Quantitative imaging of disease signatures through radioactive decay signal conversion

Abstract

In the era of personalized medicine, there is an urgent need for in vivo techniques able to sensitively detect and quantify molecular activities. Sensitive imaging of gamma rays is widely used; however, radioactive decay is a physical constant, and its signal is independent of biological interactions. Here, we introduce a framework of previously uncharacterized targeted and activatable probes that are excited by a nuclear decay-derived signal to identify and measure molecular signatures of disease. We accomplished this by using Cerenkov luminescence, the light produced by β-particle-emitting radionuclides such as clinical positron emission tomography (PET) tracers. Disease markers were detected using nanoparticles to produce secondary Cerenkov-induced fluorescence. This approach reduces background signal compared to conventional fluorescence imaging. In addition to tumor identification from a conventional PET scan, we demonstrate the medical utility of our approach by quantitatively determining prognostically relevant enzymatic activity. This technique can be applied to monitor other markers and represents a shift toward activatable nuclear medicine agents.

Figure 1. Conventional PET and Cerenkov Luminescence

PET tracer principle and the development of agents that fluorescently-convert the Cerenkov radiation optical decay signal. (a) Positron emission tomography (PET) enables high-sensitivity and quantitative imaging of whole-body tracer distribution. Unfortunately, radioactive probes are detectable regardless of their biological interactions; possibilities of which include binding, degradation or no change to the probe. This results in high background signal that is indistinguishable from accumulated probe. (b) Cerenkov luminescence (CL) is a result of charged particles moving through a dielectric medium faster than the speed of light in that medium. The high-energy particles polarize water molecules along their path. When these polarized molecules relax, they release a continuous wavelength luminescence centered in the blue. (c) This phenomenon can be harnessed to provide functional information regarding a radiotracer’s in vivo fate. We can augment the depth of penetration of the blue-weighted light by using CR to excite fluorophores. Secondary Cerenkov-induced fluorescence imaging (SCIFI) can also be used in conjunction with activatable probes. This approach affords quantitative PET radiotracer information in addition to fluorescent reports of disease specific ligands and activities.

Figure 2. Fluorescence and Secondary Cerenkov-Induced Fluorescence

Comparison of the external excitation or CL-excitation of fluorophore. (a) Quantification of background subtracted signal-to-background (SB) measurements of an in vitro phantom of fluorescein and gallium-68 (140 µCi; 5.18 MBq) in a tissue scattering and absorbing mimic at increasing depth. Schematic shown at insert. (b) White light photograph of uncovered source in phantom. Representative figures of the filtered (500–520 nm) light emitted following (c) external laser, or (d) secondary Cerenkov-induced fluorescence. Note that the images display equal dynamic ranges. (e) The same principle applies using an in vivo subcutaneous capillary tube implantation model. The removal of the reflected and background nonspecific excitation of endogenous fluorophores significantly reduces background. Representative acquisition of (f) externally laser-excited fluorophore and (g) [¹⁸F]-FDG excited fluorescein. (h) The mean SB values of SCIFI (154.1±7) are approximately six–fold greater than that of conventional fluorescence (26.7±3.2).

Figure 3. Secondary Cerenkov-Induced Fluorescence of Optical Probes

Multispectral and tandem Cerenkov-induced fluorescence emission for disease biomarker readout. (a) The absorbance and fluorescence profiles of QD605 (emission at 605 nm) are shown (upper panel) and the activation of the QD605 by [¹⁸F]-FDG (27.75 MBq) through SCIFI (lower panel). The absorbance of some of the Cerenkov radiation is indicated by the difference between luminescence spectra prior to and after addition of QD (δ). (b) Filtered SCIFI for a panel of optical probes, FITC, Cy5.5, Cy7, QD565, QD605 and QD800 excited with an [¹⁸F]-FDG source (14.8 MBq), along with a white light image as legend. See also Supplemental Figure 2. (c) Quantification of the emission intensity, relative to control (water). The ratio of Cerenkov-induced fluorescence to control was greatest for QD605, used for further targeted SCIFI studies. (d) Coronal PET images show significant uptake of [⁸⁹Zr]-DFO-trastuzumab in Her2/neu expressing xenografts. This is also visualized with CL of the animals using no filter (open channel); sagittal orientation. In the 600 nm bandpass channel, the localization of the cRGD-QD605 targeting α_vβ₃ in the tumor microenvironment can be visualized as co-localized QD is excited by the CL from the radiolabeled antibody. The control cRAD-QD605 does not target to the tumor, and therefore shows no SCIFI signal. (e) CL and SCIFI quantification time-course. The CL signal is consistent across the imaging time-points, however the 600 nm signal increases in the cRGD-QD605 group, reaching a maximum at 120 minutes. (f) Ratiometric comparison of SCIFI signal to CL for the targeted and control groups over the time-course.

Figure 4. Enzyme Activatable SCIFI

Detection and quantitation of MMP-2 enzyme action using activatable secondary Cerenkov-induced fluorescence nanoparticles. (a) Schematic of enzyme activatable SCIFI probe. Fluorescence is quenched when the fluorescein (FAM)-bearing peptide is bound to the surface of the gold nanoparticle (AuNP). Enzymatic cleavage of the peptide by MMP-2 dissociates FAM, which is no longer quenched. (b) Fluorescent measurement of probe activation in vitro demonstrates recovery of signal after cleavage of the FAM from AuNP. (c) Spectra of activation of FAM probe using [¹⁸F]-FDG by SCIFI. (d) Axial PET imaging of [¹⁸F]-FDG uptake in both tumors showing non-statistically significant difference in uptake. (e) CL imaging of the Cerenkov signal recapitulates the PET readout of [¹⁸F]-FDG uptake. (f) The activated probe is detected in the enzyme-expressing tumor through SCIFI using a filter for FAM, demonstrating concomitant determination of enzyme and glycolytic activity. (g) Schematic representation of the components of observed signal in the SCIFI channel. Quantitation of the probe activation can be accomplished by this approach because there is no non-specific autofluorescence or reflection, see also Supplemental Figure 5. (h) The amount of activated probe in the control tumor is 1.56 pmol, while in the enzyme expressing tumor the average activity is 6.23 pmol (p<0.001). (i) Quantitative western blot of the activated MMP-2 enzyme expression in cell culture supernatant and from tumor lysate. We observe greater production of the active enzyme in the supernatant (left scale) than in vivo (right) from these representative samples. (j) Assessing the gelatinolytic activity of the enzyme (to degrade a gelatin containing gel) was determined by zymography, recapitulating the SCIFI findings.