种方法取得了一定的成功，但传统的“单一化学增敏剂发现”方法本身就是低通量的，不允许对不同的增敏剂进行比较。我们假设，筛选小分子文库可以快速识别新的联合治疗策略和候选药物，并能够对不同的分子进行比较。在这项建议中，我们描述了一种新的基于癌细胞的筛选范例，在该范例中，我们集成了传统的基于孔板的筛选和微制造/微流体技术，以识别和机制评估使癌细胞对死亡受体介导的凋亡敏感的小分子。井板筛选方法将用于对FDA批准的约2500种药物进行初步筛选，以确定候选铅。然后，将使用微流控癌细胞阵列对从初筛中选择的线索进行严格的二次筛选；假设使用微流控以组合刺激询问细胞的能力可以导致二次筛查步骤的并行化和小型化。一次筛查所需的较高产量和二次筛查的组合剂量要求使我们有理由选择前者使用传统的96孔板，而后者使用基于微流体的癌细胞阵列。预计拟议的研究将导致开发一种基于孔板和微流控细胞阵列的新的综合筛选范式，用于发现用于联合治疗的小分子，并识别和机制评估大约20-50个与死亡受体激动剂联合增强癌细胞死亡的新候选药物。拟议的筛选平台具有超越目前工作的影响；该平台可扩展到筛选/调查影响联合疗法的各种因素，包括其他候选治疗因素(例如多肽、siRNA等)、可溶性因素(例如细胞因子、代谢物等)和肿瘤微环境效应。项目简介本提案旨在利用高通量筛查确定癌症治疗的有效组合治疗。将使用一种基于传统孔板筛选和微流控癌细胞阵列相结合的新型HTS范式。微流控细胞阵列将被开发为研究候选分子之间协同作用的平台。这项工作的成功完成可以导致新的联合抗癌疗法的开发，并在其他基于细胞的高通量筛查应用中。公共卫生相关性：这项提案旨在利用高通量筛查确定癌症治疗的有效组合疗法。将使用一种基于传统孔板筛选和微流控癌细胞阵列相结合的新型HTS范式。微流控细胞阵列将被开发为研究候选分子之间协同作用的平台。这项工作的成功完成可以导致新的联合抗癌疗法的开发，并在其他基于细胞的高通量筛选应用中。

DESCRIPTION (provided by applicant): The transformation of neoplastic disease to a highly tumorigenic, metastatic, and radio / chemotherapy resistant phenotype is a result of the accumulation of a number of genetic changes. Combination therapies have emerged as powerful alternatives to `single-agent therapies' since they enhance cell death by overcoming multiple resistance mechanisms in cancer cells. Tumor Necrosis Factor-1 Related Apoptosis Inducing Ligand (TRAIL) and agonistic antibodies to Death Receptor (DR) 4 and 5 have attracted significant attention due to their ability to selectively induce apoptosis in transformed (malignant) cells while demonstrating little cytotoxicity in normal cells. Despite this promise, many tumor cells are resistant to TRAIL and as a result, radiation and chemotherapeutic genotoxins have been employed to sensitize cancer cells to death receptor- mediated apoptosis. While this approach has been moderately successful, traditional `single-chemosensitizer discovery' approaches are inherently low-throughput and do not allow for comparison of different sensitizing agents. We hypothesize that screening small molecule libraries can result in the rapid identification of new combination treatment strategies and candidates and enable comparison of different molecules. In this proposal, we describe a novel cancer cell-based screening paradigm in which we integrate traditional well plate based screening and microfabrication / microfluidics technologies for the identification and mechanistic evaluation of small-molecules that sensitize cancer cells to death receptor-mediated apoptosis. Well-plate screening methods will be employed in the primary screening of a library of ~2,500 FDA approved drugs for identifying lead candidates. Microfluidic cancer cell arrays will then be employed to carry out stringent secondary screening of leads selected from the primary screen; it is hypothesized that the ability to interrogate cells with combinatorial stimuli using microfluidics can lead to the parallelization and miniaturization of the secondary screening step. The higher throughput required in the primary screening and the combinatorial dosing requirements in the secondary screening justify our choices of using conventional 96-well plates for the former and a microfluidcs-based cancer cell array for the latter. It is anticipated that the proposed research will lead to the development of a novel integrated screening paradigm based on well-plates and microfluidic cell arrays for the discovery of small molecules for combination treatments and the identification and mechanistic evaluation of approximately 20-50novel candidates that enhance cancer cell death in combination with death receptor agonists. The proposed screening platform has implications beyond the present work; the platform can be extended to screen/investigate various factors influencing combination therapies including, other therapeutic candidates (e.g. peptides, siRNA, etc.), soluble factors (e.g. cytokines, metabolites, etc.), and tumor microenvironment effects. Project Narrative This proposal aims to identify effective combination treatments for cancer therapy using high-throughput screening. A novel HTS paradigm based on the integration of traditional well-plate screening and a microfluidic cancer cell array will be used. The microfluidic cell array will be developed as a platform for investigating synergistic interactions between candidate molecules. Successful completion of this work can lead to the development of new combination anti-cancer therapies and in other cell-based high- throughput screening applications. PUBLIC HEALTH RELEVANCE: This proposal aims to identify effective combination treatments for cancer therapy using high-throughput screening. A novel HTS paradigm based on the integration of traditional well-plate screening and a microfluidic cancer cell array will be used. The microfluidic cell array will be developed as a platform for investigating synergistic interactions between candidate molecules. Successful completion of this work can lead to the development of new combination anti-cancer therapies and in other cell-based high through put screening applications.