Microfluidics-Based Platform for Screening Combinatorial Drug Treatments

描述（由申请人提供）：肿瘤疾病向高度致瘤性、转移性和放射/化疗耐药表型的转变是许多遗传变化积累的结果。联合疗法已成为“单药疗法”的有力替代品，因为它们通过克服癌细胞中的多种耐药机制来增强细胞死亡。肿瘤坏死因子-1相关凋亡诱导配体（TRAIL）和死亡受体（DR） 4和5的激动抗体引起了人们的极大关注，因为它们能够选择性地诱导转化（恶性）细胞凋亡，而在正常细胞中显示出很少的细胞毒性。尽管有这样的前景，但许多肿瘤细胞对TRAIL具有耐药性，因此，放疗和化疗基因毒素已被用于使癌细胞对死亡受体介导的细胞凋亡敏感。虽然这种方法已经取得了一定的成功，但传统的“单一化学增敏剂发现”方法本质上是低通量的，并且不允许比较不同的增敏剂。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，并可以比较不同的分子。在这篇论文中，我们描述了一种新的基于癌细胞的筛选范例，我们将传统的基于孔板的筛选和微加工/微流体技术结合起来，用于鉴定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。孔板筛选方法将用于约2,500种FDA批准的药物库的初步筛选，以确定主要候选药物。然后将采用微流控癌细胞阵列对从初级筛选中选择的导联进行严格的二次筛选；据推测，利用微流体的组合刺激来询问细胞的能力可以导致二次筛选步骤的并行化和小型化。初级筛选需要更高的通量，二级筛选需要组合给药，因此我们选择前者使用传统的96孔板，后者使用基于微流体的癌细胞阵列。预计所提出的研究将导致基于孔板和微流控细胞阵列的新型综合筛选范例的发展，用于发现用于联合治疗的小分子，以及对大约20-50种新型候选药物的鉴定和机制评估，这些候选药物可与死亡受体激动剂联合促进癌细胞死亡。拟议的筛选平台的影响超出了目前的工作；该平台可以扩展到筛选/研究影响联合疗法的各种因素，包括其他候选治疗药物（如肽、siRNA等）、可溶性因子（如细胞因子、代谢物等）和肿瘤微环境效应。本项目旨在通过高通量筛选确定有效的癌症联合治疗方案。将使用基于传统孔板筛选和微流控癌细胞阵列集成的新型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相互作用的平台。这项工作的成功完成将导致新的联合抗癌疗法和其他基于细胞的高通量筛选应用的发展。公共卫生相关性：本提案旨在通过高通量筛选确定癌症治疗的有效联合治疗。将使用基于传统孔板筛选和微流控癌细胞阵列集成的新型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相互作用的平台。这项工作的成功完成可能会导致新的联合抗癌疗法的发展和其他基于细胞的高通量筛选应用。

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.