Standardizing ex vivo CAR-T cell cytotoxicity evaluation via multimodal 2-D and 3-D imaging of CAR-T target dual reporter cell lines
Society for Immunotherapy of Cancer (SITC) 2025 Annual Meeting
National Harbor, Maryland, United States
November 08, 2025Abstract
Background: Chimeric Antigen Receptor T (CAR-T) cell therapy has emerged as an advanced and highly effective approach for treating hematologic malignancies with its scope rapidly expanding to encompass diverse applications including solid tumors. This fast-paced development calls for highly sensitive and quantitative ex vivo assays for assessing CAR-T cell cytotoxicity, particularly in models that recapitulate 3-D tumor environments.
Methods: To address this need, we generated GFP-luciferase dual reporter cancer cell lines that endogenously express high levels of key CAR-T target antigens, CD19 and BCMA. Using both antigen-specific and mock-engineered CAR-T cells, we assessed cytotoxicity via multi-modal imaging strategy, integrating luciferase-based bioluminescence assay with phase contrast and fluorescence live cell imaging. To further demonstrate dynamic visualization of CAR-T activity in an environment that mimics in vivo conditions, we embedded dual reporter spheroids in 3-D matrices which allowed for time-lapse imaging of CAR-T cell infiltration.
Results: Our multimodal imaging approach revealed significantly enhanced cancer cell killing by targeted CAR-T cells compared to controls. Employing 2-D and 3-D co-culture assays of dual reporter cancer cells with targeted and mock-engineered CAR-T cells, we found that targeted CAR-T cells exhibited higher killing of cancer cells than mock CAR-T cells in both live imaging and luciferase assays. In 3-D fluorescence imaging experiments, we identified a reduction in both the size of cancer cell spheroids and in the GFP signal in response to targeted CAR-T cells as compared to mock CAR-T cells. Additionally, in both 2-D and 3-D co-culture assays, we noticed a significant decrease in relative luciferase signal from reporter cancer cells in conditions with targeted CAR-T cells, implying targeted cancer cell killing by CAR-T cells. Moreover, time-lapse confocal imaging of spheroids in 3-D matrices captured active infiltration of CAR-T cells into tumor spheroids over time.
Conclusions: These results showcase the benefit of multimodal methodology combining bioluminescent and live fluorescence imaging to quantitatively examine CAR-T cytotoxicity as well as visualize the spatial and temporal interplay of CAR-T cells and cancer cells in both 2-D and 3-D co-culture systems. The scalability and sensitivity of this assay platform makes it a versatile tool for standardizing CAR-T cytotoxicity evaluation, advancing CAR-T therapeutic development in industrial and translational research settings.
Download the poster to explore the application of dual reporter cell lines in a multimodal imaging approach for evaluating CAR-T cytotoxicity.
DownloadPresenters
Meghan Sikes, MS
Senior Biologist, ATCC
Meghan joined ATCC in April of 2024 as a Senior Biologist in the Immuno-oncology group of the Cell Biology R&D department. She previously worked in vaccine formulation development at Novavax and in the retrovirology lab at MilliporeSigma. Meghan received her Bachelor's of Science in Biology from Shepherd University and Master's of Science in Microbiology and Cell Science at the University of Florida.
Catherine McManus, PhD
Scientist, Immuno-oncology, ATCC
Catherine McManus, PhD, is a Scientist in the Immuno-Oncology group in Cell Biology R&D at ATCC. Prior to joining ATCC, Dr. McManus received her PhD in Genetics from Yale University and completed her postdoctoral training at NIH. She has expertise in engineering reporter constructs for use in and ex vivo, identifying reporter construct integration sites, and assaying reporter expression.
Immuno-oncology reporter models
Cancer immunotherapy has emerged as an exciting new approach for cancer treatment, and immuno-oncology is one of the fastest growing fields in oncology.
The development of immunomodulatory drugs and biologics dictates a clear need for human cell-based models to evaluate immune activation. To answer this need, ATCC provides a growing collection of reporter models, including checkpoint luciferase reporter cells, CAR-T luciferase reporter cells, and THP-1 reporter cells.
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