Interrogating Cellular Communication in Cancer with Genetically Encoded Imaging Reporters

Abstract

Cells continuously communicate changes in their microenvironment, both locally and globally, with other cells in the organism. Integration of information arising from signaling networks impart continuous, time-dependent changes of cell function and phenotype. Use of genetically encoded reporters enable researchers to noninvasively monitor time-dependent changes in intercellular and intracellular signaling, which can be interrogated by macroscopic and microscopic optical imaging, nuclear medicine imaging, MRI, and even photoacoustic imaging techniques. Reporters enable noninvasive monitoring of changes in cell-to-cell proximity, transcription, translation, protein folding, protein association, protein degradation, drug action, and second messengers in real time. Because of their positive impact on preclinical research, attempts to improve the sensitivity and specificity of these reporters, and to develop new types and classes of reporters, remain an active area of investigation. A few reporters have migrated to proof-of-principle clinical demonstrations, and recent advances in genome editing technologies may enable the use of reporters in the context of genome-wide analysis and the imaging of complex genomic regulation in vivo that cannot be readily investigated through standard methodologies. The combination of genetically encoded imaging reporters with continuous improvements in other molecular biology techniques may enhance and expedite target discovery and drug development for cancer interventions and treatment. © RSNA, 2020.

Figure 1:

Clustering of imaging modalities by resolution and energy. Imaging modalities span a range of resolutions and energies, which both contribute to differences in tissue penetration. Very high and very low energy imaging modalities yield high tissue penetration and, in most cases, macroscopic imaging resolution. At intermediate energies, such as fluorescence and bioluminescence, both macroscopic and microscopic imaging resolutions are achievable, but tissue penetration is limited. NIR = near-infrared, UV = ultraviolet, Vis = visible.

Figure 2:

Typical configurations of genetically encoded imaging reporters. A, Constitutive reporter. The reporter is under the control of a constitutive promoter such as simian virus 40, chicken β-actin, or cytomegalovirus promoter. This design is most often utilized for cell tracking or trafficking experiments. B, Regulated transcriptional reporter. This design is utilized to monitor promoter activity in various cell types under various stimuli. Control constructs such as (A) or constructs with empty promoter regions are required to demonstrate specificity of signal induction. C, Translational or posttranscriptional reporters. The untranslated region (UTR) of interest is included either upstream or downstream of the reporter under the control of a constitutive promoter. Constructs such as (A) or mutated UTR are required to demonstrate specificity of signal change. D, Posttranscriptional reporters. The reporter gene is fused to a protein of interest typically through a glycine-serine linker region. As the protein of interest is degraded or trafficked, the reporter is concomitantly degraded or trafficked through the cell. Key controls involve fusing to a mutated form of the protein of interest that is no longer degraded or appropriately trafficked through the cell. E, Feedback-regulated reporter. In this case, the fusion reporter from (D) is also controlled by the promoter of the gene of interest. The feedback-regulated dynamics of degradation and synthesis can then be studied. F, Biosensor. A modified luciferase or fluorophore that changes either brightness or spectral output in response to changes in the local environment is developed. This construct is placed under the control of a constitutive promoter. Constructs such as (A) or modified biosensors with point mutations no longer capable of sensing environmental changes are required to demonstrate specificity.

Figure 3:

Luciferase complementation imaging. Genetically encoded luciferase complementation strategies enable noninvasive imaging of reversible protein-protein interactions and protein folding events in cellulo and in vivo (44,106). Multiple protein-protein interactions can be monitored through the use of multicolor click beetle luciferases by combining complementation strategies and spectral unmixing (45). A, The two interacting proteins are fused to the N-terminal fragment of the luciferase and the C-terminal fragment of the luciferase, respectively, with an interposed flexible glycine-serine linker. In this example, the two interacting proteins are the rapamycin-binding protein (FKBP) and FKBP rapamycin binding domain (FRB) that associate in the presence of rapamycin. When they associate, the luciferase active site is reconstituted, and light is produced. B, Rapamycin-induced light production is specific both in cellulo (top) and in vivo (bottom). A mutation known to abrogate the binding of rapamycin (S2035I) inhibits light production both in cellulo and in vivo. (Reprinted, with permission, from reference .) ATP = adenosine triphosphate, CFLuc = C domain of the luciferase, NFLuc = N domain of the luciferase, Rap = rapamycin.

Figure 4:

Multispectral luciferase complementation. A, The C-terminal fragment of click beetle green (CBG-C) was fused to β transducin repeats-containing proteins (βTrCP). The N-terminal fragment of click beetle red (CBR-N) was fused to IκBα. The N-terminal fragment of click beetle green was fused to β-catenin. The spectral emission of the reconstituted click-beetle luciferases maps to the N-terminal portion. Thus, light produced from the β-catenin/βTrCP interaction (green) can be resolved from the IκBa/βTrCP interaction (red) through spectral unmixing. B–D, The simultaneous quantification of the real-time switching of protein-protein interactions with βTrCP can be measured, depending on the exogenous stimulus or small molecule inhibitor. Data in red indicate IκBα/βTrCP interaction, and data in green indicate β-catenin/βTrCP interaction. (Reprinted, with permission, from reference .) GSK3β = glycogen synthase kinese 3 beta, SB-216763 = 3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione.

Figure 5:

Dual enzyme–activated proximity sensor. A, Activator cells (expressing β-galactosidase) catalyze the cleavage of lugal, ultimately releasing d-luciferin. The liberated substrate enters nearby reporter cells, where it is used by luciferase to produce light. B, Reporter cells surrounding either control (left) or activator (right) cells were incubated with X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) for 4 hours and imaged. The blue color correlates with β-gal activity. Representative bioluminescence image of cocultures after 48 hours of incubation and subsequent incubation with lugal. Each dish was incubated with lugal (100 μg/mL) for 1 hour before image acquisition. (Reprinted, with permission, from reference .) ATP = adenosine triphosphate, RLU = relative light units.

Figure 6:

Reversible notch signaling reporter. A, In the endogenous Notch pathway, ligand binding leads to ectodomain shedding due to cleavage at the S2 site by ADAM, followed by intramembrane proteolysis at the S3 site by γ-secretase. The released Notch intracellular domain (NICD) translocates to the nucleus and interacts with the DNA-binding protein CSL to recruit MAML proteins and other coactivators to activate gene expression. In the split luciferase construct, each half of the luciferase is kept separated and dark until the pathway is fully engaged in the nucleus. B, The reporter recapitulates the dose response curve of the Notch pathway to DAPT both linearly and over a large dynamic range. This inhibition could be specifically sensitized with siRNA targeting NCSTN. (Reprinted, with permission, from reference .) CSL = CBF1/RBPjκ/Su(H)/Lag1, DMSO = dimethyl sulfoxide, LCI = luciferase complementation imaging, MAML = mastermind like.

Figure 7:

Transcriptional TGF reporter. A, TβRII = TGF-β-type II receptor, TF = transcription factor. B, SBE-luc reporter gene construct consisting of 12 SBE repeats, a herpes simplex virus thymidine kinase minimal promoter (TK), firefly luciferase or SEAP, and an SV40 late 5-bromo-4-chloro-3-inodolyl-β-d-galactopyranoside signal (A). B, LPS administration results in tissue-specific activation of the SBE-luc reporter in vivo, reflecting in vivo induction of SMAD pathways by the innate immune system. (Reprinted, with permission, from reference .) SBE = Smad-binding element, SEAP = secreted alkaline phosphatase, TGF = transforming growth factor.

Figure 8:

Acidic pH specifically and reversibly stimulates the STM Tn:1787 promoter. A, Bacteria were cultured in media of different pH values, and reporter activation by Salmonella library clones in low pH media (pH 6) were compared with reporter activation in normal pH (pH 7.5). Genes identified in the tumor cell coculture screen were activated in the context of acidic pH compared with pH 7.5. pMAAC001 and luxCDABE constitutively express plasmid-encoded and chromosomally encoded luxCDABE imaging reporters, respectively. Data were normalized as the ratio of the signal in media pH 6.0 to signal in media pH 7.5. Error bars correspond to standard error of the mean. B, Mice bearing B16F10 flank tumor xenografts were injected intratumorally with tumor-activated bioluminescent (Tn:1787+pluxCDE) or constitutively bioluminescent (Tn:27.8+pluxCDE) Salmonella. The excised tumors were then imaged hourly, and data are presented as the normalized signal at each time point. The normalized signal represents the ratio of the mean of the fold-initial signal of two Tn:1787+pluxCDE-colonized tumors to the mean of the fold-initial signal of two constitutive Tn:27.8+pluxCDE-colonized tumors. C, Representative tumor imaging ex vivo shows reversibility of the bioluminescent signal in the tumor-activated Salmonella. Images on the left show Salmonella-infected tumor explants after 6 hours of incubation at the indicated pH (pH 6.0, top; pH 7.5, bottom). Two hours later (8 hours total), media were removed and replaced with media of the indicated pH (pH 7.5, top; pH 6.0, bottom). Images on the right show Salmonella-infected tumor explants 4 hours after the pH of the media was changed. Note the reversibility of the bioluminescent signal (107). (Reprinted, with permission, from reference .)