Rapid and efficient delivery of radioactive metal complexes to the cell interior would enable novel applications in medical imaging and radiotherapy. Membrane permeant peptide conjugates incorporating HIV-1 Tat transactivation protein sequences (GRKKRRQRRR) and an appropriate peptide-based motif (epsilon-KGC) that provides an N(3)S donor core for chelating technetium and rhenium were synthesized. Oxotechnetium(V) and oxorhenium(V) Tat-peptide complexes were prepared by facile transchelation reactions with permetalates, tin(II) chloride and sodium glucoheptonate. RP-HPLC showed two major [(99m)Tc]Tat-peptide species (4) that differed in retention time by approximately 2 min corresponding to two [Re]Tat-peptide species (7) shown to have identical mass, consistent with formation of two isomers, likely the oxo-metal diastereomers. [(99m)Tc]Tat-peptides were stable to transchelation in vitro. In human Jurkat cells, [(99m)Tc]Tat-peptide 4 showed concentrative cell accumulation (30-fold greater than extracellular concentration) and rapid uptake kinetics (t(1/2) < 2 min) in a diastereomeric-comparable manner. Paradoxically, uptake was enhanced in 4 degrees C buffer compared to 37 degrees C, while depolarization of membrane potential as well as inhibition of microtubule function and vesicular trafficking showed no inhibitory effect. Cells preloaded with 4 showed rapid washout kinetics into peptide-free solution. Modification of [(99m)Tc]Tat-peptide by deletion of the N-terminus Gly with or without biotinylation minimally impacted net cell uptake. In addition, the C-terminus thiol of the prototypic Tat-peptide was labeled with fluorescein-5-maleimide to yield conjugate 8. Fluorescence microscopy directly localized conjugate 8 to the cytosol and nuclei (possibly nucleolus) of human Jurkat, KB 3-1 and KB 8-5 tumor cells. Preliminary imaging studies in mice following intravenous administration of prototypic [(99m)Tc]Tat-peptide 4 showed an initial whole body distribution and rapid clearance by both renal and hepatobiliary excretion. Analysis of murine blood in vivo and human serum ex vivo revealed >95% intact complex, while murine urine in vivo showed 65% parent complex. Thus, these novel Tat-peptide chelate conjugates, capable of forming stable [Tc/Re(V)]complexes, rapidly translocate across cell membranes into intracellular compartments and can be readily derivatized for further targeted applications in molecular imaging and radiotherapy.

The advent of immune-checkpoint inhibitors (ICI) in modern oncology has significantly improved survival in several cancer settings. A subgroup of women with breast cancer (BC) has immunogenic infiltration of lymphocytes with expression of programmed death-ligand 1 (PD-L1). These patients may potentially benefit from ICI targeting the programmed death 1 (PD-1)/PD-L1 signaling axis. The use of tumor-infiltrating lymphocytes (TILs) as predictive and prognostic biomarkers has been under intense examination. Emerging data suggest that TILs are associated with response to both cytotoxic treatments and immunotherapy, particularly for patients with triple-negative BC. In this review from The International Immuno-Oncology Biomarker Working Group, we discuss (a) the biological understanding of TILs, (b) their analytical and clinical validity and efforts toward the clinical utility in BC, and (c) the current status of PD-L1 and TIL testing across different continents, including experiences from low-to-middle-income countries, incorporating also the view of a patient advocate. This information will help set the stage for future approaches to optimize the understanding and clinical utilization of TIL analysis in patients with BC.

Molecular imaging is broadly defined as the characterization and measurement of biological processes in living animals, model systems, and humans at the cellular and molecular level using remote imaging detectors. One underlying premise of molecular imaging is that this emerging field is not defined by the imaging technologies that underpin acquisition of the final image per se, but rather is driven by the underlying biological questions. In practice, the choice of imaging modality and probe is usually reduced to choosing between high spatial resolution and high sensitivity to address a given biological system. Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) inherently use image-enhancing agents (radiopharmaceuticals) that are synthesized at sufficiently high specific activity to enable use of tracer concentrations of the compound (picomolar to nanomolar) for detecting molecular signals while providing the desired levels of image contrast. The tracer technologies strategically provide high sensitivity for imaging small-capacity molecular systems in vivo (receptors, enzymes, transporters) at a cost of lower spatial resolution than other technologies. We review several significant PET and SPECT advances in imaging receptors (somatostatin receptor subtypes, neurotensin receptor subtypes, ␣ v ␤ 3 integrin), enzymes (hexokinase, thymidine kinase), transporters (MDR1 P-glycoprotein, sodium-iodide symporter), and permeation peptides (human immunodeficiency virus type 1 (HIV-1) Tat conjugates), as well as innovative reporter gene constructs (herpes simplex virus 1 thymidine kinase, somatostatin receptor subtype 2, cytosine deaminase) for imaging gene promoter activation and repression, signal transduction pathways, and protein-protein interactions in vivo.

To enable concurrent whole body scintigraphy and direct imaging of subcellular localization of permeation peptides, dual-labeled Tat-peptides useful for both radiometric analysis and fluorescence microscopy are desired for molecular imaging applications. Thus, novel dual-labeled D-Tat-peptides comprising Tat-basic domain (hgrkkrrqrrrgc), C-terminus conjugated with fluorescein-5-maleimide (FM) and N-terminus chelated with [(99m)Tc(CO)(3)] via histidine coordination, were synthesized and characterized. In human Jurkat cells, radiotracer uptake and washout studies revealed concentration-dependent accumulation of the dual-labeled Tat-peptide within cells. Subcellular localization of Tat-peptide was confirmed by fluorescence microscopy using an analogous [Re(CO)(3)] dual-labeled Tat-peptide. As seen with C-terminus single-labeled Tat-peptides, localization to the nucleoli was observed with the dual-labeled Tat-peptide, suggesting that the mechanism of Tat-peptide uptake and localization was not dependent on free peptide termini at either end. In Balb/c mice, biodistribution studies performed with the dual-labeled Tat-peptide showed fluorescence intensity by microscopic analysis that visually confirmed and correlated directly with scintigraphic and radiometric data. Of note, following intravenous administration, little brain penetration of these permeation sequences was observed in vivo. His[(99m)Tc(CO)(3)]-, DTPA[(99m)Tc(CO)(3)]-, and epsilon-lys-gly-cys[(99m)Tc(O)]-labeled Tat-peptides showed significant pharmacokinetic differences in liver and kidney depending on labeling strategy, indicating that Tat-peptide biodistribution can be impacted by the chelation moiety coordinated with (99m)Tc. Thus, we have shown that dual-labeled (99m)Tc-tricarbonyl Tat-peptide-FM conjugates can be conveniently synthesized and enable direct comparison of quantitative radiometric and qualitative fluorescence data both in vitro as well as in vivo.

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