Slusny Benedikt, Zimmer Vanessa, Nasiri Elena, Lutz Veronika, Huber Magdalena, Buchholz Malte, Gress Thomas M, Roth Katrin, Bauer Christian
Department of Gastroenterology, Endocrinology, Infectious Diseases and Metabolism, University Hospital Marburg, 35043 Marburg, Germany.
Institute of Systems Immunology, Center for Tumor Biology and Immunology, Philipps University Marburg, 35043 Marburg, Germany.
Cancers (Basel). 2024 Dec 27;17(1):51. doi: 10.3390/cancers17010051.
Most spheroid models use size measurements as a primary readout parameter; some models extend analysis to T cell infiltration or perform caspase activation assays. However, to our knowledge, T cell motility analysis is not regularly included as an endpoint in imaging studies on cancer spheroids.
Here, we intend to demonstrate that motility analysis of macrophages and T cells is a valuable functional endpoint for studies on molecular interventions in the tumor microenvironment. In particular, T cell migration analysis represents the final step of effector function, as T cells engage with targets cells upon cytotoxic interaction, which is represented by an arrest within the spheroid volume. Therefore, T cell arrest is a novel readout parameter of T cell effector function in spheroids.
Here, we demonstrate that incubation of macrophages with nigericin for NLRP3 activation increases T cell velocity, but results in decreased T cellular arrest. This is paralleled by reduced rejection kinetics of pancreatic cancer spheroids in the presence of antigen-dependent T cells and nigericin-treated macrophages. Our model demonstrates consistent changes in T cell motility upon coculturing of T cells and tumors cells with macrophages, including influences of molecular interventions such as NLRP3 activation.
Motility analysis using a spheroid model of pancreatic cancer is a more sophisticated alternative to in vitro cytotoxicity assays measuring spheroid size. Ultimately, an optimized spheroid model might replace at least some aspects of animal experiments investigating T cell effector function.
大多数球体模型将大小测量作为主要的读出参数；一些模型将分析扩展到T细胞浸润或进行半胱天冬酶激活测定。然而，据我们所知，T细胞运动分析在癌症球体成像研究中并未经常作为终点指标。
在此，我们旨在证明巨噬细胞和T细胞的运动分析对于肿瘤微环境分子干预研究是一个有价值的功能终点。特别是，T细胞迁移分析代表效应子功能的最后一步，因为T细胞在细胞毒性相互作用时与靶细胞结合，这表现为在球体内停滞。因此，T细胞停滞是球体中T细胞效应子功能的一个新的读出参数。
在此，我们证明用尼日利亚菌素孵育巨噬细胞以激活NLRP3可增加T细胞速度，但会导致T细胞停滞减少。在存在抗原依赖性T细胞和经尼日利亚菌素处理的巨噬细胞的情况下，胰腺癌球体的排斥动力学降低也与之平行。我们的模型表明，T细胞与肿瘤细胞和巨噬细胞共培养时，T细胞运动性会发生一致变化，包括NLRP3激活等分子干预的影响。
使用胰腺癌球体模型进行运动分析是一种比测量球体大小的体外细胞毒性测定更为复杂的替代方法。最终，优化后的球体模型可能至少替代动物实验中研究T细胞效应子功能的某些方面。
