A novel (19)F agent for detection and quantification of human dendritic cells using magnetic resonance imaging

Abstract

Monitoring of cell therapeutics in vivo is of major importance to estimate its efficacy. Here, we present a novel intracellular label for (19)F magnetic resonance imaging (MRI)-based cell tracking, which allows for noninvasive, longitudinal cell tracking without the use of radioisotopes. A key advantage of (19)F MRI is that it allows for absolute quantification of cell numbers directly from the MRI data. The (19)F label was tested in primary human monocyte-derived dendritic cells. These cells took up label effectively, resulting in a labeling of 1.7 ± 0.1 × 10(13) (19)F atoms per cell, with a viability of 80 ± 6%, without the need for electroporation or transfection agents. This results in a minimum detection sensitivity of about 2,000 cells/voxel at 7 T, comparable with gadolinium-labeled cells. Comparison of the detection sensitivity of cells labeled with (19)F, iron oxide and gadolinium over typical tissue background showed that unambiguous detection of the (19)F-labeled cells was simpler than with the contrast agents. The effect of the (19)F agent on cell function was minimal in the context of cell-based vaccines. From these data, we calculate that detection of 30,000 cells in vivo at 3 T with a reasonable signal to noise ratio for (19)F images would require less than 30 min with a conventional fast spin echo sequence, given a coil similar to the one used in this study. This is well within acceptable limits for clinical studies, and thus, we conclude that (19)F MRI for quantitative cell tracking in a clinical setting has great potential.

Figure 1

Labeling primary human DCs with ¹⁹F has minimal effect on cell functionality. Cells were labeled with CS-1000 by coincubation, at the concentrations indicated. They were then washed and processed for MRS or other assays. (a) ¹⁹F uptake (open circles, right y-axis) was measured using MRS on cell pellets with a calibrated reference. Viability after labeling (full squares, left y-axis) was measured using a trypan blue exclusion assay. We observed toxicity only at high label concentrations. The optimum selected concentration is indicated in the figure. (b) Flow cytometric analysis on the untreated controls (left) shows a clear difference in side scatter relative to labeled cells (right). Gating was on live cells. (c) The plot on the left shows no change in expression of standard DC markers after labeling (grey) relative to untreated cells (black). Cells were labeled and then electroporated with tumor-derived mRNA (right). Protein expression was analyzed using intracellular staining for flow cytometry and plotted in the histogram. ¹⁹F labeling did not affect mRNA uptake relative to nonlabeled controls.

Figure 2

MR images of labeled cells in phantoms with increasing cell densities. Cells were labeled with ¹⁹F, Gd or SPIO and suspended in gelatin at various cell densities. Axial slices were then acquired. (a) The panel shows MR images for ¹⁹F and Gd-labeled cells at 500–8,000 cells/voxel. Images of the Gd-labeled cells were normalized to a reference consisting of nonlabeled cells in gelatin. (b) ${\text{T}}_2^{★}$-weighted axial images of cells labeled with SPIO at 0–1000 cells/voxel in phantoms.

Figure 3

Cell quantification from MR images data. Cells were labeled, suspended at various densities in gelatin and imaged using the appropriate spin-density, T₁ or ${\text{T}}_2^{★}$-weighted sequence. (a) The signal intensity of the labeled cells relative to a calibrated ¹⁹F reference with constant ¹⁹F content was calculated and plotted, showing the linear relationship between cell density and signal intensity. The plots show the SNR (upper panel) and signal intensity relative to the reference (lower panel) for the ¹⁹F-labeled cells and ¹⁹F reference (closed and open circles respectively). Relative intensity here is the ratio between the SNR of the labeled cells (full circles in the upper panel) and SNR of the reference sample (open circles in the upper panel). (b) Similar data were plotted for cells labeled with Gd and SPIO. Cells labeled with Gd (left) were imaged using a T₁-weighted scan, and the relative signal intensity (lower panel) and T₁ plotted with increasing cell density (upper panel). Relative intensity is the change in contrast due to the presence of label. Similar plots for SPIO-labeled cells (right) show the relative $T_2^{★}$ (upper panel) and intensity (lower panel). All values for the Gd and SPIO cells are normalized to the relaxation parameters and signal intensity over a reference containing nonlabeled cells. All plots in Figure 3b are presented in a logarithmic scale. The observed depart from the linear behavior is clearly showing the difficulties of quantification process when contrast agents are used.

Figure 4

Detection and identification of labeled DCs in muscle tissue. 1.5 million cells were labeled with SPIO, Gd or ¹⁹F (false color) and injected as a bolus in bovine muscle tissue. (a) Unlabeled cells were used as a control, which shows the typical nonhomogenous ¹H background of tissue. The cells labeled with SPIO were imaged with both an spin-echo (SE) and a gradient-echo (GE) sequence. ¹⁹F-labeled cells are clearly visible (false color). The lower panels show the corresponding intensity profiles for a horizontal line through center of the sample, showing the intensity of the pixels at a given position. All plots are set to the same scale. (b) A cell number map of the ¹⁹F-labeled cells in the slice was calculated from the image data. A projection of the calculated slice is shown, which corresponds with the original ¹⁹F image.

Figure 5

In vivo detection of labeled human DCs in a xenograft mouse model. CS-1000 labeled DCs were injected subcutaneously in an NOD-SCID mouse. Shown is fused ¹⁹F/¹H image, where the ¹⁹F is rendered in false color and the ¹H is in grayscale. The DCs are visible as a “hot spot” in an anatomical location consistent with the draining inguinal lymph node on the side of the cell injection.