3D Live Imaging and Phenotyping of the Subcellular Cytotoxicity in CAR-T Cell Immunotherapy
Authors: Chen Liting , Wang Zhaofei , Wang Jie , Jin Jin , Wei Jia , Zhang Yicheng , Fei Peng 
Published: December 2024

Introduction
Chimeric antigen receptor-engineered T cells (CAR-T cells) have ushered in a new era for treating hematological malignancies. However, highly selected patients still have overall response rates (ORRs) ranging from 50-90%. Current functional studies of CAR-T cells have focused mostly on the typical attributes of cell populations and lack long-term phenotyping of antitumor cytotoxicity at the subcellular level; the latter has great potential for the identification of the efficacy-related biomarkers. In contrast to classical fluorescence microscopy techniques, light-sheet fluorescence microscopy (LSFM) can rapidly image live cells in three dimensions (3D) with low phototoxicity, thereby allowing for a more physiologically relevant assessment of T-cell functionality. Due to the heterogeneity of CAR-T cells, the results from the analysis of the individual cells potentially exhibit significant discrepancies in overall performance. Therefore, a high-throughput long-term live-cell imaging system is needed for analyzing the CAR-T-cell cytotoxicity.
Methods
In this study, we developed a high-throughput, high-resolution, multidimensional live-cell phenotyping pipeline consisting of the following: a dedicated microfluidic chip for large-scale scouting over 400 CAR-T/tumor cell pairs; an high-throughput Bessel oblique plane microscopy (HBOPM) that could automatically detect immune cytotoxicity events and three-dimensionally image the subcellular interactions with an isotropic subcellular resolution of 320 nm over hours; and a comprehensive spatial-spectrum cell phenotyping algorithm that could specifically identify and quantitatively analyze organelle phenotypes that were strongly correlated with the killing efficacy.
Results
Using this advanced microscopy platform, several key subcellular events in CAR-T cells associated with their cytolytic capacity were captured and comprehensively analyzed; these events included the instantaneous formation of immune synapses and the sustained changes in the microtubing morphology. Furthermore, we identified the actin retrograde flow speed, the actin depletion coefficient, the microtubule polarization and the contact area of the CAR-T/target cell conjugates as essential parameters strongly correlated with CAR-T-cell cytotoxic function. The visualization and statistical results were significantly different between the dasatinib-treated CAR-T cells and controls, validating the effectiveness of our platform. With this microfluidics-enabled intelligent HBOPM platform, we revealed the dynamics of immune synapses as key factors for evaluating the cytotoxicity of CAR-T cells.
Conclusions
In summary, based on the new development of intelligent HBOPM and T-cell behavior analysis, we accomplished high-throughput, long-term 3D imaging and phenotyping of CAR-T/Nalm6 interactions to comprehensively analyze the antitumor cytotoxic function of CAR-T cells. The advanced imaging technique in conjunction with high-dimensional image-based analysis formed an automated and scalable cell observation and analysis pipeline well suited for diverse types of cell research. Our approach will be useful for establishing criteria for quantifying T-cell function in individual patients for all T-cell-based immunotherapies.