Secretion responsive Hydrogels for Identification of Functional single T cells (SHIFT)

项目摘要/摘要 过继细胞疗法有可能通过使用患者来源的、 肿瘤特异性细胞毒性T细胞攻击癌症。虽然晚期病例对癌症的反应前景看好 免疫疗法增强了围绕这些治疗策略的乐观情绪，患者之间的治疗差异 为了更好和安全地应对，需要一种新的方法来选择高效的肿瘤特异性细胞毒T细胞 免疫疗法。 我们提出了一种高通量、单细胞水平的鉴定和恢复高功能T细胞的方法 在肿瘤存在的情况下分泌肿瘤杀伤信号。我们建议的技术建立在转型基础上 液滴乳状液的产生、分泌组学和基于水凝胶的新型传感技术的进展 最近开发出来的。在这项实验中，肿瘤杀伤T细胞和癌细胞将以均匀的 使用微流控液滴生成器测量分泌因子敏感水凝胶液滴的大小。由此产生的孤立 每对细胞的反应隔间将防止T细胞的多种分泌因子混合，并将 将它们集中起来，以便进行敏感的检测。这种水凝胶将对功能T细胞分泌的一种因子产生反应 通过显著的尺寸变化，允许具有活性T细胞的水凝胶可靠地从异质 使用基于流体动力大小的颗粒分选机制的混合物。已排序的对将分别 使用吸气吸管恢复，水凝胶壳溶解而不损害细胞，允许 单个T细胞功能的恢复。我们建议使用这种高通量检测方法，能够进行筛查 数以百万计的T细胞，以识别肿瘤反应性T细胞并将其回收用于下游分析或 扩散。这种分析方法简单、灵敏、通用，足以被广泛的研究团体或 最终在临床环境中分离和分析具有目标分泌表型的稀有细胞。

Project Summary/Abstract Adoptive cell therapy has the potential to circumvent cancer immune evasion by employing patient-derived, tumor-specific cytotoxic T-cells to attack cancer. While promising responses of advanced cases to cancer immunotherapy bolster the optimism surrounding these therapeutic strategies, interpatient variations in treatment responses demand a new method to select highly effective tumor-specific cytotoxic T-cells for better and safe immunotherapy. We propose a high-throughput, single-cell level method for identifying and recovering highly functional T cells that secrete tumor-killing signals in the presence of tumors. Our proposed technology builds on transformational advances in the generation of droplet emulsions, secretomics, and new hydrogel-based sensing techniques we recently developed. In this assay, tumor-killing T cells and cancer cells will be encapsulated in pairs in a uniformly sized, secreted factor-sensitive hydrogel droplet using a microfluidic droplet generator. The resulting isolated reaction compartments for each cell pair will prevent the mixing of T cells' multiple secreted factors and will concentrate them to allow sensitive detection. The hydrogel will respond to a factor secreted by functional T cells via a dramatic size change, allowing hydrogels with active T cells to be reliably isolated from the heterogeneous mixture using a hydrodynamic size-based particle sorting mechanism. The sorted pairs will be individually recovered using an aspiration pipette and the hydrogel shell dissolved without cell damage, allowing for the recovery of functional individual T cells. We propose to use this high-throughput assay, capable of screening millions of T cells, to identify tumor-responsive T cells and to recover them for downstream analysis or proliferation. This assay is simple, sensitive, and versatile enough to be used by a wide research community or eventually within clinical settings for isolating and profiling rare cells with target secretory phenotypes.