In Vitro Evaluation of Adenosine 5′-Monophosphate as an Imaging Agent of Tumor Metabolism

³H-AMP is converted to ³H-adenosine before tumor cell uptake via nucleoside transporter. (A) Inhibition of CD73 (ecto-5′-nucleotidase) with APCP (α,β-methylene adenosine-5′-diphosphate) blocks uptake of ³H-AMP (black bars), but not same dose of ³H-FDG (white bars), in dose-dependent manner. (B) ³H-AMP uptake in SKOV-3 cells is competitively inhibited by exposure to increasing concentrations of extracellular unlabeled adenosine.

(A) Significantly more ³H-AMP than ³H-FDG accumulates in U251 human glioblastoma cells (P < 0.05). ³H-AMP is dephosphorylated by CD73, and ³H-adenosine is taken up by ENT nucleoside transporter in U251 cells. Cells were exposed to 3.7 kBq (0.1 μCi) of radiotracer in vitro. (B) Significantly more ³H-AMP than ³H-FDG accumulates in U87 human anaplastic astrocytoma cells (P < 0.05). Dephosphorylation to ³H-adenosine is required, but transport is predominantly via non–dipyridamole-inhibited ENT route. Cells were exposed to 3.7 kBq of radiotracer in vitro. Dipyr = dipyridamole.

Low uptake of ³H-AMP by Raji cells because of lack of CD73 expression. (A) In Raji cells, uptake of ³H-AMP (*) is very low and uptake of ³H-adenosine is high (P < 0.05). ³H-Adenosine uptake is only partially inhibited by dipyridamole and is competitively inhibited by unlabeled adenosine. CD73 inhibition by APCP does not affect ³H-adenosine uptake. Cells were exposed to 3.7 kBq of radiotracer in vitro. (Daudi cells also showed similarly low uptake of ³H-AMP and high uptake of ³H-adenosine—data not shown.) (B) Extracellular CD73 is expressed on SKOV-3 cells but not on Raji cells. Viable cells were selected by physical parameters, size, and internal complexity (upper left graphs). Immunophenotyping of these viable cells was compared using cells labeled with phycoerythrin-conjugated CD73 antibody and control cells without antibody labeling. Cell fluorescence intensity is graphed on y-axis and cell physical parameter on x-axis for control cells (lower left graphs) and CD73-labeled cells (lower right graphs). These graphs are also represented as number of cells on y-axis versus fluorescence intensity on x-axis (upper right graphs). Consistent with CD73 expression, intensity of fluorescence is greater on SKOV-3 cells labeled with CD73 than on control cells. However, Raji cells did not show difference in fluorescence intensity between cells labeled with CD73 and control cells. (Daudi cells were also negative for CD73—data not shown.)

Nucleoside transporter characterization reveals that SKOV-3 cells possess primarily hENT1s. (A) ³H-Adenosine uptake is inhibited by low nanomolar concentrations of NBMPR in Na⁺-free physiologic solution. (B) No evidence is seen of concentrative Na⁺-dependent ³H-adenosine transport in SKOV-3 cells in physiologic or Na⁺-free physiologic buffer.

Nucleoside transporter characterization of U87 cells. U87 cells possess both ENTs and CNTs. ENT appears to be NBMPR resistant. (A) ³H-Adenosine uptake is not inhibited by nanomolar or micromolar concentrations of NBMPR in Na⁺-free physiologic solution. (B) Evidence is seen of concentrative Na⁺-dependent ³H-adenosine transport in SKOV-3 cells in physiologic or Na⁺-free physiologic buffer.