Scott E. Harpstrite

Cerenkov Radiation Energy Transfer (CRET) Imaging: A Novel Method for Optical Imaging of PET Isotopes in Biological Systems

Background Positron emission tomography (PET) allows sensitive, non-invasive analysis of the distribution of radiopharmaceutical tracers labeled with positron (β+)-emitting radionuclides in small animals and humans. Upon β+ decay, the initial velocity of high-energy β+ particles can momentarily exceed the speed of light in tissue, producing Cerenkov radiation that is detectable by optical imaging, but is highly absorbed in living organisms. Principal Findings To improve optical imaging of Cerenkov radiation in biological systems, we demonstrate that Cerenkov radiation from decay of the PET isotopes 64Cu and 18F can be spectrally coupled by energy transfer to high Stokes-shift quantum nanoparticles (Qtracker705) to produce highly red-shifted photonic emissions. Efficient energy transfer was not detected with 99mTc, a predominantly γ-emitting isotope. Similar to bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET), herein we define the Cerenkov radiation energy transfer (CRET) ratio as the normalized quotient of light detected within a spectral window centered on the fluorophore emission divided by light detected within a spectral window of the Cerenkov radiation emission to quantify imaging signals. Optical images of solutions containing Qtracker705 nanoparticles and [18F]FDG showed CRET ratios in vitro as high as 8.8±1.1, while images of mice with subcutaneous pseudotumors impregnated with Qtracker705 following intravenous injection of [18F]FDG showed CRET ratios in vivo as high as 3.5±0.3. Conclusions Quantitative CRET imaging may afford a variety of novel optical imaging applications and activation strategies for PET radiopharmaceuticals and other isotopes in biomaterials, tissues and live animals.

The neurofibromatosis type 1 tumor suppressor controls cell growth by regulating signal transducer and activator of transcription-3 activity in vitro and in vivo

Neurofibromatosis type 1 (NF1) is a common cancer predisposition syndrome in which affected individuals develop benign and malignant nerve tumors. The NF1 gene product neurofibromin negatively regulates Ras and mammalian target of rapamycin (mTOR) signaling, prompting clinical trials to evaluate the ability of Ras and mTOR pathway inhibitors to arrest NF1-associated tumor growth. To discover other downstream targets of neurofibromin, we performed an unbiased cell-based high-throughput chemical library screen using NF1-deficient malignant peripheral nerve sheath tumor (MPNST) cells. We identified the natural product, cucurbitacin-I (JSI-124), which inhibited NF1-deficient cell growth by inducing apoptosis. We further showed that signal transducer and activator of transcription-3 (STAT3), the target of cucurbitacin-I inhibition, was hyperactivated in NF1-deficient primary astrocytes and neural stem cells, mouse glioma cells, and human MPNST cells through Ser(727) phosphorylation, leading to increased cyclin D1 expression. STAT3 was regulated in NF1-deficient cells of murine and human origin in a TORC1- and Rac1-dependent manner. Finally, cucurbitacin-I inhibited the growth of NF1-deficient MPNST cells in vivo. In summary, we used a chemical genetics approach to reveal STAT3 as a novel neurofibromin/mTOR pathway signaling molecule, define its action and regulation, and establish STAT3 as a tractable target for future NF1-associated cancer therapy studies.

Second-Generation Triple Reporter for Bioluminescence, Micro-Positron Emission Tomography, and Fluorescence Imaging

Bioluminescence, positron emission tomography (PET), and fluorescence modalities are currently available for noninvasive imaging in vivo, each with its own merits. To exploit the combined strengths of each and facilitate multimodality imaging, we engineered a dual-reporter construct in which firefly luciferase (FLuc) and a 12–amino acid nonstructural linker were fused in frame to the N-terminus of a mutant herpes simplex virus thymidine kinase (mNLS-SR39TK) kinetically enhanced for positron emission tomography (PET). Furthermore, a triple-reporter construct was developed in which monster green fluorescent protein (MGFP), a recently available enhanced fluorescent protein, was introduced into the fusion vector downstream of an internal ribosome entry site (IRES) to allow analysis by fluorescence microscopy or flow cytometry without compromising the specific activities of the upstream fusion components. FLuc bioluminescence was measured with a cooled charge-coupled device camera and mNLS-SR39TK activity by 9-[4-[18F]fluoro-3-(hydroxymethyl) butyl guanine (18F-FHBG) microPET or 3H-penciclovir net accumulation. Importantly, HeLa cells transiently transfected with the FLuc-mNLS-SR39TK-IRES-MGFP triple reporter retained the same specific activities of the FLuc-mNLS-SR39TK heteroenzyme and the individual unfused enzymes with no change in protein half-lives. The presence of the IRES-MGFP modestly decreased upstream heteroprotein expression. In living mice, somatic gene transfer of a ubiquitin promoter-driven FLuc-mNLS-SR39TK-IRES-MGFP plasmid showed a > 1,000-fold increase in liver photon flux and a > 2-fold increase in liver retention of 18F-FHBG by microPET compared with mice treated with control plasmid. Multifocal hepatocellular fluorescence was readily observed by standard confocal microscopy. This second-generation triple reporter incorporating enhanced components enables bioluminescence, PET, and fluorescence imaging of cells and living animals.

Targeted chemotherapy in drug-resistant tumors, noninvasive imaging of P-glycoprotein-mediated functional transport in cancer, and emerging role of Pgp in neurodegenerative diseases.

Multidrug resistance (MDR) mediated by overexpression of P-glycoprotein (Pgp) is one of the best characterized transporter-mediated barriers to successful chemotherapy in cancer patients and is also a rapidly emerging target in the progression of neurodegenerative disorders such as Alzheimers and Parkinsons diseases. Therefore, strategies capable of delivering chemotherapeutic agents into drug-resistant tumors and targeted radiopharmaceuticals acting as ultrasensitive molecular imaging probes for detecting functional Pgp expression in vivo could be expected to play a vital role in systemic biology as personalized medicine gains momentum in the twenty-first century. While targeted therapy could be expected to deliver optimal doses of chemotherapeutic drugs into the desired targets, the interrogation of Pgp-mediated transport activity in vivo via noninvasive imaging techniques (SPECT and PET) would be beneficial in stratification of patient populations likely to benefit from a given therapeutic treatment, thereby assisting management of drug resistance in cancer and treatment of neurodegenerative diseases. Both strategies could play a vital role in advancement of personalized treatments in cancer and neurodegenerative diseases. Via this tutorial, authors make an attempt in outlining these strategies and discuss their strengths and weaknesses.

Synthesis, Characterization, and Antimalarial Activity of Novel Schiff-Base-Phenol and Naphthalene-Amine Ligands

Emergence of chloroquine (CQ) resistant Plasmodium falciparum strains necessitates discovery of inexpensive antimalarial drugs capable of targeting CQ-resistant strains. Towards this objective, herein we have synthesized and characterized naphthalene-Schiff bases or naphthalene-amine phenols. Among these compounds, 7 demonstrated a significant bioactivity with a half-maximal inhibitory concentration (IC(50)) of 1.7 microM against CQ-resistant Dd2 strains.

A Generator-produced gallium-68 radiopharmaceutical for PET imaging of myocardial perfusion

Lipophilic cationic technetium-99m-complexes are widely used for myocardial perfusion imaging (MPI). However, inherent uncertainties in the supply chain of molybdenum-99, the parent isotope required for manufacturing 99Mo/99mTc generators, intensifies the need for discovery of novel MPI agents incorporating alternative radionuclides. Recently, germanium/gallium (Ge/Ga) generators capable of producing high quality 68Ga, an isotope with excellent emission characteristics for clinical PET imaging, have emerged. Herein, we report a novel 68Ga-complex identified through mechanism-based cell screening that holds promise as a generator-produced radiopharmaceutical for PET MPI.

Synthesis, characterization, and molecular structure of a gallium(III) complex of an amine-phenol ligand with activity against chloroquine-sensitive Plasmodium falciparum strains

Emergence of chloroquine-resistant Plasmodium falciparum strains necessitates discovery of novel antimalarial drugs, especially if the agents can be synthesized from commercially available, inexpensive precursors via short synthetic routes. While exploring structure-activity relationships, we found a gallium(III) complex, [(1,12-bis(2-hydroxy-5-methoxybenzyl)-1,5,8,12-tetraazadodecane)-gallium(III)](+) [Ga-5-Madd](+), 1, that possessed antimalarial efficacy. Like previously reported complexes, the crystal structure of 1 revealed gallium(III) in a symmetrical octahedral environment surrounded by four secondary amine nitrogen atoms in equatorial plane and two axial oxygen atoms. In contrast to a previously reported complex, [Ga-3-Madd](+), this novel metallo-antimalarial 1 possessed modest efficacy against chloroquine-sensitive HB3 Plasmodium lines. Thus, slight variation in the positions of methoxy functionalities on the aromatic rings of the organic scaffold dramatically altered specificity thereby suggesting a targeted (e.g., transporter- or receptor-mediated) rather than non-specific (e.g., pH or other gradient-mediated) mechanism of action for these agents.

Gold Nanoparticles Doped with (199) Au Atoms and Their Use for Targeted Cancer Imaging by SPECT.

Gold nanoparticles have been labeled with various radionuclides and extensively explored for single photon emission computed tomography (SPECT) in the context of cancer diagnosis. The stability of most radiolabels, however, still needs to be improved for accurate detection of cancer biomarkers and thereby monitoring of tumor progression and metastasis. Here, the first synthesis of Au nanoparticles doped with (199)Au atoms for targeted SPECT tumor imaging in a mouse triple negative breast cancer (TNBC) model is reported. By directly incorporating (199)Au atoms into the crystal lattice of each Au nanoparticle, the stability of the radiolabel can be ensured. The synthetic procedure also allows for a precise control over both the radiochemistry and particle size. When conjugated with D-Ala1-peptide T-amide, the Au nanoparticles doped with (199)Au atoms can serve as a C-C chemokine receptor 5 (CCR5)-targeted nanoprobe for the sensitive and specific detection of both TNBC and its metastasis in a mouse tumor model.

Interrogation of multidrug resistance (MDR1) P-glycoprotein (ABCB1) expression in human pancreatic carcinoma cells: correlation of 99mTc-Sestamibi uptake with western blot analysis.

Histopathological studies indicate that ∼63% of pancreatic tumors express multidrug resistance (MDR1) P-glycoprotein (Pgp) and its polymorphic variants. However, Pgp expression detected at the mRNA or protein level does not always correlate with functional transport activity. Because Pgp transport activity is affected by specific mutations and the phosphorylation state of the protein, altered or less active forms of Pgp may also be detected by PCR or immunohistochemistry, which do not accurately reflect the status of tumor cell resistance. To interrogate the status of the functional expression of MDR1 Pgp in MiaPaCa-2 and PANC-1 cells, cellular transport studies using 99mTc-Sestamibi were performed and correlated with western blot analysis. Biochemical transport assays in human pancreatic carcinoma MiaPaCa-2 and PANC-1 cells, human epidermal carcinoma drug-sensitive KB-3-1 cells, and human breast carcinoma MCF-7 cells (negative controls), and human epidermal carcinoma drug-resistant KB-8-5 cells, human breast carcinoma stably transfected with Pgp MCF-7/MDR1Pgp cells, and liver carcinoma HepG2 cells (positive controls) were performed. Protein levels were determined using a monoclonal antibody C219. 99mTc-Sestamibi demonstrates accumulation in human pancreatic carcinoma MiaPaCa-2 and PANC-1 cells. Uptake profiles are not affected by treatment with LY335979, a Pgp inhibitor, and correlate with western blot analysis. These cellular transport studies indicate an absence of Pgp at a functional level in MiaPaCa-2 and PANC-1 cells. Because major pancreatic tumors originate from the pancreatic duct and 99mTc-Sestamibi undergoes a dominant hepatobiliary mode of excretion, it would not be a sensitive probe for imaging pancreatic adenocarcinomas. Following interrogation of the functional status of Pgp in other pancreatic carcinoma cells, chemotherapeutic drugs that are also MDR1 substrates could offer alternative therapeutics for treating pancreatic adenocarcinomas.

67/68Galmydar: A metalloprobe for monitoring breast cancer resistance protein (BCRP)-mediated functional transport activity

INTRODUCTION For stratification of chemotherapeutic choices, radiopharmaceuticals capable of imaging breast cancer resistance protein (BCRP/ABCG2)-mediated functional transport are desired. To accomplish this objective, Galmydar, a fluorescent and moderately hydrophobic Ga(III) cationic complex and its (67/68)Ga-radiolabeled counterparts were interrogated in HEK293 cells stably transfected with BCRP and their WT counterparts transfected with empty vector. Additionally, the sensitivity and specificity of (68)Ga-Galmydar to evaluate functional expression of BCRP at the blood-brain barrier (BBB) was investigated in gene-knockout mdr1a/1b(-/-) (double knockout, dKO) and mdr1a/1b(-/-)ABCG2(-/-) (triple knockout, tKO) mouse models. METHODS For radiotracer uptake assays and live cell fluorescence imaging, either (67)Ga-Galmydar or its unlabeled counterpart was incubated in HEK293 cells transfected with BCRP (HEK293/BCRP) and their WT counterparts at 37°C under a continuous flux of CO2 (5%) in the presence or absence of Ko143, a potent BCRP antagonist, and cellular uptake was measured to assess the sensitivity of Galmydar to probe BCRP-mediated functional transport activity in cellulo. For assessing the potential of Galmydar to enable diagnostic imaging of targeted tissues in vivo, the (67)Ga-radiolabeled counterpart was incubated in either human serum albumin or human serum at 37°C and the percentage of unbound (67)Ga-Galmydar was determined. To evaluate the sensitivity of (68)Ga-Galmydar for molecular imaging of BCRP-mediated efflux activity in vivo, microPET/CT brain imaging was performed in dKO and tKO mice and their age-matched WT counterparts, 60min post-intravenous injection. RESULTS (67)Ga-Galmydar shows uptake profiles in HEK293 cells inversely proportional to BCRP expression, and antagonist (Ko143) induced accumulation in HEK293/BCRP cells, thus indicating target sensitivity and specificity. Furthermore, employing the fluorescent characteristics of Galmydar, optical imaging in HEK293/BCRP cells shows an excellent correlation with the radiotracer cellular accumulation data. (67)Ga-Galmydar shows > 85% unbound fraction and presence of parental compound in human serum. Finally, microPET/CT imaging shows higher retention of (68)Ga-Galmydar in brains of dKO and tKO mice compared to their age-matched WT counterparts, 60min post-intravenous tail-vein injection. CONCLUSIONS Combined data indicate that Galmydar could provide a template scaffold for development of a PET tracer for imaging BCRP-mediated functional transport activity in vivo.