Ex vivo cell labeling with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for imaging cell trafficking in mice with positron-emission tomography

Abstract

We have used copper-64-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (64Cu-PTSM) to radiolabel cells ex vivo for in vivo positron-emission tomography (PET) imaging studies of cell trafficking in mice and for eventual application in patients. 2-[18F]-Fluoro-2-deoxy-d-glucose (FDG) cell labeling also was evaluated for comparison. 64Cu-PTSM uptake by C6 rat glioma (C6) cells increased for 180 min and then stabilized. The labeling efficiency was directly proportional to 64Cu-PTSM concentration and influenced negatively by serum. Label uptake per cell was greater with 64Cu-PTSM than with FDG. However, both 64Cu-PTSM- and FDG-labeled cells showed efflux of cell activity into supernatant. The 64Cu-PTSM labeling procedure did not interfere significantly with C6 cell viability and proliferation rate. MicroPET images of living mice indicate that tail-vein-injected labeled C6 cells traffic to the lungs and liver. In addition, transient splenic accumulation of radioactivity was clearly detectable in a mouse scanned at 3.33 h postinfusion of 64Cu-PTSM-labeled lymphocytes. In contrast, the liver was the principal organ of tracer localization after tail-vein administration of 64Cu-PTSM alone. These results indicate that in vivo imaging of cell trafficking is possible with 64Cu-PTSM-labeled cells. Given the longer t(1/2) of 64Cu (12.7 h) relative to 18F (110 min), longer cell-tracking periods (up to 24-36 h) should be possible now with PET.

Figure 1

Schematic of Cu–PTSM uptake and retention by cells. PTSM has a high binding affinity for divalent rather than monovalent copper. Cu(II)–PTSM is very stable as a complex (K_a = 10¹⁸, pH 7.4). Cu(II)-PTSM acts as a lipophilic, redox-active transporter of Cu(II) ions that passively diffuses across the cell membrane and delivers copper into the cells. The retention of copper is governed by the reduction of the stable Cu(II)–PTSM complex to a labile Cu(I)–PTSM complex, trapping the dissociated Cu(I) ion in the cell because of charge. Intracellular macromolecules capture the monovalent copper, and the neutral PTSM molecule is able to diffuse back out. The bioreductive mechanism of Cu–PTSM trapping varies with cell type (9).

Figure 2

C6 cell-labeling efficiency of ⁶⁴Cu–PTSM and FDG as a function of time. C6 cells were labeled with ⁶⁴Cu–PTSM for up to 480 min or with FDG for up to 90 min in 1 ml of serum-supplemented medium. Note the influx of ⁶⁴Cu–PTSM plateaus after 3 h. The graph shows the mean percentage uptake per microgram of protein (±SE) of triplicates.

Figure 3

⁶⁴Cu–PTSM and FDG retention by labeled C6 cells as a function of time. This graph reflects the intracellular stability of ⁶⁴Cu–PTSM and FDG as a function of time. FDG efflux was measured at 4 rather than 5 h on this graph. Rapid tracer efflux indicates slow trapping. Each bar represents the mean percentage label retained ± SE of triplicate wells.

Figure 4

In vivo microPET imaging of ⁶⁴Cu–PTSM-labeled C6 cells post i.v. injection into a mouse. This mouse was microPET-scanned at 0.45 h postinjection of C6 cells (4.27 μCi). Immediately after the scan, the mouse was killed (at 1.45 h) for DWBA. (A) The average of nine coronal planes from the WB microPET image. (B) Photo of the DWBA section shown in C. Concordance between location of activity in the microPET image and DWBA section demonstrates that cells are trapped initially in the lungs. The %ID/g scale is shown for quantification of the microPET signal.

Figure 5

In vivo microPET imaging of ⁶⁴Cu–PTSM biodistribution post i.v. injection into a mouse. The mouse was microPET-scanned once on day 1 at 0.10 h (3.48 μCi) and then on day 2 at 20.3 h (1.16 μCi) after tail-vein injection of free ⁶⁴Cu–PTSM. The mouse was killed at 21.8 h for DWBA. A and B show the average of five coronal planes from the day 1 and 2 WB microPET images, respectively. (C) Photo of the DWBA section shown in D. Location of activity in the last microPET image (B) correlates with the DWBA image (D). Comparison with the photo shown in C reveals that liver is the primary organ of radiocopper accumulation as seen in the corresponding DWBA section and microPET image. The %ID/g scale quantifies the magnitude of signal observed in each microPET image. Lu, lungs; Li, liver.

Figure 6

In vivo microPET imaging of ⁶⁴Cu–PTSM-labeled lymphocytes post i.v. injection into a mouse. The mouse was microPET-scanned 0.12 h (A, 11.2 μCi), 3.12 h (B, 9.48 μCi), and 18.9 h (C, 4.01 μCi) postinjection of lymphocytes. Each microPET image shown here (A–C) is an average of 5–6 coronal slices. After the last microPET scan, this mouse was killed for DWBA (20.7 h). The location of activity in the last microPET image (C) clearly correlates with the DWBA image (E). The photo (D) provides the anatomic map necessary to resolve the source of activity in the microPET image from the DWBA section. Note that splenic lymphocytes initially traffic through lungs (A) and then accumulate in liver and spleen (B and C). The %ID/g scale quantifies the magnitude of signal observed in each microPET image. Lu, lungs; Li, liver; Sp, spleen; In, intestine.