Influence of bioluminescence imaging dynamics by D-luciferin uptake and efflux mechanisms

Abstract

Bioluminescence imaging (BLI) detects light generated by luciferase-mediated oxidation of substrate and is used widely for evaluating transgene expression in cell-based assays and in vivo in relevant preclinical models. The most commonly used luciferase for in vivo applications is firefly luciferase (fLuc), for which D-luciferin serves as the substrate. We demonstrated previously that the expression of the ABCG2 efflux transporter can significantly reduce BLI signal output and that HhAntag-691 can inhibit the efflux of D-luciferin, thereby enhancing BLI signal. Here we show that an HhAntag-691-sensitive uptake mechanism facilitates the intracellular concentration of D-luciferin and that the BLI dynamics of different cell lines are coregulated by this uptake mechanism in conjunction with ABCG2-mediated efflux. After administration of D-luciferin, the HhAntag-691-sensitive uptake mechanism generates a rapid increase in BLI signal that decreases over time, whereas ABCG2-mediated efflux stably reduces signal output. We implicate SLC22A4 (OCTN1), a member of the organic cation/zwitterion uptake transporter family, as one potential mediator of the HhAntag-691-sensitive D-luciferin uptake. These findings provide insight into mechanisms that contribute to the cellular uptake kinetics and in vivo biodistribution of D-luciferin.

Figure 1

Time course of BLI signal in 22Rv1 cells treated with HhAntag-691 (A) or fumitremorgan C (FTC) (B). Cells were transiently transfected with a cytomegalovirus promoter–controlled trifusion reporter gene that produces fLuc, red fluorescent protein (RFP), and a mutated form of herpes simplex virus 1 thymidine kinase (sr39ttk), as previously described. HhAntag-691 or FTC was added at the indicated concentration, followed by D-luciferin, which was at a final concentration of 150 mg/mL. Imaging commenced immediately after adding D-luciferin. HhAntag-691 caused a transient, dose-dependent signal decrease before a dose-dependent signal increase became evident, whereas FTC caused enhanced BLI signal from the outset.

Figure 2

Time course of BLI signal in HEK293 cells treated with HhAntag-691. Cells were transfected with (A) empty vector; (B) wild-type ABCG2; (C) G2 (R482G); or (D) T10 (R482T) mutant ABCG2. In HEK293/empty cells, a dose-dependent initial BLI signal decrease was observed and became less prominent over time (A). When wild-type ABCG2 was overexpressed, the transient signal decrease was abolished and signal was enhanced from the beginning of the imaging session (B). The overexpression of G2 mutant ABCG2 did not abolish the initial signal decrease (C). The overexpression of T10 mutant ABCG2 abolished the initial signal decrease but did not cause immediate signal enhancement. Cells were transiently transfected with the CMV-driven trifusion reporter, and BLI was performed identically as in Figure 1.

Figure 3

Time course of BLI signal in HhAntag-691-treated MDCK II parent control cells (A) or MDCK II cells expressing ABCB1/Pgp (B), ABCC1/MRP1 (C), or ABCC2/MRP2 (D). In control cells, a dose-dependent initial BLI signal decrease was observed and lasted over time (A). The overexpression of ABCB1/Pgp (B), ABCC1/MRP1 (C), or ABCC2/MRP2 (D) did not affect the signal decrease. Cells were transiently transfected with the CMV-driven trifusion reporter, and BLI was performed identically as in Figure 1 and Figure 2.

Figure 4

HhAntag-691 inhibits the initial high BLI signal produced by MDCK II cells over the time course of imaging. MDCK II/cytomegalovirus-driven trifusion reporter–expressing cells were imaged for 90 minutes in the presence or absence of HhAntag-691 with increasing concentrations of D-luciferin.

Figure 5

BLI signal dynamics in different cell lines are affected by the level of ABCG2 expression. A, BLI signal from HEK293/empty cells started from a higher level, while decreasing smoothly over time, than did signal from ABCG2-overexpressing HEK293 cells, which remained constant in BLI signal output. B, The BLI/luciferase assay (in vitro) signal output ratio in ABCG2-overexpressing HEK293 cells was significantly lower than that produced by four different HEK293/empty colonies, confirming that the lower BLI signal from the HEK293/ABCG2 cells was caused by the relatively lower availability of intracellular D-luciferin in this cell line. C, Quantitative RT-PCR indicated that ABCG2 is expressed at very low levels in the HEK293/empty cells but is elevated in HEK293/ABCG2 cells, while being intermediate in expression in the 22Rv1 cell line. D, The BLI signal dynamics of 22Rv1 cells is relatively stable, consistent with its moderate expression of ABCG2. MDCK II cells demonstrated an initial high BLI signal, followed by a continuously decreasing signal, consistent with a high level of expression of the HhAntag-691-sensitive uptake pump and little expression of ABCG2.

Figure 6

Schematic diagram of proposed transporter involvement in D-luciferin transport across the cell membrane and the effect on BLI signal output. Soon after (early phase) D-luciferin administration and imaging, there is little intracellular D-luciferin, such that the uptake process dominates the overall signal, and inhibiting that uptake, such as with HhAntag-691, decreases the signal. By later time points (late phase), there has been sufficient time for D-luciferin to accumulate within cells, such that efflux (ABCG2) dominates and inhibition of ABCG2 enhances signal.

Figure 7

The expression levels of the SLC22A4 and SLC22A2 uptake pumps in different cell lines revealed by quantitative RT-PCR. RNA was extracted from HEK293 parent cells (HEK293/par) or from cells stably transfected with an ABCG2-expressing plasmid (HEK293/ABCG2). 22Rv1 cells and MDCK II cells were similarly treated.