Molecular Imaging with Reporter Genes: Has Its Promise Been Delivered?

Abstract

The first reporter systems were developed in the early 1980s and were based on measuring the activity of an enzyme-as a surrogate measure of promoter-driven transcriptional activity-which is now known as a reporter gene system. The initial objective and application of reporter techniques was to analyze the activity of a specific promoter (namely, the expression of a gene that is under the regulation of the specific promoter that is linked to the reporter gene). This system allows visualization of specific promoter activity with great sensitivity. In general, there are 2 classes of reporter systems: constitutively expressed (always-on) reporter constructs used for cell tracking, and inducible reporter systems sensitive to endogenous signaling molecules and transcription factors that characterize specific tissues, tumors, or signaling pathways.This review traces the development of different reporter systems, using fluorescent and bioluminescent proteins as well as radionuclide-based reporter systems. The development and application of radionuclide-based reporter systems is the focus of this review. The question at the end of the review is whether the "promise" of reporter gene imaging has been realized. What is required for moving forward with radionuclide-based reporter systems, and what is required for successful translation to clinical applications?

FIGURE 1.

The first reporter gene systems with constitutive or inducible regulatory elements, based on measuring activity of the enzyme chloramphenicol acetyltransferase and visualizing reporter activity. Reporter systems can be placed under constitutive or inducible promoters. Constitutive promoters such as CMV, elongation factor 1α, and others are always “on.” Inducible promoters are activated by specific transcriptional factors and can function as endogenous molecular–genetic sensors. mRNA = messenger RNA; poly-A = the addition of a poly tail to mRNA; TATA box-DNA sequence in the core promoter region.

FIGURE 2.

Histologic (A) and autoradiographic (B) images of HSV1-tk expression in RG2TK-positive rat brain tumor with stably expressed HSV1-tk in left hemisphere and wild-type (nontransduced) RG2 tumor in right hemisphere. Both tumors are clearly seen in toluidine blue–stained histologic section. Autoradiographic imaging was performed 24 h after intravenous administration of ¹⁴C-FIAU. RG2TK-positive tumor is clearly visualized in autoradiographic image, whereas RG2 tumor is barely detectable. (C) PET imaging of HSV1-tk expression. Three tumors were generated in rnu rats: W256TK-positive (positive control) tumor in left flank and 2 wild-type W256 tumors in dorsum of neck (test) and right flank (negative control). Neck tumor was inoculated with 10⁶ gp-STK-A2 vector-producer cells (retroviral titer, 10⁶–10⁷ cfu/mL) to induce HSV1-tk transduction of wild-type tumor in vivo. Fourteen days after inoculation, ¹²⁴I-FIAU (925 kBq) was injected intravenously. PET was performed 30 h after injection. Radioactivity is clearly localized in left flank tumor and neck tumor but is only at low background level in right flank tumor (C). (Adapted with permission of (1).)

FIGURE 3.

(A) HSV1-tk processing for imaging with different reporter probes (¹²⁴I- or ¹³¹I-FIAU and ¹⁸F-FHBG). (B and C) Chemical structures of pyrimidine (B) and acycloguanosine (C) nucleoside probes. Highlighted nucleosides have been determined to be most effective imaging probes for HSV1-tk (pyrimidines) and HSV1-sr39tk (acycloguanosines). Adapted from (96).

FIGURE 4.

Development of fusion gene to code fusion protein for multimodality imaging. Fusion protein contains HSV1-tk for imaging with reporter probes ¹²⁴I- or ¹³¹I-FIAU and ¹⁸F-FHBG; GFP component is used for fluorescence-activated cell sorting and fluorescence imaging; FLuc component is used for BLI.

FIGURE 5.

Validation of first inducible reporter system for p53 pathway in cells and tumors. (A) PET imaging of endogenous p53 activation. Transaxial PET images through shoulder (top 2 panels) and pelvis (bottom 2 panels) of 2 rats are shown. Both nontreated and treated animals had 3 subcutaneous tumor xenografts: U87p53TKGFP (test) in right shoulder, U87 wild-type (negative control) in left shoulder, and RG2TKGFP (positive control) in left thigh. Treated animal shows significant radioactivity localization in test tumor and in positive control but no radioactivity above background in negative control. (B) Structure of p53TKGFP retroviral vector with reporter system. Expression of TKGFP gene is regulated by artificial promoter containing multiple tandem repeats of p53-specific DNA-binding motif. Constitutive expression of neomycin-resistance gene (Neo) is driven by SV40 (simian virus 40 early) promoter, allowing for selection of stably transduced cells. LTR = Long Terminal Repeat; dLTR = deleted LTR. This vector has a mutation in the 3′LTR that renders the silencing of its promoter activity after duplication as 5′LTR during integration. Amp = ampicillin; EcoRI, NheI = are restriction enzymes that cleaves DNA. (C) U87p53TKGFP subcutaneous tumor samples assessed for levels of activated p53 (Ser15 phosphorylated), total p53 protein, p21, and TKGFP proteins by immunoblot analysis obtained from nontreated and treated rats. (D) Microscopic fluorescence images of same U87p53TKGFP subcutaneous tumor samples. (Adapted with permission of (153).)

FIGURE 6.

Validation of first dual-reporter system (hypoxia-inducible and constitutive reporter system in cells and tumors). (A) HIF-1 (hypoxia inducible factor 1)–mediated TKGFP expression was studied in model with subcutaneous 4C6 xenograft bilaterally in forelimbs before and after tourniquet was applied to left forelimb proximal to tumor. ¹⁸F-FEAU uptake (arrows) is seen only in tourniquet-applied limb. (B) HIF-1 response in growing spheroids. (C and D) Confirmation of integrity of HIF-1 signaling pathway and reporter systems in C6 rat glioma cells before (C) and after (D) HIF-1 upregulation by 100 μM of CoCl₂ (hypoxia mimetic). (E) Western blotting for TKGFP and reverse-transcriptase polymerase chain reaction for VEGF164 and VEGF120, 24 h after exposure to different concentrations of CoCl₂. (F) Structure of cis-HRE/TKGFP retroviral vector with dual-reporter systems. Expression of HSV1-tk/eGFP gene is regulated by artificial promoter containing 8 tandem repeats for HIF-1 DNA-binding motif. Constitutive expression of XPRT/dsRFP is driven by CMV promoter (pCMV), allowing for selection of stably transduced cells. (Adapted with permission of (156).)

FIGURE 7.

Diagnostic adoptive cell transfer. (A) Macrophages are genetically engineered to secrete synthetic biomarker on adopting tumor-associated metabolic profile. (B and C) Engineered macrophages are injected intravenously into syngeneic hosts (B) and allowed to home to existing sites of disease (C). (D) Blood test can then be used to monitor for secretion of biomarker that would indicate presence of disease. (E) This system can also provide spatial information on immune cell activation with use of imageable synthetic biomarker. RBC = red blood cell; Macs = macrophages. (Reprinted with permission of (167).)

FIGURE 8.

Imaging sensitivity for reporter-transduced T cells. (A) Sensitivity of hNET-¹⁸F-MFBG reporter system for imaging human T cells. (Reprinted from (188).) (B) hNET reporter-transduced T cells imaged 4 h after intravenous injection of ¹⁸F-MFBG. Number of imageable T cells (above background level) is more than 31,515 (R = 0.80). ID = injected dose; hsvTK = herpes simplex virus type 1 thymidine kinase (hsvTK); dCK = human deoxycytidine kinase double mutant (hdCKDM).

FIGURE 9.

(A) Sequential small-animal PET images of ¹²⁴I-MIBG-hNET over 72 h in same mouse bearing transduced C6/hNET-IRES-GFP xenograft and C6 wild-type xenograft in opposite shoulders, after intravenous injection of 7.4 MBq (200 μCi) of ¹²⁴I-MIBG. (B) Time–activity profiles of ¹²⁴I-MIBG accumulation. Tumor radioactivity (percentage dose/mL; maximum pixel value) was determined from sequential small-animal PET images. (C) Background-corrected radioactivity profile (transduced xenograft-to-nontransduced xenograft) for xenograft. (D) Time profile of percentage of measured radioactivity that represents background-corrected radioactivity for xenograft. (E) Time profile of transduced-to-nontransduced (background) xenograft ratio. Values are mean ± SD (n = 10). ID = injected dose. (Adapted with permission of (188).)

FIGURE 10.

(A) HSV1-tk reporter gene imaging in GBM patient. Liposomal (LIPO) encapsulated HSV1-tk DNA was injected directly into tumor cavity. PET reporter imaging was performed (at 1 and 68 h after ¹²⁴I-FIAU administration) before and after ganciclovir administration. (Reprinted with permission of (220).) (B) ¹⁸F-FHBG PET of liver cancer patient treated with direct intratumor injection of adenoviral HSV1-sr39tk (Adv HSV1-tk_sr39). Arrow is showing tumor localized radioactivity seen only on day 2, not on days 9 or 30. Radioactive background was detected in intestine (I) and bladder (B). (Reprinted with permission of (222).)

FIGURE 11.

Reporter gene imaging of targeted T-cell immunotherapy in recurrent glioma. (A and B) ¹⁸F-FHBG PET imaging was performed on patient with recurrent glioma before (A) and 7 d after (B) CTL infusions directly into tumor cavity. Tumor recurrence was monitored by T1-weighted (T₁W) MRI. ¹⁸F-FHBG PET images were fused with MR images. (C) Voxelwise analysis of ¹⁸F-FHBG SUV in pre- and post-CTL infusion scans was performed. (D) Small but significant changes (paired Wilcoxon test) in ¹⁸F-FHBG total activity were observed after CTL infusions. (Adapted with permission of (224).)

FIGURE 12.

Ex vivo transduction of target cells with reporter genes under constitutive or inducible promoters. Sequential enrichment and functional assessment of transduced cells in good-manufacturing-practice (GMP) facility is critical, followed by injection and imaging in living subject. LTR = long terminal repeat.