Multimodal In Vivo Tracking of Chimeric Antigen Receptor T Cells in Preclinical Glioblastoma Models

Abstract

Objectives: Iron oxide nanoparticles have been used to track the accumulation of chimeric antigen receptor (CAR) T cells with magnetic resonance imaging (MRI). However, the only nanoparticle available for clinical applications to date, ferumoxytol, has caused rare but severe anaphylactic reactions. MegaPro nanoparticles (MegaPro-NPs) provide an improved safety profile. We evaluated whether MegaPro-NPs can be applied for in vivo tracking of CAR T cells in a mouse model of glioblastoma multiforme.

Materials and methods: We labeled tumor-targeted CD70CAR (8R-70CAR) T cells and non-tumor-targeted controls with MegaPro-NPs, followed by inductively coupled plasma optical emission spectroscopy, Prussian blue staining, and cell viability assays. Next, we treated 42 NRG mice bearing U87-MG/eGFP-fLuc glioblastoma multiforme xenografts with MegaPro-NP-labeled/unlabeled CAR T cells or labeled untargeted T cells and performed serial MRI, magnetic particle imaging, and histology studies. The Kruskal-Wallis test was conducted to evaluate overall group differences, and the Mann-Whitney U test was applied to compare the pairs of groups.

Results: MegaPro-NP-labeled CAR T cells demonstrated significantly increased iron uptake compared with unlabeled controls ( P < 0.01). Cell viability, activation, and exhaustion markers were not significantly different between the 2 groups ( P > 0.05). In vivo, tumor T2* relaxation times were significantly lower after treatment with MegaPro-NP-labeled CAR T cells compared with untargeted T cells ( P < 0.01). There is no significant difference in tumor growth inhibition between mice injected with labeled and unlabeled CAR T cells.

Conclusions: MegaPro-NPs can be used for in vivo tracking of CAR T cells. Because MegaPro-NPs recently completed phase II clinical trial investigation as an MRI contrast agent, MegaPro-NP is expected to be applied to track CAR T cells in cancer immunotherapy trials in the near future.

Figure 1.. In vivo study timeline.

Forty-two NRG mice received two fractionated irradiation doses (2 × 4.5Gy), followed by intra-cardiac injection of 12 × 10⁶ CAR T-cells or untargeted T-cells. The mice underwent BLI, MRI and MPI before, on D1, D3 and D5 after T-cell treatment (n=5). 3 mice/group/time point were sacrificed for histology (Nine additional mice without treatment served as controls). (Created using BioRender.com).

Figure 2.. Evaluating the in vitro uptake of MegaPro-NP by CAR T-cells.

(A) Intracellular iron content of CAR T-cells, as determined by inductively coupled plasma optical emission spectroscopy (ICP-OES) after incubation with increasing concentrations of MegaPro-NP for increasing time periods. Data are displayed as means and standard deviations of triplicate experiments for each group. (B) Representative prussian blue stains of CAR T-cells at 1 hour as well as 1, 2, 3 and 4 weeks after labeling with MegaPro-NP at a concentration of 500 μg/mL for 4h. (scale bars, 100 μm) C) Corresponding intracellular iron content of CAR T-cells, as determined by ICP-OES. (D) Corresponding axial T2-weighted MRI images and T2* relaxation times of 2 million CAR T-cells at different time points after labeling with MegaPro-NP at a concentration of 500 μg/mL for 4 hours. (E) Corresponding MPI images of 2 million CAR T-cells at different time points after labeling with MegaPro-NP at a concentration of 500 μg/mL for 4 hours. The Kruskal–Wallis test was used to test the statistical difference among different time points, a p < 0.05 was considered statistically significant. The Kruskal-Wallis test was conducted to evaluate the overall group difference, and if any group difference was detected (p<0.05), then the Mann-Whitney U test was applied to compare the pairs of groups. A value of p < 0.01 was considered statistically significant and denoted with an asterisk.

Figure 3.. Functional in vitro evaluation of CAR T-cells after MegaPro-NP labeling.

U87-MG cells (2 × 10⁴ cells/well) were co-cultured with CAR T-cells at the indicated effector-to-target ratios and analyzed for various factors. (A) The morphologies of the cells were observed under a light microscope (scale bars, 100 μm). (B) Cancer cell viabilities, as quantified with a Luciferase-based cytotoxicity assay, after co-incubation with CAR T-cells at the indicated effector-to-target ratios. (C) Fold change of expression of various cytokines by labeled and unlabeled CAR T-cells, normalized to untargeted T-cells. (D) Relative number of labeled and unlabeled CAR T-cells which express exhaustion and activation markers. (E) Relative change in proliferation of labeled and unlabeled CAR T-cells at different time points after labeling (D = day). Comparisons between labeled and unlabeled cells were performed using Mann–Whitney U test (Figure 3B, 3C and 3D). For comparisons of three or more groups (Figure E), the values were analyzed with the Kruskal–Wallis test. There were no statistically significant differences between labeled and unlabeled CAR T-cells for any of the data shown.

Figure 4.. Bioluminescence Imaging (BLI) before and after treatment with CAR T-cells or untargeted controls.

(A) Top row: BLI signal of a representative mouse infused with MegaPro-NP-labeled CAR T-cells increases on day 1 (D1) and decreases on day 3 and 5. Lower row: By contrast, BLI signal of a representative mouse infused with untargeted CAR T-cells increases continuously. (B) Corresponding flux of the tumor tissue demonstrates a significant decrease in tumor burden at day 3 and 5 after CAR T-cell therapy, while the tumor burden continuously increases after treatment with untargeted T-cells. (C) The body weight of mice infused with labeled CAR T-cells remained unchanged while the body weight of mice infused with labeled untargeted T-cells decreased over time. The Kruskal-Wallis test was conducted to evaluate the overall group difference, and if any group difference was detected (p<0.05), then the Mann-Whitney U test was applied to compare the pairs of groups. A value of p < 0.01 was considered statistically significant and denoted with an asterisk.

Figure 5.. In Vivo detection of MegaPro-NP labeled CAR T-cells using MRI and MPI.

(A) T2-weighted FSE- and T2*-MGE image shows hyperintense U87-MG tumor before therapy (BT). Left: After infusion of MegaPro-NP-labeled CAR T-cells, the tumor demonstrates a signal decline at day 1 (D1), 3 (D3) and 5 (D5). T2* GE scan with superimposed T2* color map demonstrate T2* shortening of the tumor tissue. Right: After infusion of MegaPro-NP-labeled untargeted T-cells, a representative control tumor shows no signal change on T2- and T2*- weighted MRI scans or T2* color maps. (B) Corresponding mean T2* relaxation times of tumors treated with MegaPro-NP-labeled CAR T-cells or MegaPro-NP-labeled untargeted T-cells. Tumors treated with MegaPro-NP-labeled CAR T-cells demonstrated a significant decline in T2* relaxation times. (C) Mean tumor volume of mice treated with MegaPro-NP-labeled CAR T-cells or MegaPro-NP-labeled untargeted T-cells. (D) MPI images of mice treated with MegaPro-NP-labeled CAR T-cells demonstrate marked nanoparticle signal, while MPI images of mice treated with MegaPro-NP-labeled untargeted T-cells demonstrate minor MPI signal. (E) Corresponding quantitative MPI signal of tumors treated with MegaPro-NP-labeled CAR T-cells or MegaPro-NP-labeled untargeted T-cells. D = day. The Kruskal-Wallis test was conducted to evaluate the overall group difference, and if any group difference was detected (p<0.05), then the Mann-Whitney U test was applied to compare the pairs of groups. A value of p < 0.01 was considered statistically significant and denoted with an asterisk.

Figure 6.. Histology demonstrated the accumulation of MegaPro labeled CAR T-cells in GBM.

(A) Tumor specimen before and after treatment with MegaPro-NP-labeled CAR T-cells. Prussian blue staining (upper row) showed iron containing cells. Hematoxylin and eosin (H&E) staining (lower row) showed the tumor morphology and vascular niches (scale bars, 100 μm). (B) Corresponding quantitative data show increasing quantities of prussian-blue positive cells with increasing time after infusion of MegaPro-NP-labeled CAR T-cells. (C) Tumor specimen before and after treatment with MegaPro-NP-labeled untargeted T-cells. Prussian blue staining (upper row) showed no iron containing cells. Hematoxylin and eosin (H&E) staining (lower row) showed the tumor morphology (scale bars, 100 μm). (D) Corresponding quantitative data show few prussian-blue positive cells. (E) CD45 immunostaining of representative brain tumors at different time points before and after infusion of either MegaPro-NP-labeled CAR T-cells (upper row) or MegaPro-NP-labeled untargeted T-cells (lower row). (F) Corresponding quantitative data show significantly more CD45 positive cells (red) in tumors treated with MegaPro-NP-labeled CAR T-cells than MegaPro-NP-labeled untargeted T-cells (scale bars, 100 μm). The Kruskal-Wallis test was conducted to evaluate the overall group difference, and if any group difference was detected (p<0.05), then the Mann-Whitney U test was applied to compare the pairs of groups. A value of p < 0.01 was considered statistically significant and denoted with an asterisk.