Examination of the PET in vivo generator 134Ce as a theranostic match for 225Ac.

PURPOSE: The radionuclide pair cerium-134/lanthanum-134 (134Ce/134La) was recently proposed as a suitable diagnostic counterpart for the therapeutic alpha-emitter actinium-225 (225Ac). The unique properties of 134Ce offer perspectives for developing innovative in vivo investigations that are not possible with 225Ac. In this work, 225Ac- and 134Ce-labelled tracers were directly compared using internalizing and slow-internalizing cancer models to evaluate their in vivo comparability, progeny meandering, and potential as a matched theranostic pair for clinical translation. Despite being an excellent chemical match, 134Ce/134La has limitations to the setting of quantitative positron emission tomography imaging. METHODS: The precursor PSMA-617 and a macropa-based tetrazine-conjugate (mcp-PEG8-Tz) were radiolabelled with 225Ac or 134Ce and compared in vitro and in vivo using standard (radio)chemical methods. Employing biodistribution studies and positron emission tomography (PET) imaging in athymic nude mice, the radiolabelled PSMA-617 tracers were evaluated in a PC3/PIP (PC3 engineered to express a high level of prostate-specific membrane antigen) prostate cancer mouse model. The 225Ac and 134Ce-labelled mcp-PEG8-Tz were investigated in a BxPC-3 pancreatic tumour model harnessing the pretargeting strategy based on a trans-cyclooctene-modified 5B1 monoclonal antibody. RESULTS: In vitro and in vivo studies with both 225Ac and 134Ce-labelled tracers led to comparable results, confirming the matching pharmacokinetics of this theranostic pair. However, PET imaging of the 134Ce-labelled precursors indicated that quantification is highly dependent on tracer internalization due to the redistribution of 134Ce's PET-compatible daughter 134La. Consequently, radiotracers based on internalizing vectors like PSMA-617 are suited for this theranostic pair, while slow-internalizing 225Ac-labelled tracers are not quantitatively represented by 134Ce PET imaging. CONCLUSION: When employing slow-internalizing vectors, 134Ce might not be an ideal match for 225Ac due to the underestimation of tumour uptake caused by the in vivo redistribution of 134La. However, this same characteristic makes it possible to estimate the redistribution of 225Ac's progeny noninvasively. In future studies, this unique PET in vivo generator will further be harnessed to study tracer internalization, trafficking of receptors, and the progression of the tumour microenvironment.

Ultrasmall Downconverting Nanoparticle for Enhanced Cerenkov Imaging.

Cerenkov imaging provides an opportunity to expand the application of approved radiotracers and therapeutic agents by utilizing them for optical approaches, which opens new avenues for nuclear imaging. The dominating Cerenkov radiation is in the UV/blue region, where it is readily absorbed by human tissue, reducing its utility in vivo. To solve this problem, we propose a strategy to shift Cerenkov light to the more penetrative red-light region through the use of a fluorescent down-conversion technique, based upon europium oxide nanoparticles. We synthesized square-shape ultrasmall Eu2O3 nanoparticles, functionalized with polyethylene glycol and chelate-free radiolabeled for intravenous injection into mice to visualize the lymph node and tumor. By adding trimethylamine N-oxide during the synthesis, we significantly increased the brightness of the particle and synthesized the (to-date) smallest radiolabeled europium-based nanoparticle. These features allow for the exploration of Eu2O3 nanoparticles as a preclinical cancer diagnosis platform with multimodal imaging capability.

Exploring the PET in vivo generator 134Ce as a theranostic match for 225Ac.

Purpose: The radionuclide pair cerium-134/lanthanum-134 (134Ce/134La) was recently proposed as a suitable diagnostic counterpart for the therapeutic alpha-emitter actinium-225 (225Ac). The unique properties of 134Ce offer perspectives for developing innovative in vivo investigations not possible with 225Ac. In this work, 225Ac- and 134Ce-labeled tracers were directly compared using internalizing and slow-internalizing cancer models to evaluate their in vivo comparability, progeny meandering, and potential as a matched theranostic pair for clinical translation. Despite being an excellent chemical match, 134Ce/134La has limitations to the setting of quantitative positron emission tomography imaging. Methods: The precursor PSMA-617 and a macropa-based tetrazine-conjugate (mcp-PEG8-Tz) were radiolabelled with 225Ac or 134Ce and compared in vitro and in vivo using standard (radio)chemical methods. Employing biodistribution studies and positron emission tomography (PET) imaging in athymic nude mice, the radiolabelled PSMA-617 tracers were evaluated in a PC3/PIP (PC3 engineered to express a high level of prostate-specific membrane antigen) prostate cancer mouse model. The 225Ac and 134Ce-labeled mcp-PEG8-Tz were investigated in a BxPC-3 pancreatic tumour model harnessing the pretargeting strategy based on a trans-cyclooctene-modified 5B1 monoclonal antibody. Results: In vitro and in vivo studies with both 225Ac and 134Ce-labelled tracers led to comparable results, confirming the matching pharmacokinetics of this theranostic pair. However, PET imaging of the 134Ce-labelled precursors indicated that quantification is highly dependent on tracer internalization due to the redistribution of 134Ce's PET-compatible daughter 134La. Consequently, radiotracers based on internalizing vectors like PSMA-617 are suited for this theranostic pair, while slow-internalizing 225Ac-labelled tracers are not quantitatively represented by 134Ce PET imaging. Conclusion: When employing slow-internalizing vectors, 134Ce might not be an ideal match for 225Ac due to the underestimation of tumour uptake caused by the in vivo redistribution of 134La. However, this same characteristic makes it possible to estimate the redistribution of 225Ac's progeny noninvasively. In future studies, this unique PET in vivo generator will further be harnessed to study tracer internalization, trafficking of receptors, and the progression of the tumour microenvironment.

Radiances of Cerenkov-Emitting Isotopes on the IVIS.

Cerenkov (or Cherenkov) luminescence occurs when charged particles exceed the phase velocity of a given medium. Cerenkov has gained interest in preclinical space as well as in clinical trials for optical visualization of numerous radionuclides. However, Cerenkov intensity has to be inferred from alternative databases with energy emission spectra, or theoretical fluence estimates. Here we present the largest experimental dataset of Cerenkov emitting isotopes recorded using the IVIS optical imaging system. We report Cerenkov measurements spanning orders of magnitude normalized to the activity concentration for 21 Cerenkov emitting isotopes, covering electron, alpha, beta minus, and positron emissions. Isotopes measured include Carbon-11, Fluorine-18, Phosphorous-32, Scandium-47, Copper-64, Copper-67, Gallium-68, Arsenic-72, Bromine-76, Yttrium-86, Zirconium-89, Yttrium-90, Iodine-124, Iodine-131, Cerium-134, Lutetium-177, Lead-203, Lead-212, Radium-223, Actinium-225, and Thorium-227. We hope this updating resource will serve as a rank ordering for comparing isotopes for Cerenkov luminescence in the visible window and serve as a rule of thumb for comparing Cerenkov intensities in vitro and in vivo. Methods: All Cerenkov emitting radionuclides were either produced at Memorial Sloan Kettering Cancer Center (Carbon-11, 11 C; Fluorine-18, 18 F; Iodine-124, 124 I), from commercial sources such as Perkin Elmer (Phosphorous-32, 32 P; Yttrium-90, 90 Y), Bayer (Radium-223, 223 Ra, Xofigo), 3D-Imaging (Zirconium-89, 89 Zr), Nuclear Diagnostic Products (Iodine-131, 131 I), or from academic collaborators at Washington University at St. Louis (Copper-64, 64 Cu), University of Wisconsin (Bromine-76, 76 Br), MD Anderson Cancer Center (Yttrium-86, 86 Y), Brookhaven National Laboratory (Arsenic-72, 72 As; Thorium-227, 227 Th), or Oak Ridge National Laboratory (Cerium-134, 134 Ce, Actinium-225, 225 Ac), and Viewpoint Molecular Targeting (Lead-203, 203 Pb; Lead 212, 212 Pb). All isotopes were diluted in triplicate on a black bottomed corning 96 well plate to several activity concentrations ranging from 0.1-250 µCi in 100-200 µL of Phosphate Buffered Saline. Cerenkov imaging was acquired on a single Perkin-Elmer Spectrum In-Vivo Imaging System (IVIS) at field of view c with exposures ranging up to 15 minutes or lower provided no part of the image intensity was saturated, or that the activity significantly changed during the exposure. Experimental radiances on the IVIS were calculated from regions of interest drown over each 96 well, and then normalized for the activity present in the well, and the volume the isotope was diluted into.

Detection of Shortwave-Infrared Cerenkov Luminescence from Medical Isotopes.

Medical radioisotopes produce Cerenkov luminescence (CL) from charged subatomic particles (ß+/-) traveling faster than light in dielectric media (e.g., tissue). CL is a blue-weighted and continuous emission, decreasing proportionally to increasing wavelength. CL imaging (CLI) provides an economic PET alternative with the advantage of also being able to image ß- and α emitters. Like any optical modality, CLI is limited by the optical properties of tissue (scattering, absorption, and ambient photon removal). Shortwave-infrared (SWIR, 900-1700 nm) CL has been detected from MeV linear accelerators but not yet from keV medical radioisotopes. Methods: Indium-gallium-arsenide sensors and SWIR lenses were mounted onto an ambient light-excluding preclinical enclosure. An exposure and processing pipeline was developed for SWIR CLI and then performed across 6 radioisotopes at in vitro and in vivo conditions. Results: SWIR CL was detected from the clinical radioisotopes 90Y, 68Ga, 18F, 89Zr, 131I, and 32P (biomedical research). SWIR CLI's advantage over visible-wavelength (VIS) CLI (400-900 nm) was shown via increased light penetration and decreased scattering at depth. The SWIR CLI radioisotope sensitivity limit (8.51 kBq/µL for 68Ga), emission spectrum, and ex vivo and in vivo examples are reported. Conclusion: This work shows that radioisotope SWIR CLI can be performed with unmodified commercially available components. SWIR CLI has significant advantages over VIS CLI, with preserved VIS CLI features such as radioisotope radiance levels and dose response linearity. Further improvements in SWIR optics and technology are required to enable widespread adoption.

Ambient Light Resistant Shortwave Infrared Fluorescence Imaging for Preclinical Tumor Delineation via the pH Low-Insertion Peptide Conjugated to Indocyanine Green.

Shortwave infrared (900-1,700 nm) fluorescence imaging (SWIRFI) has shown significant advantages over visible (400-650 nm) and near-infrared (700-900 nm) fluorescence imaging (reduced autofluorescence, improved contrast, tissue resolution, and depth sensitivity). However, there is a major lag in the clinical translation of preclinical SWIRFI systems and targeted SWIRFI probes. Methods: We preclinically show that the pH low-insertion peptide conjugated to indocyanine green (pHLIP ICG), currently in clinical trials, is an excellent candidate for cancer-targeted SWIRFI. Results: pHLIP ICG SWIRFI achieved picomolar sensitivity (0.4 nM) with binary and unambiguous tumor screening and resection up to 96 h after injection in an orthotopic breast cancer mouse model. SWIRFI tumor screening and resection had ambient light resistance (possible without gating or filtering) with outstanding signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) values at exposures from 10 to 0.1 ms. These SNR and CNR values were also found for the extended emission of pHLIP ICG in vivo (>1,100 nm, 300 ms). Conclusion: SWIRFI sensitivity and ambient light resistance enabled continued tracer clearance tracking with unparalleled SNR and CNR values at video rates for tumor delineation (achieving a tumor-to-muscle ratio above 20). In total, we provide a direct precedent for the democratic translation of an ambient light resistant SWIRFI and pHLIP ICG ecosystem, which can instantly improve tumor resection.

Simultaneous quantitative imaging of two PET radiotracers via the detection of positron-electron annihilation and prompt gamma emissions.

In conventional positron emission tomography (PET), only one radiotracer can be imaged at a time, because all PET isotopes produce the same two 511 keV annihilation photons. Here we describe an image reconstruction method for the simultaneous in vivo imaging of two PET tracers and thereby the independent quantification of two molecular signals. This method of multiplexed PET imaging leverages the 350-700 keV range to maximize the capture of 511 keV annihilation photons and prompt γ-ray emission in the same energy window, hence eliminating the need for energy discrimination during reconstruction or for signal separation beforehand. We used multiplexed PET to track, in mice with subcutaneous tumours, the biodistributions of intravenously injected [124I]I-trametinib and 2-deoxy-2-[18F]fluoro-D-glucose, [124I]I-trametinib and its nanoparticle carrier [89Zr]Zr-ferumoxytol, and the prostate-specific membrane antigen (PSMA) and infused PSMA-targeted chimaeric antigen receptor T cells after the systemic administration of [68Ga]Ga-PSMA-11 and [124I]I. Multiplexed PET provides more information depth, gives new uses to prompt γ-ray-emitting isotopes, reduces radiation burden by omitting the need for an additional computed-tomography scan and can be implemented on preclinical and clinical systems without any modifications in hardware or image acquisition software.

Membrane-derived particles shed by PSMA-positive cells function as pro-angiogenic stimuli in tumors.

Cell membrane-derived particles (Mp) are rounded membrane-enclosed particles that are shed from tumor cells. Mp are formed from tumor membranes and are capable of tumor targeting and immunotherapeutic agents because they share membrane homology with parental cells; thus, they are under consideration as a drug delivery vehicle. Prostate-specific membrane antigen (PSMA), a transmembrane glycoprotein with enzymatic functionality, is highly expressed in Mp and extracellular vesicles (EV) from prostate cancer (PCa) with poor clinical prognosis. Although PSMA expression was previously shown in EV and Mp isolated from cell lines and from the blood of patients with high-grade PCa, no pathophysiological effects have been linked to PCa-derived Mp. Here, we compared Mp from PSMA-expressing (PSMA-Mp) and PSMA-non-expressing (WT-Mp) cells side by side in vitro and in vivo. PSMA-Mp can transfer PSMA and new phenotypic characteristics to the tumor microenvironment. The consequence of PSMA transfer to cells and increased secretion of vascular endothelial growth factor-A (VEGF-A), pro-angiogenic and pro-lymphangiogenic mediators, with increased 4E binding protein 1 (4EBP-1) phosphorylation.

Prospective testing of clinical Cerenkov luminescence imaging against standard-of-care nuclear imaging for tumour location.

In oncology, the feasibility of Cerenkov luminescence imaging (CLI) has been assessed by imaging superficial lymph nodes in a few patients undergoing diagnostic 18F-fluoro-2-deoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT). However, the weak luminescence signal requires the removal of ambient light. Here we report the development of a clinical CLI fiberscope with a lightproof enclosure, and the clinical testing of the setup using five different radiotracers. In an observational prospective trial (ClinicalTrials.gov identifier NCT03484884 ) involving 96 patients with existing or suspected tumours, scheduled for routine clinical FDG PET or 131I therapy, the level of agreement of CLI with standard-of-care imaging (PET or planar single-photon emission CT) for tumour location was 'acceptable' or higher (≥3 in the 1-5 Likert scale) for 90% of the patients. CLI correlated with the concentration of radioactive activity, and captured therapeutically relevant information from patients undergoing targeted radiotherapy or receiving the alpha emitter 223Ra, which cannot be feasibly imaged clinically. CLI could supplement radiological scans, especially when scanner capacity is limited.

Synthesis of the PET Tracer 124I-Trametinib for MAPK/ERK Kinase Distribution and Resistance Monitoring.

Trametinib is an extremely potent allosteric inhibitor of mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) (MEK) 1/2, which has been approved for treatment of metastatic melanoma and anaplastic thyroid cancer in patients with confirmed BRAFV600E/K mutations. Though trametinib is highly efficacious, adverse side effects, including skin, gastrointestinal, and hepatic toxicity, are dose-limiting and can lead to treatment termination. Development of a noninvasive tool to visualize and quantify the delivery and distribution of trametinib (either as a single agent or in combination with other therapeutics) to tumors and organs would be helpful in assessing therapeutic index, personalizing individual dose, and potentially predicting resistance to therapy. Methods: To address these issues, we have developed a radiolabeled trametinib and evaluated the in vitro and in vivo properties. 123I-, 124I-, and 131I-trametinib, pure tracer analogs to trametinib, were synthesized in more than 95% purity, with an average yield of 69.7% and more than 100 GBq/µmol specific activity. Results: Overall, 124I-trametinib uptake in a panel of cancer cell lines can be blocked with cold trametinib, confirming specificity of the radiotracer in vitro and in vivo. 124I-trametinib was taken up at higher rates in KRAS and BRAF mutant cell lines than in wild-type KRAS cancer cell lines. In vivo, biodistribution revealed high uptake in the liver 2 h after injection, followed by clearance through the gastrointestinal tract over 4 d. Importantly, uptake higher than expected was observed in the lung and heart for up to 24 h. Peak uptake in the skin and gastrointestinal tract was observed between 6 and 24 h, whereas in B16F10 melanoma-bearing mice peak tumor concentrations were achieved between 24 and 48 h. Tumor uptake relative to muscle and skin was relatively low, peaking at 3.4- to 8.1-fold by 72 h, respectively. The biodistribution of 124I-trametinib was significantly reduced in mice on trametinib therapy, providing a quantitative method to observe MEK inhibition in vivo. Conclusion:124I-trametinib serves as an in vivo tool to personalize the dose instead of using the current single-fixed-dose scheme and, when combined with radiomic data, to monitor the emergence of therapy resistance. In addition, the production of iodinated trametinib affords researchers the ability to measure drug distribution for improved drug delivery studies.

FDA-approved ferumoxytol displays anti-leukaemia efficacy against cells with low ferroportin levels.

Acute myeloid leukaemia is a fatal disease for most patients. We have found that ferumoxytol (Feraheme), an FDA-approved iron oxide nanoparticle for iron deficiency treatment, demonstrates an anti-leukaemia effect in vitro and in vivo. Using leukaemia cell lines and primary acute myeloid leukaemia patient samples, we show that low expression of the iron exporter ferroportin results in a susceptibility of these cells via an increase in intracellular iron from ferumoxytol. The reactive oxygen species produced by free ferrous iron lead to increased oxidative stress and cell death. Ferumoxytol treatment results in a significant reduction of disease burden in a murine leukaemia model and patient-derived xenotransplants bearing leukaemia cells with low ferroportin expression. Our findings show how a clinical nanoparticle previously considered largely biologically inert could be rapidly incorporated into clinical trials for patients with leukaemia with low ferroportin levels.

Innovations in Nuclear Imaging Instrumentation: Cerenkov Imaging.

Cerenkov luminescence (CL) is blue glow light produced by charged subatomic particles travelling faster than the phase velocity of light in a dielectric medium such as water or tissue. CL was first discovered in 1934, but for biomedical research it was recognized only in 2009 after advances in optical camera sensors brought the required high sensitivity. Recently, applications of CL from clinical radionuclides have been rapidly expanding to include not only preclinical and clinical biomedical imaging but also an approach to therapy. Cerenkov Luminescence Imaging (CLI) utilizes CL generated from clinically relevant radionuclides alongside optical imaging instrumentation. CLI is advantageous over traditional nuclear imaging methods in terms of infrastructure cost, resolution, and imaging time. Furthermore, CLI is a truly multimodal imaging method where the same agent can be detected by two independent modalities, with optical (CL) imaging and with positron emission tomography (PET) imaging. CL has been combined with small molecules, biomolecules and nanoparticles to improve diagnosis and therapy in cancer research. Here, we cover the fundamental breakthroughs and recent advances in reagents and instrumentation methods for CLI as well as therapeutic application of CL.

Nanoparticles as multimodal photon transducers of ionizing radiation.

In biomedical imaging, nanoparticles combined with radionuclides that generate Cerenkov luminescence are used in diagnostic imaging, photon-induced therapies and as activatable probes. In these applications, the nanoparticle is often viewed as a carrier inert to ionizing radiation from the radionuclide. However, certain phenomena such as enhanced nanoparticle luminescence and generation of reactive oxygen species cannot be completely explained by Cerenkov luminescence interactions with nanoparticles. Herein, we report methods to examine the mechanisms of nanoparticle excitation by radionuclides, including interactions with Cerenkov luminescence, ß particles and γ radiation. We demonstrate that ß-scintillation contributes appreciably to excitation and reactivity in certain nanoparticle systems, and that excitation by radionuclides of nanoparticles composed of large atomic number atoms generates X-rays, enabling multiplexed imaging through single photon emission computed tomography. These findings demonstrate practical optical imaging and therapy using radionuclides with emission energies below the Cerenkov threshold, thereby expanding the list of applicable radionuclides.

Sonophore-enhanced nanoemulsions for optoacoustic imaging of cancer.

Optoacoustic imaging offers the promise of high spatial resolution and, at the same time, penetration depths well beyond the conventional optical imaging technologies, advantages that would be favorable for a variety of clinical applications. However, similar to optical fluorescence imaging, exogenous contrast agents, known as sonophores, need to be developed for molecularly targeted optoacoustic imaging. Despite numerous optoacoustic contrast agents that have been reported, there is a need for more rational design of sonophores. Here, using a library screening approach, we systematically identified and evaluated twelve commercially available near-infrared (690-900 nm) and highly absorbing dyes for multi-spectral optoacoustic tomography (MSOT). In order to achieve more accurate spectral deconvolution and precise data quantification, we sought five practical mathematical methods, namely direct classical least squares based on UV-Vis (UV/Vis-DCLS) or optoacoustic (OA-DCLS) spectra, non-negative LS (NN-LS), independent component analysis (ICA) and principal component analysis (PCA). We found that OA-DCLS is the most suitable method, allowing easy implementation and sufficient accuracy for routine analysis. Here, we demonstrate for the first time that our biocompatible nanoemulsions (NEs), in combination with near-infrared and highly absorbing dyes, enable non-invasive in vivo MSOT detection of tumors. Specifically, we found that NE-IRDye QC1 offers excellent optoacoustic performance and detection compared to related near-infrared NEs. We demonstrate that when loaded with low fluorescent or dark quencher dyes, NEs represent a flexible and new class of exogenous sonophores suitable for non-invasive pre-clinical optoacoustic imaging.

Utilizing the power of Cerenkov light with nanotechnology.

The characteristic blue glow of Cerenkov luminescence (CL) arises from the interaction between a charged particle travelling faster than the phase velocity of light and a dielectric medium, such as water or tissue. As CL emanates from a variety of sources, such as cosmic events, particle accelerators, nuclear reactors and clinical radionuclides, it has been used in applications such as particle detection, dosimetry, and medical imaging and therapy. The combination of CL and nanoparticles for biomedicine has improved diagnosis and therapy, especially in oncological research. Although radioactive decay itself cannot be easily modulated, the associated CL can be through the use of nanoparticles, thus offering new applications in biomedical research. Advances in nanoparticles, metamaterials and photonic crystals have also yielded new behaviours of CL. Here, we review the physics behind Cerenkov luminescence and associated applications in biomedicine. We also show that by combining advances in nanotechnology and materials science with CL, new avenues for basic and applied sciences have opened.

Nanoparticles and radiotracers: advances toward radionanomedicine.

In this study, we cover the convergence of radiochemistry for imaging and therapy with advances in nanoparticle (NP) design for biomedical applications. We first explore NP properties relevant for therapy and theranostics and emphasize the need for biocompatibility. We then explore radionuclide-imaging modalities such as positron emission tomography (PET), single-photon emission computed tomography (SPECT), and Cerenkov luminescence (CL) with examples utilizing radiolabeled NP for imaging. PET and SPECT have served as diagnostic workhorses in the clinic, while preclinical NP design examples of multimodal imaging with radiotracers show promise in imaging and therapy. CL expands the types of radionuclides beyond PET and SPECT tracers to include high-energy electrons (ß- ) for imaging purposes. These advances in radionanomedicine will be discussed, showing the potential for radiolabeled NPs as theranostic agents. WIREs Nanomed Nanobiotechnol 2016, 8:872-890. doi: 10.1002/wnan.1402 For further resources related to this article, please visit the WIREs website.