Theranostics 2022; 12(11):4834-4850. doi:10.7150/thno.68966 This issue

Research Paper

A multimodal imaging workflow for monitoring CAR T cell therapy against solid tumor from whole-body to single-cell level

Rita Pfeifer1*, Janina Henze1,2*, Katharina Wittich1, Andre Gosselink1,3, Ali Kinkhabwala1, Felix Gremse4, Cathrin Bleilevens1, Kevin Bigott1, Melanie Jungblut1, Olaf Hardt1, Frauke Alves2,5, Wa'el Al Rawashdeh1,6✉

1. Miltenyi Biotec B.V. & Co. KG, R&D Reagents, Bergisch Gladbach, North Rhine-Westphalia, Germany.
2. University Medical Center Göttingen, Translational Molecular Imaging, Institute for Diagnostic and Interventional Radiology & Clinic for Haematology and Medical Oncology, Göttingen, Lower Saxony, Germany.
3. Institute of Medical Statistics and Computational Biology, University of Cologne, Cologne, North Rhine-Westphalia, Germany.
4. Gremse-IT GmbH, Aachen, North Rhine-Westphalia, Germany.
5. Max-Planck-Institute for Multidisciplinary Science, Translational Molecular Imaging, Göttingen, Lower Saxony, Germany.
6. Ossium Health Inc, Indianapolis, Indiana, United States of America.
*Shared first authorship.

This is an open access article distributed under the terms of the Creative Commons Attribution License ( See for full terms and conditions.
Pfeifer R, Henze J, Wittich K, Gosselink A, Kinkhabwala A, Gremse F, Bleilevens C, Bigott K, Jungblut M, Hardt O, Alves F, Al Rawashdeh W. A multimodal imaging workflow for monitoring CAR T cell therapy against solid tumor from whole-body to single-cell level. Theranostics 2022; 12(11):4834-4850. doi:10.7150/thno.68966. Available from

File import instruction


Graphic 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.

Keywords: CAR T Cells, Cell Tracking, Optical tomography, 3D µCT/BLT, Light-Sheet Fluorescence microscopy