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

项目总结/摘要 免疫细胞疗法具有通过采用患者来源的， 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很有希望 免疫疗法支持了对这些治疗策略的乐观态度， 免疫应答需要一种新的方法来选择高效的肿瘤特异性细胞毒性T细胞， 免疫疗法 我们提出了一种高通量、单细胞水平的方法来鉴定和回收高功能的T细胞 在肿瘤存在时分泌肿瘤杀伤信号。我们提出的技术建立在变革性的 液滴乳剂、分泌组学和新的基于水凝胶的传感技术的发展， 最近开发的。在该测定中，肿瘤杀伤性T细胞和癌细胞将以均匀的方式成对包封。 使用微流控液滴发生器，对大小合适的分泌因子敏感的水凝胶液滴进行微流控。由此产生的孤立 每个细胞对的反应区室将防止T细胞的多种分泌因子的混合， 将其浓缩以进行灵敏的检测。这种水凝胶会对功能性T细胞分泌的一种因子产生反应 通过戏剧性的大小变化，允许具有活性T细胞的水凝胶可靠地从异质细胞中分离出来。 使用基于流体动力学尺寸的颗粒分选机制对混合物进行分选。排序后的对将分别 使用抽吸移液管回收，并且水凝胶壳溶解而没有细胞损伤，从而允许 恢复功能性单个T细胞。我们建议使用这种高通量检测，能够筛选 数以百万计的T细胞，以识别肿瘤反应性T细胞并回收它们用于下游分析， 增殖该测定方法简单、灵敏且通用性强，足以被广泛的研究团体或组织使用。 最终在临床环境中用于分离和分析具有靶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.