Determining treatment sensitivity in B cell lymphoma by novel microfluidics-based NK cell immunogenicity platform

摘要 B细胞性非霍奇金淋巴瘤(BNHL)是最常见的淋巴瘤亚型，占 全是NHL。BNHL通常单独使用抗CD20抗体(如利妥昔单抗)或与其联合治疗 化疗。然而，目前还没有生物学方法或标记来预测敏感性或 对利妥昔单抗(或任何其他)抗体疗法的抗药性。抗体活性的一个关键特征是通过天然的 然而，杀伤(NK)细胞介导的抗体包裹的靶肿瘤细胞的杀伤活性和随后的 抵抗，人们对此知之甚少。在这一应用中，我们建议开发并验证一种高通量液滴 基于微流控平台研究NK细胞与快、慢或不活跃相关的关键特征 非霍奇金淋巴瘤的肿瘤杀伤动力学。我们将首先采用一种新的方法，将生物兼容的声流控 液滴微阵列形成过程中的液滴分选仪用于确定免疫靶细胞的表型 微流控液滴中的相互作用。我们将验证一种基于液滴的微流控设备来询问单细胞 完整液滴内的动态响应和细胞-细胞相互作用。接下来，我们将展示一种高纯度的 (&gt；95%)，高通量(&gt；10,000事件/S)，四通道声流液滴分选器，与液滴集成 分析数组。在建立相互作用的动力学曲线之后，下游4通道分选将允许， 识别含有活跃淋巴细胞的液滴并将其分类到一个独特的池中；分离基础淋巴细胞 放入另一个基于荧光的池中。基于图像分析选择分类标准的独特功能 可以通过液滴成像阵列和声流控液滴分类器的组合来提供， 对于传统的荧光激活液滴分类器(FADS)是无法实现的，因为成像跟踪本质上是 在高速流动中很棘手。因此，我们的方法作为一种“自下而上”的分类方法，首先确定 区分不同的功能类别，然后探索单个细胞类别的内容以确定关键 与靶细胞杀伤相关的NK细胞异质性免疫功能分子分类的影响因素。 此外，我们还将鉴定NK细胞的异质性和生物功能特性，以发现新药 通过集成的声流控液滴分选平台进行NK细胞依赖免疫疗法的组合。我们 将证明我们的设备的表型识别的准确性及其临床适用性 在单抗存在下监测和分类NK/NHL单细胞相互作用的应用 抗体，并执行生化分泌组分析从‘高度活跃’，‘基础’和无反应池。 通过将这些发现与药物筛选和表型改变药物的鉴定结合起来，我们将 演示该技术在个性化医疗和合理临床中的适用性 免疫治疗的应用。我们设想我们的平台可以用于各种单细胞分析 在免疫治疗中的应用，将对生物工程、生物医学和治疗具有很高的价值。 研究社区。

Abstract B cell non-Hodgkin lymphomas (bNHL) are the most common lymphoma subtype representing >85% of all NHLs. bNHL are typically treated the anti-CD20 antibody (e.g., rituximab) alone or in combination with chemotherapy. There are currently, however, no biological methods or markers to predict the sensitivity or resistance to rituximab (or any other) antibody therapy. A key feature of antibody activity occurs through natural killer (NK) cell-mediated killing of antibody-coated target tumor cells, however, antitumor activity and subsequent resistance, is poorly understood. In this application, we propose to develop and validate a high throughput droplet based microfluidic platform to investigate the key features of NK cells associated with rapid, slow or inactive tumor killing kinetics in NHL. We will first adapt a novel approach and integrate the biocompatible acoustofluidic droplet sorter during the droplet microarray formation to determine the phenotypes of immune-target cell interaction in microfluidic droplets. We will validate a droplet-based microfluidic device to interrogate single-cell dynamic responses and cell-cell interactions within intact droplets. Next, we will demonstrate a high-purity (>95%), high-throughput (>10,000 events/s), four-channel acoustofluidic droplet sorter to integrate with droplet analysis array. The downstream 4-channel sorting will allow, after establishing the kinetic profiles of interactions, to identify and sort droplets containing active lymphocytes into a distinctive pool; separate basal lymphocytes into another pool based on fluorescence. A unique function of selecting sorting criteria based on imaging analysis can be provided by the combination of droplet imaging array and acoustofluidic droplet sorters, which is unachievable for conventional fluorescence activated droplet sorters (FADS) since imaging tracking is inherently tricky in high-speed flow. Thus, our approach serves as a “bottom-up” method of classification, by first identifying distinct functional categories and then probing the content of the individual cell category to determine the key factors for the molecular classification of heterogeneous immune functions of NK cells related to target cell kill. In addition, we will identify NK cell heterogeneity and bio-functional characteristics to discover novel drug combinations for NK cell dependent immunotherapy via an integrated acoustofluidic droplet sorting platform. We will demonstrate that the accuracy of phenotype identification of our device and its suitability for clinical applications by monitoring and classifying NK/NHL single cell interactions in the presence of monoclonal antibodies and performing biochemical secretome assay from ‘hyperactive’, ‘basal’ and non-responsive pools. By combing these findings with drug screening and identification of phenotype altering drugs, we will demonstrate the applicability of this technology for personalized medicine and rational clinical immunotherapeutic applications. We envision our platform may be leveraged in a variety of single-cell analysis applications in immunotherapy and it will provide high value to the bioengineering, biomedical, and therapeutic research communities.