Department of Mechanical Engineering, University of Maryland, College Park, MD 20742, United States.
Fischell Institute for Biomedical Devices, University of Maryland, College Park, MD 20742, United States.
Integr Biol (Camb). 2024 Jan 23;16. doi: 10.1093/intbio/zyae014.
Immune responses against cancer are inherently stochastic, with small numbers of individual T cells within a larger ensemble of lymphocytes initiating the molecular cascades that lead to tumor cytotoxicity. A potential source of this intra-tumor variability is the differential ability of immune cells to respond to tumor cells. Classical microwell co-cultures of T cells and tumor cells are inadequate for reliably culturing and analyzing low cell numbers needed to probe this variability, and have failed in recapitulating the heterogeneous small domains observed in tumors. Here we leverage a membrane displacement trap array technology that overcomes limitations of conventional microwell plates for immunodynamic studies. The microfluidic platform supports on-demand formation of dense nanowell cultures under continuous perfusion reflecting the tumor microenvironment, with real-time monitoring of T cell proliferation and activation within each nanowell. The system enables selective ejection of cells for profiling by fluorescence activated cell sorting, allowing observed on-chip variability in immune response to be correlated with off-chip quantification of T cell activation. The technology offers new potential for probing the molecular origins of T cell heterogeneity and identifying specific cell phenotypes responsible for initiating and propagating immune cascades within tumors. Insight Box Variability in T cell activation plays a critical role in the immune response against cancer. New tools are needed to unravel the mechanisms that drive successful anti-tumor immune response, and to support the development of novel immunotherapies utilizing rare T cell phenotypes that promote effective immune surveillance. To this end, we present a microfluidic cell culture platform capable of probing differential T cell activation in an array of nanoliter-scale wells coupled with off-chip cell analysis, enabling a high resolution view of variable immune response within tumor / T cell co-cultures containing cell ensembles orders of magnitude smaller than conventional well plate studies.
肿瘤的免疫反应本质上是随机的，在淋巴细胞的较大集合中，只有少数个体 T 细胞启动导致肿瘤细胞毒性的分子级联反应。这种肿瘤内变异性的一个潜在来源是免疫细胞对肿瘤细胞反应的能力差异。T 细胞和肿瘤细胞的经典微孔共培养不足以可靠地培养和分析探测这种变异性所需的低细胞数量，并且未能重现肿瘤中观察到的异质小域。在这里，我们利用膜置换陷阱阵列技术克服了传统微孔板在免疫动力学研究中的局限性。该微流控平台支持在连续灌注下按需形成密集纳米孔培养物，反映肿瘤微环境，实时监测每个纳米孔内 T 细胞的增殖和激活。该系统允许选择细胞进行荧光激活细胞分选分析，从而可以将芯片上观察到的免疫反应变异性与芯片外 T 细胞激活的定量分析相关联。该技术为探索 T 细胞异质性的分子起源以及鉴定负责启动和传播肿瘤内免疫级联反应的特定细胞表型提供了新的潜力。洞察框 T 细胞激活的变异性在抗肿瘤免疫反应中起着关键作用。需要新的工具来揭示驱动成功抗肿瘤免疫反应的机制，并支持利用促进有效免疫监测的罕见 T 细胞表型的新型免疫疗法的开发。为此，我们提出了一种能够在纳米升规模的阵列孔中探测差异 T 细胞激活的微流控细胞培养平台，结合芯片外细胞分析，能够对包含比传统微孔板研究小几个数量级的细胞集合的肿瘤/T 细胞共培养物中的可变免疫反应进行高分辨率观察。
