In vivo imaging of nanoparticle-labeled CAR T cells

Abstract

Metastatic osteosarcoma has a poor prognosis with a 2-y, event-free survival rate of ∼15 to 20%, highlighting the need for the advancement of efficacious therapeutics. Chimeric antigen receptor (CAR) T-cell therapy is a potent strategy for eliminating tumors by harnessing the immune system. However, clinical trials with CAR T cells in solid tumors have encountered significant challenges and have not yet demonstrated convincing evidence of efficacy for a large number of patients. A major bottleneck for the success of CAR T-cell therapy is our inability to monitor the accumulation of the CAR T cells in the tumor with clinical-imaging techniques. To address this, we developed a clinically translatable approach for labeling CAR T cells with iron oxide nanoparticles, which enabled the noninvasive detection of the iron-labeled T cells with magnetic resonance imaging (MRI), photoacoustic imaging (PAT), and magnetic particle imaging (MPI). Using a custom-made microfluidics device for T-cell labeling by mechanoporation, we achieved significant nanoparticle uptake in the CAR T cells, while preserving T-cell proliferation, viability, and function. Multimodal MRI, PAT, and MPI demonstrated homing of the T cells to osteosarcomas and off-target sites in animals administered with T cells labeled with the iron oxide nanoparticles, while T cells were not visualized in animals infused with unlabeled cells. This study details the successful labeling of CAR T cells with ferumoxytol, thereby paving the way for monitoring CAR T cells in solid tumors.

Keywords: CAR T cells; ferumoxytol; magnetic resonance imaging; mechanoporation; multimodal imaging.

Fig. 1.

The expression profile and labeling efficiency of B7-H3 CAR T cells. (A) Representative flow cytometry histograms obtained on Day 10 post transduction displaying the mean transduction efficiency of anti-B7-H3 CAR T cells (74.72 ± 3.23%) and NT T cells (0.94 ± 0.26%). (B) Measurements of the iron per cell levels using ICP-OES revealed significantly higher (P < 0.0001) uptake of ferumoxytol in the labeled T cells than unlabeled T cells. (C) Prussian blue staining showing greater accumulation of iron in the T cells labeled with ferumoxytol than the unlabeled T cells (Scale bar, 50 µm). (D) Trypan blue exclusion method showed no significant difference in the cell viability of the unlabeled or ferumoxytol-labeled anti-B7-H3 CAR T cells. (E) Proliferation of the unlabeled or ferumoxytol-labeled anti-B7-H3 CAR T cells was not significantly different on Day 1, 3, and 5, as determined by the CCK-8 assay. Data represents mean ± SEM, and n is ≥ 3 per group. Statistical comparisons were performed using an unpaired Student t test.

Fig. 2.

Function of the CAR T cells was maintained in vitro. (A) The CCK-8 assay indicated that the proliferation of the T cells labeled with ferumoxytol and the unlabeled T cells was not significantly different (P > 0.99), while the coculture of the anti-B7-H3 CAR T cells with the target tumor cells at a 1:1 ratio revealed a 1.2-fold decrease in the viability of MG63.3 cells cocultured with the labeled anti-B7-H3 CAR T cells compared to the unlabeled cells. (B) Cytokine release was maintained following mechanoporation. The release of IFNγ in the T cells labeled with ferumoxytol and coincubated with or without tumor cells was not significantly different from the unlabeled cells. (C) Quantitative ROI analysis of T2* images demonstrated that the limit of detection for MRI was 2 × 10⁴ pelleted T cells labeled with ferumoxytol, while (D) the limit of detection for MPI was 2 × 10³ pelleted T cells. Data represents mean ± SEM, and n is ≥ 4 per group. Statistical comparisons were performed using the one-way ANOVA with the Tukey’s honest significant difference test.

Fig. 3.

Detection of ferumoxytol-labeled CAR T cells using MRI. (A) Representative coronal T2*-weighted MR images of the osteosarcomas in the tibia of experimental mice fused with superimposed, color-coded T2* maps. (B) Corresponding quantitative tumor T2* relaxation times at different time points before and after infusion of the anti-B7-H3 CAR T cells. There was significant enhancement of the tumor on Day 1 (P = 0.04) and Week 1 (W1, P < 0.0001) in the labeled tumor + CAR + iron group compared to the pretreatment images. (C) Serial tumor-size (millimeters²) measurements on the MRI images noted a decrease in tumor size at W2 and W3 (P < 0.0001) in the labeled tumor + CAR + iron group. (D) Representative bioluminescence images acquired 1 h post injection confirmed the viability of the anti-B7-H3 CAR-nLuc T cells and coronal gradient echo MR images of animals injected with either 2 × 10⁷ or 1 × 10⁷ B7-H3 CAR-nLuc T cells visualized the colocalization of the iron-labeled CAR T cells to off-target sites (orange arrows). (E–G) Quantitative analysis indicated significantly greater and dose-dependent accumulation of the ferumoxytol-labeled anti-B7-H3 CAR-nLuc T cells in the spleens and livers of the animals treated with 2 × 10⁷ T cells compared to animals injected with 1 × 10⁷ T cells on Day 1 (spleen, P = 0.04; liver, P = 0.049). Alternatively, there was a dose-dependent enhancement of the signal in the tumor in the animals administered with 2 × 10⁷ T cells compared to the animals injected with 1 × 10⁷ T cells on Day 3 (P = 0.04). (H) Representative histological images of paraffin-embedded tissue sections obtained from mice on D3 post treatment stained with Prussian blue DAP (Left) as well as the anti-CD3 primary antibody and cy-5-conjugated secondary antibody (Right). (Scale bars, 50 μm.) Magnification is ×20. (I) Quantitatively, the localization of the iron-labeled B7-H3 CAR-nLuc T cells in the spleen, liver, and tumor was significantly greater in the animals administered with 2 × 10⁷ T cells compared to the animals injected with 1 × 10⁷ T cells (spleen, P = 0.011; liver, P = 0.00; and tumor, P = 0.020). Tumor + CAR + iron group (n = 5); animals injected with ferumoxytol-labeled CAR T cells, tumor + CAR group (n = 5); animals injected with unlabeled CAR T cells, tumor + NT + iron group (n = 5); animals injected with NT ferumoxytol-labeled T cells and CAR + iron group (n = 3); and animals without tumors injected with ferumoxytol-labeled CAR T cells. Tumor + CAR + iron 2 × 10⁷ group (n = 5); animals injected with 2 × 10⁷ ferumoxytol-labeled B7-H3 CAR-nLuc T cells, Tumor + CAR + iron 1 × 10⁷ group (n = 4); animals injected with 1 × 10⁷ ferumoxytol-labeled B7-H3 CAR-nLuc T cells, Tumor + CAR 2 × 10⁷ group (n = 3); and animals injected with 2 × 10⁷ unlabeled B7-H3 CAR-nLuc T cells. D = day, W = week, and NT = nontransduced. Data represents mean ± SEM. Statistical comparisons were performed using the one-way ANOVA with the Tukey’s honest significant difference test.

Fig. 4.

Visualization of CAR T cells labeled with ferumoxytol using PAT and MPI. (A) Representative photoacoustic images showing the accumulation of CAR T cells labeled with ferumoxytol in the tumor + CAR + iron group (green). (Scale bar, 10 mm.) (B) Quantitative photoacoustic data demonstrating the percentage photoacoustic signal (% PA). (C) Representative 3D rendering of the photoacoustic images of osteosarcomas at different time points before and after CAR T-cell treatment. (Scale bars, 4 mm.) (D) Tumor volume (millimeters³) was acquired by drawing ROI on the 3D-rendered photoacoustic images of osteosarcomas. At W2, there was a significant decrease in the tumor volume in both the tumor + CAR + iron (P < 0.05) and tumor + CAR (P < 0.001) groups when compared to the tumor + NT + iron group. (E) Representative bright-field images acquired on W2 postinfusion of the T cells showing the tibiae of the animals from the tumor + CAR + iron, tumor + CAR and tumor + NT + iron groups. (F) Representative MPI showed the detection of iron in the animals administered with CAR T cells labeled with ferumoxytol. (G) Quantification of the MPI data indicated that the level of iron was significantly greater (P < 0.0001) in the labeled tumor + CAR + iron group than in the unlabeled tumor + CAR and the untreated tumor + NT + iron groups. Tumor + CAR + iron group (n = 5); animals injected with ferumoxytol-labeled CAR T cells, tumor + CAR group (n = 5); animals injected with unlabeled CAR T cells, tumor + NT + iron group (n = 5); and animals injected with NT ferumoxytol-labeled T cells. D = day, W = week, and NT = nontransduced. Data represents mean ± SEM. Statistical comparisons were performed using the one-way ANOVA with the Tukey’s honest significant difference test.