基于微流控芯片的单细胞平台用于白血病细胞异质性和耐药性分析

Tumor heterogeneity and the resulted drug resistance make tumor curing a significant challenge. Recent achievements from single-cell studies have demonstrated that future cancer research should mainly be single-cell based analysis. Current microfluidic-based platforms can achieve good performance for single-cell capture and short-term cellular function analysis. However, it is challenging to do long-term culture in the devices and recover the cells of interest to perform downstream analysis, which hence limits their applications in deep analysis of tumor characteristics. Previously, we developed a microfluidic strategy to perform long-term cell culture and cell recovery and validated its performance. In this project, we propose to develop a high-throughput microfluidic single-cell platform based on our prior work, to analyze leukemia (or blood cancer) heterogeneity and its drug resistance. This platform has the ability to control cells in one-, two- and three-dimensional (3D) microchambers, which allows us to analyze the heterogeneity and drug resistance of tumor cells at three levels of single cell, single-cell clone and 3D sphere. In addition, it provides a way to combine the laser capture microdissection system to recover the drug-resistant cells for downstream analysis. The achievements of this project will benefit the fundamental researches including single-cell analysis, cancer drug-resistance studies and precise medicine. We hope that it will greatly promote the development of Chinese Biomedical Engineering and/or other related disciplines.

肿瘤的高度异质性和由此而引起的耐药性是导致治疗失败的主要原因。近年来基于单细胞技术的研究结果表明单细胞水平的肿瘤研究是未来主要发展方向。现有微流控平台可以完成单细胞的有效捕获和短时间的功能分析，但难以进行细胞的长期培养及目标细胞的选择性回收，因此难以实现肿瘤耐药性的深度分析。我们在前期研究中摸索了用于细胞长期培养与回收的微流控方法，并初步验证了其功效。本项目拟在已有研究的基础上，提出建立基于微流控芯片的高通量单细胞平台以用于白血病（血癌）细胞异质性和耐药性分析：在线形、二维和三维微腔室对单细胞进行操控、培养和梯度药物浓度在线刺激，在单细胞、单克隆和三维细胞团水平对血癌细胞的耐药性进行研究，进一步通过激光显微切割将耐药目标细胞取出以用于下游耐药机制的深度解析。本项目成果可广泛应用于单细胞研究、肿瘤药物筛选以及肿瘤精准医疗等领域，并促进生物医学及相关交叉学科的发展。

耐药引起的肿瘤复发仍然是当前肿瘤治疗的瓶颈。单细胞水平耐药性分析对于开发个性化肿瘤精准治疗具有显著指导意义。然而，目前高通量单细胞水平动态药物分析仍然是一重大技术挑战。近年来，微流控芯片技术的快速发展为这一问题的解决提供了可能。该项目首先设计、制作了一种包含>3000个单细胞微腔室阵列与6个浓度梯度的微流控芯片，并对该芯片进行了表征，包括单细胞与单克隆阵列的行成、化学浓度梯度的生成等。在此基础上，分析测试了白血病细胞系K562细胞对抗癌药物伊马替尼（imatinib）、白藜芦醇（resveratrol）及二者组合后的耐药性，发现单细胞和单克隆中的细胞均表现出明显的异质性，且药物组合比单个药物对癌细胞具有更为明显的杀伤效果，其IC50降低近一半；同时单克隆阵列中细胞的IC50比单细胞阵列的IC50有所提高，猜测癌细胞间相互作用可能有利于提高细胞耐药性。最后，为操控尺寸更小的原代细胞（来自于临床白血病患者骨髓样本），对芯片微腔室结构进行了进一步优化，可实现4000个以上原代细胞的捕获；基于此研究了病人细胞对临床药物Ara-C/DNR的耐药性，其结果进一步展现了细胞的异质性和单细胞水平研究的必要性。该高通量微流控芯片平台有望广泛用于单细胞水平肿瘤性分析。

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