Related Experiment Video
Updated: Sep 4, 2025
![Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography](/_next/image?url=https%3A%2F%2Fcloudfront.jove.com%2FCDNSource%2Fteasers%2F62334.jpg&w=3840&q=50)
Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography
Published on: February 17, 2022
A multimodal imaging workflow for monitoring CAR T cell therapy against solid tumor from whole-body to single-cell
Rita Pfeifer1, Janina Henze1,2, Katharina Wittich1
1Miltenyi Biotec B.V. & Co. KG, R&D Reagents, Bergisch Gladbach, North Rhine-Westphalia, Germany.
Abstract:
CAR T cell research in solid tumors often lacks spatiotemporal information and therefore, there is a need for a molecular tomography to facilitate high-throughput preclinical monitoring of CAR T cells. Furthermore, a gap exists between macro- and microlevel imaging data to better assess intratumor infiltration of therapeutic cells. We addressed this challenge by combining 3D µComputer tomography bioluminescence tomography (µCT/BLT), light-sheet fluorescence microscopy (LSFM) and cyclic immunofluorescence (IF) staining. Methods: NSG mice with subcutaneous AsPC1 xenograft tumors were treated with EGFR CAR T cell (± IL-2) or control BDCA-2 CAR T cell (± IL-2) (n = 7 each). Therapeutic T cells were genetically modified to co-express the CAR of interest and the luciferase CBR2opt. IL-2 was administered s.c. under the xenograft tumor on days 1, 3, 5 and 7 post-therapy-initiation at a dose of 25,000 IU/mouse. CAR T cell distribution was measured in 2D BLI and 3D µCT/BLT every 3-4 days. On day 6, 4 tumors were excised for cyclic IF where tumor sections were stained with a panel of 25 antibodies. On day 6 and 13, 8 tumors were excised from rhodamine lectin-preinjected mice, permeabilized, stained for CD3 and imaged by LSFM. Results: 3D µCT/BLT revealed that CAR T cells pharmacokinetics is affected by antigen recognition, where CAR T cell tumor accumulation based on target-dependent infiltration was significantly increased in comparison to target-independent infiltration, and spleen accumulation was delayed. LSFM supported these findings and revealed higher T cell accumulation in target-positive groups at day 6, which also infiltrated the tumor deeper. Interestingly, LSFM showed that most CAR T cells accumulate at the tumor periphery and around vessels. Surprisingly, LSFM and cyclic IF revealed that local IL-2 application resulted in early-phase increased proliferation, but long-term overstimulation of CAR T cells, which halted the early added therapeutic effect. Conclusion: Overall, we demonstrated that 3D µCT/BLT is a valuable non-isotope-based technology for whole-body cell therapy monitoring and investigating CAR T cell pharmacokinetics. We also presented combining LSFM and MICS for ex vivo 3D- and 2D-microscopy tissue analysis to assess intratumoral therapeutic cell distribution and status.
Insights
This study introduces a multimodal imaging approach combining 3D micro-CT/BLT, LSFM, and cyclic IF for preclinical CAR T cell monitoring in solid tumors. It reveals how antigen recognition impacts CAR T cell distribution and the unexpected long-term effects of IL-2 on T cell function.
Area of Science:
- Oncology
- Immunology
- Biomedical Imaging
Background:
- CAR T cell therapy for solid tumors requires precise monitoring of cell distribution and behavior.
- Existing imaging techniques lack the spatiotemporal resolution and multiscale integration needed for comprehensive preclinical assessment.
- Bridging macro- and microlevel imaging is crucial for understanding intratumoral infiltration of therapeutic cells.
Purpose of the Study:
- To develop and validate a multimodal imaging strategy for high-throughput preclinical monitoring of CAR T cells in solid tumors.
- To investigate the impact of antigen recognition on CAR T cell pharmacokinetics and intratumoral distribution.
- To assess the effects of local IL-2 administration on CAR T cell proliferation and therapeutic efficacy.
Main Methods:
- Combined 3D micro-Computer Tomography/Bioluminescence Tomography (µCT/BLT), light-sheet fluorescence microscopy (LSFM), and cyclic immunofluorescence (IF) staining.
- Utilized NSG mice with AsPC1 xenograft tumors treated with EGFR CAR T cells (± IL-2) or control BDCA-2 CAR T cells.
- Measured CAR T cell distribution using 2D BLI and 3D µCT/BLT; performed cyclic IF and LSFM on excised tumors for detailed cellular analysis.
Main Results:
- 3D µCT/BLT demonstrated that antigen recognition significantly increased CAR T cell tumor accumulation and delayed spleen accumulation.
- LSFM confirmed higher T cell infiltration in target-positive tumors, primarily at the periphery and near vessels.
- Local IL-2 application initially boosted proliferation but led to long-term overstimulation, negating early therapeutic benefits.
Conclusions:
- 3D µCT/BLT is a powerful non-isotope tool for whole-body CAR T cell therapy monitoring and pharmacokinetic studies.
- Integrated LSFM and cyclic IF provide detailed ex vivo analysis of intratumoral CAR T cell distribution and status.
- The findings highlight the complex interplay between CAR T cell targeting, tumor microenvironment, and cytokine administration, informing future therapeutic strategies.
