Intracellular Protein Binding of Zr-89 Oxine Cell Labeling for PET Cell Tracking Studies

Abstract

Background/Objectives: ⁸⁹Zr-oxine is an ex vivo cell labeling agent that enables cells to be tracked in vivo by positron emission tomography (PET) over a period of up to two weeks. To better understand where ⁸⁹Zr-oxine binds within cellular components, factors affecting labeling and intracellular distribution of ⁸⁹Zr were examined. Methods: Mouse primary T cells, natural killer cells, dendritic cells, and monocytes, and cell lines EL4 (mouse lymphoma), DC2.4 (mouse dendritic cell), Kit225K6 (human T cell leukemia) and MC38 (mouse colon adenocarcinoma) were labeled with ⁸⁹Zr-oxine or ¹¹¹In-oxine and protein binding within the cellular compartments, the labeling thresholds, and radioactivity retention were subsequently determined. Results: Cell incorporation of ⁸⁹Zr-oxine (27.8-71.8 kBq/10⁶ cells) positively correlated with cellular size and protein mass. Most (>97%) ⁸⁹Zr was protein-bound and primarily localized in the cytoplasm, membrane, and nuclear fractions (>81%) with distribution patterns varying by cell type. By contrast, ¹¹¹In-oxine showed lower protein-binding activity of approximately 59-65%, with 62-65% of ¹¹¹In localized in the cytoplasm. Autoradiography of electrophoresed subcellular fractionated cell samples indicated stable binding by ⁸⁹Zr-oxine to proteins in all subcellular fractions but unstable protein binding by ¹¹¹In. Saturation studies showed that ⁸⁹Zr-oxine labeling was saturable, and further labeling reduced cellular retention. Biodistribution of dendritic cells labeled with either ⁸⁹Zr-oxine or ¹¹¹In-oxine indicated greater retention of ⁸⁹Zr in the labeled cells in vivo than ¹¹¹In. Conclusions: ⁸⁹Zr-oxine stably binds many intracellular proteins and shows much higher and more stable protein binding than ¹¹¹In-oxine. Intracellular protein binding of ⁸⁹Zr accounts for the ability of ⁸⁹Zr-oxine labeling to successfully track cells in vivo long-term on PET.

Keywords: cell labeling; cell tracking; positron emission tomography; protein labeling; radiolabeling; zirconium-89 oxine.

Figure 1

⁸⁹Zr-oxine cell labeling positively correlates with the protein mass and cell size. (A) Various cell types incubated with 37 kBq/10⁶ cells of ⁸⁹Zr-oxine incorporated different amounts of activity (n = 3–6). (B) Labeling efficiency (percentage of incorporated activity vs. incubation activity) was calculated for each cell type (n = 3–6). (C) Flow cytometry was performed to determine the forward scatter (FSC) values that reflect cellular size (n = 3–6). (D) Various cell types were lysed at 100 µL/10⁶ cells, and total protein concentration of each lysate was measured as a readout for protein mass of a cell (n = 3–6). (E) Using flow cytometry, the side scatter (SSC) value reflecting vesicular contents was obtained for each cell type (n = 3–6). All data in (A–E) are represented as mean ± standard deviation. (F) Correlation analysis using the values shown in (A–E) indicated strong positive correlation between ⁸⁹Zr activity incorporation or labeling efficiency with protein concentration and cell size (FSC). Pearson r values are shown.

Figure 2

⁸⁹Zr incorporated into cells is protein-bound. Trichloroacetic acid (TCA) protein precipitation of lysates generated from 2 × 10^{6 89}Zr-oxine labeled EL4, DC2.4, and Kit225K6 cells demonstrated that virtually all activity was protein-bound (n = 3). By contrast, more than one-third of the activity was non-protein-bound in the cells labeled with ¹¹¹In-oxine (n = 4), demonstrating significant differences in % protein binding with ⁸⁹Zr-oxine labeling. PBS containing either ⁸⁹Zr-oxine alone or ¹¹¹In-oxine alone was used as a control in the relevant assays, and the addition of TCA to these control samples did not precipitate the activity. Data are represented as mean ± standard deviation. ****: p < 0.0001 by two-way ANOVA. Radioactivity of the protein-bound fraction in each cell lysate was significantly higher than that in the relevant control (p < 0.0001, not indicated in the graph).

Figure 3

⁸⁹Zr-oxine cell labeling leads to distinct intracellular distribution of ⁸⁹Zr by cell type, predominantly in the cytoplasm, membranes, and nucleus, whereas ¹¹¹In-oxine labeling results in the activity limited to the cytoplasm. (A) ⁸⁹Zr-oxine labeled EL4, DC2.4, and Kit225K6 cells were fractionated into the subcellular compartments indicated, and ⁸⁹Zr activity in each fraction was measured. ⁸⁹Zr primarily localized in the cytoplasm, membrane, and soluble nuclear fractions, showing different distribution patterns among the cell types (n = 4 for EL4 and DC2.4 cells, n = 3 for Kit225K6 cells). (B) Subcellular fractionation of ¹¹¹In-oxine labeled EL4 and DC2.4 cells was performed, followed by activity measurement for each fraction. ¹¹¹In primarily localized in the cytoplasm, membrane, and soluble nuclear fractions, showing different distribution patterns among the cell types (n = 3). All data are represented as mean ± standard deviation.

Figure 4

⁸⁹Zr-oxine labels multiple cellular proteins of different sizes in various subcellular compartments. (A–C) Autoradiography of the membranes obtained from western blotting of subcellular fractions of ⁸⁹Zr-oxine labeled EL4 (A), DC2.4 (B), and Kit225K6 (C) cells. Multiple bands in each fraction indicate the binding of ⁸⁹Zr to multiple proteins of different sizes. The pattern of protein binding of ⁸⁹Zr differed by cell type. Representative results of more than three independent experiments showing duplicated sample preparations for each cell type. (D) Autoradiography of ¹¹¹In-oxine labeled EL4 cell subcellular fractions only showed extremely faint bands in the cytoplasmic, chromatin-bound, and cytoskeletal fractions. Representative results of two independent experiments, showing duplicated sample preparations.

Figure 5

Intracellular protein binding of ⁸⁹Zr-oxine occurs rapidly. (A) EL4 cells incubated with ⁸⁹Zr-oxine for 15 min and 5 min showed similar labeling efficiency with no statistical difference (ns: not significant by Student’s two-tailed t-test, n = 3). (B) EL4 cells incubated with ⁸⁹Zr-oxine for 5 min showed virtually all activity bound to protein(s) similar to the cells incubated for 15 min, as indicated by the TCA protein precipitation of whole cell lysates. PBS containing ⁸⁹Zr-oxine alone was used as a control, which did not precipitate the activity (n = 3, ns: not significant, ****: p < 0.0001, by one-way ANOVA). (C) Five min incubation was sufficient to similarly label intracellular proteins as 15 min incubation in EL4 cells (n = 3, ns: not significant, by two-way ANOVA). All data are represented as mean ± standard deviation.

Figure 6

⁸⁹Zr-oxine incorporation demonstrates a cell type-dependent saturation, and an activity lower than the saturation shows better retention. (A) Saturation assay shows the maximum of ⁸⁹Zr-oxine incorporation in EL4, DC2.4, and Kit225K6 cells that were incubated with increasing doses of ⁸⁹Zr-oxine calculated from the labeling efficiency shown in Figure 1B for the following targeted incorporation doses: 18.5 KBq/10⁶, 37KBq/10⁶, 74 kBq/10⁶, 148 kBq/10⁶, 296 kBq/10⁶, and 592 kBq/10⁶ cells (broken line). Incorporated activity plateaued at higher doses, indicating the presences of saturation thresholds. EL4 and DC2.4 showed a threshold point around 90 kBq/10⁶ cells, while that of Kit225K6 was around 60 kBq/10⁶ cells (n = 4 for EL4 and Kit225K6 cells, n = 3 for DC2.4 cells). (B) Kit225K6 cells labeled with ⁸⁹Zr-oxine at 29, 55, 165, and 339 kBq/10⁶ cells were cultured, and decay-corrected cell-associated activity was examined over 48 h (also see Figure S2A). Relative ⁸⁹Zr retention compared to the activity immediately after the labeling (0 h) is plotted for each labeling dose (n = 3, *: p < 0.05, ***: p < 0.001, by repeated measure two-way ANOVA). Data are represented as mean ± standard deviation.

Figure 7

Biodistribution of ⁸⁹Zr-oxine labeled cells showed higher activity retention compared to ¹¹¹In-oxine labeled cells. (A,B) The graphs show %ID/g values of intravenously infused DCs (6 × 10⁶ cells) labeled with ⁸⁹Zr-oxine (29.6 kBq/10⁶ cells) or ¹¹¹In-oxine (76.0 kBq/10⁶ cells) in mice 1 day (A) and 7 days (B) after infusion. The cells primarily migrated to the liver and spleen, regardless of the labeling method. However, the %ID/g values of ⁸⁹Zr were significantly higher than those of ¹¹¹In in both organs, indicating greater activity retention in ⁸⁹Zr-oxine labeled cells (n = 4, ***: p < 0.001, ****: p < 0.0001, by two-way ANOVA). Data are decay corrected, normalized to a 20-g mouse, and represented as mean ± standard deviation. BM: bone marrow.