Human Organic Anion Transporting Polypeptide 1B3 Applied as an MRI-Based Reporter Gene

Abstract

Objective: Recent innovations in biology are boosting gene and cell therapy, but monitoring the response to these treatments is difficult. The purpose of this study was to find an MRI-reporter gene that can be used to monitor gene or cell therapy and that can be delivered without a viral vector, as viral vector delivery methods can result in long-term complications.

Materials and methods: CMV promoter-human organic anion transporting polypeptide 1B3 (CMV-hOATP1B3) cDNA or CMV-blank DNA (control) was transfected into HEK293 cells using Lipofectamine. OATP1B3 expression was confirmed by western blotting and confocal microscopy. In vitro cell phantoms were made using transfected HEK293 cells cultured in various concentrations of gadoxetic acid for 24 hours, and images of the phantoms were made with a 9.4T micro-MRI. In vivo xenograft tumors were made by implanting HEK293 cells transfected with CMV-hOATP1B3 (n = 4) or CMV-blank (n = 4) in 8-week-old male nude mice, and MRI was performed before and after intravenous injection of gadoxetic acid (1.2 μL/g).

Results: Western blot and confocal microscopy after immunofluorescence staining revealed that only CMV-hOATP1B3-transfected HEK293 cells produced abundant OATP1B3, which localized at the cell membrane. OATP1B3 expression levels remained high through the 25th subculture cycle, but decreased substantially by the 50th subculture cycle. MRI of cell phantoms showed that only the CMV-hOATP1B3-transfected cells produced a significant contrast enhancement effect. In vivo MRI of xenograft tumors revealed that only CMV-hOATP1B3-transfected HEK293 tumors demonstrated a T1 contrast effect, which lasted for at least 5 hours.

Conclusion: The human endogenous OATP1B3 gene can be non-virally delivered into cells to induce transient OATP1B3 expression, leading to gadoxetic acid-mediated enhancement on MRI. These results indicate that hOATP1B3 can serve as an MRI-reporter gene while minimizing the risk of long-term complications.

Keywords: Gadoxetic acid; Gene therapy; MRI; OATP1B3; Reporter gene.

Fig. 1. Generating CMV-hOATP1B3 overexpressing HEK293 cells.

A. Immunofluorescence staining and confocal microscopy of HEK293 cells passed for 25 (upper row) or 50 (middle row) cycles after transfection with CMV-hOATP1B3; (lower row) CMV-blank transfected HEK293 cells for negative control (left column: × 20, right column: × 40, hOATP1B3 [red], DAPI [blue]). B. Western blot analysis of hOATP1B3 (upper row) and β-actin (lower row) in HEK293 cells transfected with (left) CMV-blank (negative control) or with CMV-hOATP1B3 and passed for 25 (middle) or 50 (right) cycles. CMV-hOATP1B3 = CMV promoter-human organic anion transporting polypeptide 1B3

Fig. 2. 9.4T MRI T1WIs of (A) gadoxetic acid and (B) gadobenate dimeglumine based cell phantoms.

Either CMV-hOATP1B3 transfected HEK293 cells (left column) or CMV-blank transfected control HEK293 cells (right column) were pelleted. PBS = phosphate-buffered saline, T1WI = T1 weighted image

Fig. 3. 9.4T MRI T1WIs of (A) CMV-blank transfected xenograft tumor and (B) CMV-hOATP1B3 transfected xenograft tumor-bearing mice.

Pre-contrast images (left), post-contrast images obtained 30 minutes (middle), and 5 hours (right) after gadoxetic acid intravenous injection.

Fig. 4. Hematoxylin and eosin staining (upper: × 10, × 40) and hOATP1B3 immunohistochemistry (lower: × 10, × 40) of in vivo xenograft tumor specimens.

(A) Control CMV-blank transfected HEK293 xenograft tumor and (B) CMV-hOATP1B3 transfected HEK293 xenograft tumor.