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

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.

Harnessing the Transcriptional Signatures of CAR-T-Cells and Leukemia/Lymphoma Using Single-Cell Sequencing Technologies

“…On the other hand, factors associated with CAR-T-cells may also contribute to therapeutic failure [55,[68][69][70][71][72][73][74][75][76][77][78][79][121][122][123][124]. It has been proposed that CD19-negative relapse is related to immune escape.…”

Ultrasensitive and multiplexed tracking of single cells using whole-body PET/CT

“…Positron emission tomography (PET), known for its exceptional sensitivity across the entire body, offers a compelling platform for tracking cellular dynamics in human subjects and preclinical animal models (9)(10)(11). However, applications of this technique have been predominantly conducted on a population level, which is incompatible with the need to elucidate the trajectories of individual cells.…”

Efficient and multiplexed tracking of single cells using whole-body PET/CT

“…Positron emission tomography (PET), known for its exceptional sensitivity across the entire body, presents a compelling platform for tracking cellular dynamics in both human subjects and preclinical animal models. [9][10][11] However, applications of this technique have been predominantly…”

“…Numerous studies have applied PET imaging to track the spatiotemporal distribution of labeled cells in humans and small animals, albeit on a bulk population level that cannot resolve the fate of individual cells. [9][10][11] Recent work from our lab closed this gap and pushed the capabilities of PET towards the realm of single-cell imaging.…”