Tyrosinase as a multifunctional reporter gene for Photoacoustic/MRI/PET triple modality molecular imaging

Abstract

Development of reporter genes for multimodality molecular imaging is highly important. In contrast to the conventional strategies which have focused on fusing several reporter genes together to serve as multimodal reporters, human tyrosinase (TYR)--the key enzyme in melanin production--was evaluated in this study as a stand-alone reporter gene for in vitro and in vivo photoacoustic imaging (PAI), magnetic resonance imaging (MRI) and positron emission tomography (PET). Human breast cancer cells MCF-7 transfected with a plasmid that encodes TYR (named as MCF-7-TYR) and non-transfected MCF-7 cells were used as positive and negative controls, respectively. Melanin targeted N-(2-(diethylamino)ethyl)-18F-5-fluoropicolinamide was used as a PET reporter probe. In vivo PAI/MRI/PET imaging studies showed that MCF-7-TYR tumors achieved significant higher signals and tumor-to-background contrasts than those of MCF-7 tumor. Our study demonstrates that TYR gene can be utilized as a multifunctional reporter gene for PAI/MRI/PET both in vitro and in vivo.

Figure 1. Multimodality molecular imaging of the TYR reporter gene system.

TYR reporter is introduced into cells through gene transfer and the expressed tyrosinase catalyzes the oxidation of tyrosine and Dopa to synthesize melanin. Melanin then serves as a multi-functional target. First, melanin has a broad optical absorption and is an excellent agent for photoacoustic effect, which can be used to perform PAI. Second, melanin has the ability to chelate metal ions (Fe³⁺) which provides cantrast for MRI. Third, melanin can be specifically imaged by a recently established melanin avid PET probe, ¹⁸F-P3BZA. Lastly, PET probe such as ¹⁸F-P3BZA has the potential to allow Cerenkov Luminesecnce Imaging (CLI) and this application was explored in this work as well.

Figure 2. In vitro evaluation of the expression of TYR reporter.

(A) Western blot assay of tyrosinase expression in MCF-7-TYR and MCF-7 cells. β-actin was used as the control. (B) Time-response tyrosinase activity curves in MCF-7-TYR, B16F10 and MCF-7 cells. (C) Photos of the cell pellets from MCF-7-TYR, B16F10 and MCF-7 cells without (upper) or with (bottom) 2 mM L-tyrosine treatment for 24 h. (D) Melanin production in MCF-7-TYR, B16F10 and MCF-7 cells with or without tyrosine treatment for 24 h. (E) The effect of TYR expression on the growth of the melanotic (MCF-7-TYR) and amelanotic (MCF-7) cells.

Figure 3. In vitro detection of melanotic (MCF-7-TYR, B16F10) and amelanotic (MCF-7) cells.

(A) Photoacoustic imaging (top), ultrasound (middle), and PAI/US images (bottom) of the gel phantom with different concentrations of cells. Photoacoustic images were obtained using the Vevo LAZR Photoacoustic Imaging System at a wavelength of 680 nm. (B) MRI images of three concentrations of MCF-7-TYR, B16F10 and MCF-7 cells pretreated without (top row) or with (bottom row) FeCl₃. (C) Uptake of ¹⁸F-P3BZA in MCF-7-TYR, B16F10 and MCF-7 cells after incubation with ¹⁸F-P3BZA at 37°C for 0.5 and 1 h. All results, expressed as percentage of cellular uptake, are mean of triplicate measurements ± SD.

Figure 4. Quantitative analysis of in vitro cell phantom PAI and MR imaging results.

(A) Photoacoustic signal increased with the higher concentration of melanotic cells. (B) MRI signal of MCF-7-TYR and B16F10 slightly increased with the increased concentrations of cells; a dramatic increase was observed in the melanotic cells pretreated with FeCl₃. The signal of MCF-7 cells remained consistent in all concentrations without FeCl₃ pretreatment; with FeCl₃ treatment the signal increased slightly.

Figure 5. In vivo multimodality imaging of tumor bearing mice with PAI, MRI and PET.

Five tumor bearing mice were used and tumors were indicated by arrows. (A) Photographic images of tumor bearing mice (left: melanotic MCF-7-TYR tumor; right: amelanotic MCF-7 tumor). (B) PAI (top), ultrasound (middle), and PAI/US images (bottom) of the tumor mice (left: MCF-7-TYR; right: MCF-7). (C) MRI images of MCF-7-TYR (left) and MCF-7 (right) tumors. Top row shows black and white images, and bottom row shows the pseudo-colored images. (D) Representative decay-corrected coronal (top) and transaxial (bottom) small animal PET images of MCF-7-TYR (left three images) and MCF-7 (right three images) tumors acquired at 0.5, 1 and 2 h after tail vein injection of ¹⁸F-P3BZA. In all three imaging modalities, MCF-7-TYR tumor shows higher contrast than that of MCF-7 tumor.

Figure 6. Quantitative analysis of MCF-7-TYR and MCF-7 tumors images obtained from PAI (A) and MRI (B).

MCF-7-TYR tumor displays much higher PAI signal than that of MCF-7 tumor, and MRI also demonstrates that higher tumor/muscle ration in MCF-7-TYR than that in MCF-7 model (P < 0.05).