In Vivo Imaging of Glutamine Metabolism to the Oncometabolite 2-Hydroxyglutarate in IDH1/2 Mutant Tumors.

The oncometabolite 2-hydroxyglutarate (2-HG) is a signature biomarker in various cancers, where it accumulates as a result of mutations in isocitrate dehydrogenase (IDH). The metabolic source of 2-HG, in a wide variety of cancers, dictates both its generation and also potential therapeutic strategies, but this remains difficult to access in vivo. Here, utilizing patient-derived chondrosarcoma cells harboring endogenous mutations in IDH1 and IDH2, we report that 2-HG can be rapidly generated from glutamine in vitro. Then, using hyperpolarized magnetic resonance imaging (HP-MRI), we demonstrate that in vivo HP [1-13C] glutamine can be used to non-invasively measure glutamine-derived HP 2-HG production. This can be readily modulated utilizing a selective IDH1 inhibitor, opening the door to targeting glutamine-derived 2-HG therapeutically. Rapid rates of HP 2-HG generation in vivo further demonstrate that, in a context-dependent manner, glutamine can be a primary carbon source for 2-HG production in mutant IDH tumors.

Hyperpolarized 13C Spectroscopic Evaluation of Oxidative Stress in a Rodent Model of Steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) has become highly prevalent, now considered the most common liver disease in the western world. Approximately one-third of patients with NASH develop non-alchoholic steatohepatitis (NASH), histologically defined by lobular and portal inflammation, and accompanied by marked oxidative stress. Patients with NASH are at increased risk for cirrhosis and hepatocellular carcinoma, and diagnosis currently requires invasive biopsy. In animal models of NASH, particularly the methionine-choline deficient (MCD) model, profound changes are seen in redox enzymes and key intracellular antioxidants. To study antioxidant status in NASH non-invasively, we applied the redox probe hyperpolarized [1-13C] dehydroascorbic acid (HP DHA), which is reduced to Vitamin C (VitC) rapidly in the normal liver. In MCD mice, we observed a significant decrease in HP DHA to VitC conversion that accompanied hepatic fat deposition. When these animals were subsequently placed on a normal diet, resonance ratios reverted to those seen in control mice. These findings suggest that HP DHA, a potentially clinically translatable imaging agent, holds special promise in imaging NASH and other metabolic syndromes, to monitor disease progression and response to targeted therapies.

Multinuclear NMR and MRI Reveal an Early Metabolic Response to mTOR Inhibition in Sarcoma.

Biomarkers predicting rapalog responses in sarcomas where PI3K and mTOR are often hyperactivated could improve the suitable recruitment of responsive patients to clinical trials. PI3K/mTOR pathway activation drives energy production by regulating anaerobic glycolysis in cancer cells, suggesting a route toward a monitoring strategy. In this study, we took a multimodality approach to evaluate the phenotypic effects and metabolic changes that occur with inhibition of the PI3K/mTOR pathway. Its central role in regulating glycolysis in human sarcomas was evaluated by short- and long-term rapamycin treatment in sarcoma cell lines. We observed an overall decrease in lactate production in vitro, followed by cell growth inhibition. In vivo, we observed a similar quantitative reduction in lactate production as monitored by hyperpolarized MRI, also followed by tumor size changes. This noninvasive imaging method could distinguish reduced cell proliferation from induction of cell death. Our results illustrate the use of hyperpolarized MRI as a sensitive technique to monitor drug-induced perturbation of the PI3K/mTOR pathway in sarcomas. Cancer Res; 77(11); 3113-20. ©2017 AACR.

Hyperpolarized 13C pyruvate mouse brain metabolism with absorptive-mode EPSI at 1T.

The expected signal in echo-planar spectroscopic imaging experiments was explicitly modeled jointly in spatial and spectral dimensions. Using this as a basis, absorptive-mode type detection can be achieved by appropriate choice of spectral delays and post-processing techniques. We discuss the effects of gradient imperfections and demonstrate the implementation of this sequence at low field (1.05T), with application to hyperpolarized [1-13C] pyruvate imaging of the mouse brain. The sequence achieves sufficient signal-to-noise to monitor the conversion of hyperpolarized [1-13C] pyruvate to lactate in the mouse brain. Hyperpolarized pyruvate imaging of mouse brain metabolism using an absorptive-mode EPSI sequence can be applied to more sophisticated murine disease and treatment models. The simple modifications presented in this work, which permit absorptive-mode detection, are directly translatable to human clinical imaging and generate improved absorptive-mode spectra without the need for refocusing pulses.

High-Throughput Indirect Quantitation of 13C Enriched Metabolites Using 1H NMR.

Nuclear magnetic resonance (NMR) spectroscopy is widely used in metabolomics to perform quantitative profiling of low-molecular weight compounds from biological specimens. The measurement of endogenous metabolites using NMR has proven to be a powerful tool to identify new metabolic biomarkers in physiological and pathological conditions, and to study and evaluate treatment efficiency. In this study we present a rapid approach to indirectly quantify 13C enriched molecules using one-dimensional (1D) 1H NMR. We demonstrate this approach using isotopically labeled [1,6-13C]glucose and in four different cell lines. We confirm the applicability of this approach for treatment follow-up, utilizing a renal cancer cell line with rapamycin as a tool compound to study changes in metabolic profiles. Finally, we validate the applicability of this method to study metabolic biomarkers from ex vivo tumor extracts, after infusion, using isotopically enriched glucose. Given the high throughput and increased sensitivity of direct-detect 1H NMR, this analytical approach provides an avenue for simple and rapid metabolic analysis of biological samples including blood, urine, and biopsies.

Remodeling the Vascular Microenvironment of Glioblastoma with α-Particles.

Tumors escape antiangiogenic therapy by activation of proangiogenic signaling pathways. Bevacizumab is approved for the treatment of recurrent glioblastoma, but patients inevitably develop resistance to this angiogenic inhibitor. We previously investigated targeted α-particle therapy with 225Ac-E4G10 as an antivascular approach and showed increased survival and tumor control in a high-grade transgenic orthotopic glioblastoma model. Here, we investigated changes in tumor vascular morphology and functionality caused by 225Ac-E4G10. METHODS: We investigated remodeling of the tumor microenvironment in transgenic Ntva glioblastoma mice using a therapeutic 7.4-kBq dose of 225Ac-E4G10. Immunofluorescence and immunohistochemical analyses imaged morphologic changes in the tumor blood-brain barrier microenvironment. Multicolor flow cytometry quantified the endothelial progenitor cell population in the bone marrow. Diffusion-weighted MR imaged functional changes in the tumor vascular network. RESULTS: The mechanism of drug action is a combination of remodeling of the glioblastoma vascular microenvironment, relief of edema, and depletion of regulatory T and endothelial progenitor cells. The primary remodeling event is the reduction of both endothelial and perivascular cell populations. Tumor-associated edema and necrosis were lessened, resulting in increased perfusion and reduced diffusion. Pharmacologic uptake of dasatinib into tumor was enhanced after α-particle therapy. CONCLUSION: Targeted antivascular α-particle radiation remodels the glioblastoma vascular microenvironment via a multimodal mechanism of action and provides insight into the vascular architecture of platelet-derived growth factor-driven glioblastoma.

Non-invasive PET Imaging of PARP1 Expression in Glioblastoma Models.

PURPOSE: The current study presents [(18)F]PARPi as imaging agent for PARP1 expression. PROCEDURES: [(18)F]PARPi was generated by conjugating a 2H-phthalazin-1-one scaffold to 4-[(18)F]fluorobenzoic acid. Biochemical assays, optical in vivo competition, biodistribution analysis, positron emission tomography (PET)/X-ray computed tomography, and PET/magnetic resonance imaging studies were performed in subcutaneous and orthotopic mouse models of glioblastoma. RESULTS: [(18)F]PARPi shows suitable pharmacokinetic properties for brain tumor imaging (IC50 = 2.8 ± 1.1 nM; logPCHI = 2.15 ± 0.41; plasma-free fraction = 63.9 ± 12.6 %) and accumulates selectively in orthotopic brain tumor tissue. Tracer accumulation in subcutaneous brain tumors was 1.82 ± 0.21 %ID/g, whereas in healthy brain, the uptake was only 0.04 ± 0.01 %ID/g. CONCLUSIONS: [(18)F]PARPi is a selective PARP1 imaging agent that can be used to visualize glioblastoma in xenograft and orthotopic mouse models with high precision and good signal/noise ratios. It offers new opportunities to non-invasively image tumor growth and monitor interventions.

The Potential of Metabolic Imaging.

Metabolic imaging is a field of molecular imaging that focuses and targets changes in metabolic pathways for the evaluation of different clinical conditions. Targeting and quantifying metabolic changes noninvasively is a powerful approach to facilitate diagnosis and evaluate therapeutic response. This review addresses only techniques targeting metabolic pathways. Other molecular imaging strategies, such as affinity or receptor imaging or microenvironment-dependent methods are beyond the scope of this review. Here we describe the current state of the art in clinically translatable metabolic imaging modalities. Specifically, we focus on PET and MR spectroscopy, including conventional (1)H- and (13)C-MR spectroscopy at thermal equilibrium and hyperpolarized MRI. In this article, we first provide an overview of metabolic pathways that are altered in many pathologic conditions and the corresponding probes and techniques used to study those alterations. We then describe the application of metabolic imaging to several common diseases, including cancer, neurodegeneration, cardiac ischemia, and infection or inflammation.

[(11)C]5-HTP and microPET are not suitable for pharmacodynamic studies in the rodent brain.

The PET tracer [(11)C]5-hydroxytryptophan ([(11)C]5-HTP), which is converted to [(11)C]5-hydroxytryptamine ([(11)C]5-HT) by aromatic amino acid decarboxylase (AADC), is thought to measure 5-HT synthesis rates. But can we measure these synthesis rates by kinetic modeling of [(11)C]5-HTP in rat? Male rats were scanned with [(11)C]5-HTP (60 minutes) after different treatments. Scans included arterial blood sampling and metabolite analysis. 5-HT synthesis rates were calculated by a two-tissue compartment model (2TCM) with irreversible tracer trapping or Patlak analysis. Carbidopa (inhibitor peripheral AADC) dose-dependently increased [(11)C]5-HTP brain uptake, but did not influence 2TCM parameters. Therefore, 10 mg/kg carbidopa was applied in all subsequent study groups. These groups included treatment with NSD 1015 (general AADC inhibitor) or p-chlorophenylalanine (PCPA, inhibitor of tryptophan hydroxylase, TPH). In addition, the effect of a low-tryptophan (Trp) diet was investigated. NSD 1015 or Trp depletion did not affect any model parameters, but PCPA reduced [(11)C]5-HTP uptake, and the k3. This was unexpected as NSD 1015 directly inhibits the enzyme converting [(11)C]5-HTP to [(11)C]5-HT, suggesting that trapping of radioactivity does not distinguish between parent tracer and its metabolites. As different results have been acquired in monkeys and humans, [(11)C]5-HTP-PET may be suitable for measuring 5-HT synthesis in primates, but not in rodents.

Induction of ß-glucuronidase release by cytostatic agents in small tumors.

Extracellular ß-glucuronidase (ß-GUS) in tumors has been investigated as a target enzyme for prodrug therapy. However, despite encouraging preclinical results, animal studies also indicate that the success of prodrug therapy might be limited by the insufficient prodrug-converting enzyme activity, especially in small tumors. We hypothesized that a single dose of a cytostatic drug might induce the release of ß-GUS in small tumors, resulting in increased levels of extracellular ß-GUS and consequently a higher efficacy of the prodrug treatment. Here we examine the extent of ß-GUS release in small C6 glioma tumors after a single treatment of doxorubicin (DOX), carmustine (BCNU) and tumor necrosis factor α (TNF-α) with positron emission tomography (PET) and the tracer 1-O-(4-(2-fluoroethyl-carbamoyloxymethyl)-2-nitrophenyl)-O-ß-d-glucopyronuronate, [(18)F]FEAnGA, which has been proven to be selective for extracellular ß-GUS. Induction of ß-GUS release was first investigated in cultured C6 glioma cells. In addition, a [(18)F]FEAnGA PET study was performed in C6 tumor-bearing rats 48 h after a single treatment with different cytostatics to evaluate the extent of ß-glucuronidase release. The cleavage of [(18)F]FEAnGA by ß-GUS was analyzed in tumor homogenates. The induction of tumor necrosis and leukocyte infiltration was confirmed by histochemical analysis and flow cytometry. The in vitro studies indicated that all treatments resulted in a decline of viable cells and an increase of extracellular ß-GUS activity. PET studies confirmed that ß-GUS was released in vivo and the distribution volume of the PET tracer [(18)F]FEAnGA in C6 gliomas was increased significantly by 15-70%, depending on the treatment. Histochemical analysis of the tumors indicated that carmustine and TNF-α treatment caused a larger necrotic area with the absence of infiltrating immune cells, whereas doxorubicin induced an increase in leukocyte infiltration. These results were confirmed by flow cytometry. In conclusion, the present study demonstrates that a single dose of a cytostatic agent is able to increase the release of ß-GUS. The release in ß-GUS can be monitored by [(18)F]FEAnGA PET in a noninvasive manner. This study may open the way to a two-step chemotherapy-prodrug approach, in which tumors are treated with a single dose of a cytostatic drug prior to prodrug treatment.

11C-hydroxytryptophan uptake and metabolism in endocrine and exocrine pancreas.

UNLABELLED: Determination of the residual ß-cell mass using noninvasive tools might help to follow up the efficacy of new treatments in both type 1 (insulin-dependent) and type 2 (non-insulin-dependent) diabetes mellitus, including islet transplantation. ß-cells are neuroendocrine cells that can take up and metabolize the serotonin precursor 5-hydroxytryptophan. The serotonin pathway may therefore be an attractive target for the development of an imaging tracer for residual ß-cell mass. The aim of this study was to evaluate the uptake mechanism and the retention of the PET tracer (11)C-hydroxytryptophan in endocrine and exocrine pancreas in vitro and in vivo. METHODS: The exocrine human pancreas carcinoma cell line (PANC-1) and the endocrine human insulinoma cell line (CM) were applied for in vitro (11)C-hydroxytryptophan accumulation/efflux experiments and blocking studies using inhibitors of key enzymes and transporters involved in the serotonin pathway. Animal experiments were performed on normal Wistar rats and on rats pretreated with the monoamine oxidase (MAO) inhibitor clorgyline. After intravenous injection of (11)C-hydroxytryptophan, a 60-min dynamic PET scan was acquired followed by an ex vivo biodistribution study. Autoradiography and hematoxylin-eosin staining were performed on the dissected pancreas to localize the radioactivity within the pancreatic tissue. RESULTS: (11)C-hydroxytryptophan accumulated rapidly in both endocrine CM cells and exocrine PANC-1 cells. In the exocrine cells, a rapid efflux of radioactivity was observed, whereas most radioactivity remained trapped in the endocrine cells. PET images showed clear accumulation of (11)C-hydroxytryptophan in the pancreas in both animal groups, but with a significant 3-fold higher retention of the radiopharmaceutical in clorgyline-treated animals. Ex vivo biodistribution studies confirmed the results obtained by PET. Autoradiographs did not discriminate between the exocrine and endocrine pancreas in control animals, whereas autoradiographs showed intense radioactive spots colocalized with the islets of Langerhans in clorgyline-treated animals. CONCLUSION: (11)C-hydroxytryptophan is trapped in ß-cells but not in exocrine pancreatic cells. ß-cell selectivity can be strongly enhanced by inhibition of MAO-A. This observation offers perspectives for the development of a more selective PET tracer for ß-cell mass, based on an (11)C-hydroxytryptophan derivative with increased resistance toward degradation by MAO-A.

Imaging of ß-cell mass and insulitis in insulin-dependent (Type 1) diabetes mellitus.

Insulin-dependent (type 1) diabetes mellitus is a metabolic disease with a complex multifactorial etiology and a poorly understood pathogenesis. Genetic and environmental factors cause an autoimmune reaction against pancreatic ß-cells, called insulitis, confirmed in pancreatic samples obtained at autopsy. The possibility to noninvasively quantify ß-cell mass in vivo would provide important biological insights and facilitate aspects of diagnosis and therapy, including follow-up of islet cell transplantation. Moreover, the availability of a noninvasive tool to quantify the extent and severity of pancreatic insulitis could be useful for understanding the natural history of human insulin-dependent (type 1) diabetes mellitus, to early diagnose children at risk to develop overt diabetes, and to select patients to be treated with immunotherapies aimed at blocking the insulitis and monitoring the efficacy of these therapies. In this review, we outline the imaging techniques currently available for in vivo, noninvasive detection of ß-cell mass and insulitis. These imaging techniques include magnetic resonance imaging, ultrasound, computed tomography, bioluminescence and fluorescence imaging, and the nuclear medicine techniques positron emission tomography and single-photon emission computed tomography. Several approaches and radiopharmaceuticals for imaging ß-cells and lymphocytic insulitis are reviewed in detail.

Pharmacokinetic modelling of N-(4-[(18)F]fluorobenzoyl)interleukin-2 binding to activated lymphocytes in an xenograft model of inflammation.

PURPOSE: N-(4-[(18)F]Fluorobenzoyl)interleukin-2 ([(18)F]FB-IL2) specifically binds to interleukin-2 receptors (IL-2R) and thus may be used to detect inflammation processes using positron emission tomography (PET). We now validated whether [(18)F]FB-IL2 can be used to quantify activated human peripheral blood mononuclear cells (hPBMC) in rats by pharmacokinetic modelling. METHODS: Eleven Wistar rats were subcutaneously inoculated in the shoulder with different amounts of phytohaemagglutinin (PHA) activated hPBMC 15 min before i.v. injection of [(18)F]FB-IL2. A 60-min dynamic PET scan was acquired and arterial blood sampling and metabolite analysis were performed. At the end of the scan, animals were terminated and the inflammatory lesion dissected. PET data were analysed using Logan and Patlak analysis as well as one-tissue and two-tissue compartment models. Model preferences according to the Akaike information criterion (AIC) and correlation between PET measurements and the number of CD25-positive cells were evaluated. RESULTS: A high correlation between ex vivo tracer uptake (standardized uptake value) in the xenograft and the number of inoculated CD25-positive cells was observed (R (2) = 0.90). Plasma time-activity curves showed a rapid washout of the radiopharmaceutical from blood, while the time-activity curves of the inflammatory lesions showed slower washout. Time-activity curves could be fitted well by the Logan analysis method, indicating that the binding between [(18)F]FB-IL2 and CD25 is reversible. AIC indicated that data could be modelled best by a two-tissue reversible compartment model. A high correlation was observed between the binding potential and the number of CD25-positive cells (R (2) = 0.876, p < 0.0001). Based on binding potential measured by PET, the limit of detection was about 160,000 CD25-positive cells per 200 µl lesion (95 % confidence). CONCLUSION: [(18)F]FB-IL2 kinetics in this animal model of inflammation could be best described by a reversible two-tissue compartment model. The [(18)F]FB-IL2 binding potential is a suitable measure for accurate quantification of lymphocytic infiltration in pathological conditions with PET.

N-(4-18F-fluorobenzoyl)interleukin-2 for PET of human-activated T lymphocytes.

UNLABELLED: Interleukin-2 (IL2) binds with high affinity to the IL2 receptors overexpressed on activated T lymphocytes in various pathologic conditions. Radiolabeling of IL2 with a positron-emitting isotope could provide a tool for noninvasive PET of activated T cells in immune-mediated diseases. We report the labeling of IL2 with N-succinimidyl 4-(18)F-fluorobenzoate ((18)F-SFB) for the synthesis of N-(4-(18)F-fluorobenzoyl)interleukin-2 ((18)F-FB-IL2) and the in vitro and in vivo evaluation of this novel radiopharmaceutical for the detection of IL2 receptor-positive cells by PET. METHODS: (18)F-SFB was efficiently prepared using a 3-step radiochemical pathway. Purified (18)F-SFB was incubated with IL2 in borate buffer (pH 8.5) and ethanol at 50°C for 10 min. (18)F-FB-IL2 was purified by reversed-phase high-performance liquid chromatography. As in vitro quality controls, a biologic binding assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis, mass spectrometry, and 3-chloroacetic acid precipitation stability tests were performed. Biodistribution studies were performed in BALB/c mice to evaluate the distribution of the tracer in healthy animals. PET experiments were performed in severe combined immunodeficiency disease mice inoculated with phytohemoagglutinin-activated human peripheral blood mononuclear cells (hPBMc). Whole-body images were acquired 30 min after injection of 5-15 MBq of (18)F-FB-IL2. RESULTS: (18)F-SFB was produced with a 34%-38% radiochemical yield. The radiochemical purity after solid-phase extraction purification ranged from 93% to 96%. Conjugation of (18)F-SFB to IL2 yielded (18)F-FB-IL2 as the major product. The radiochemical yield of (18)F-FB-IL2 after high-performance liquid chromatography purification was 25%-35% based on (18)F-SFB. (18)F-FB-IL2 was stable in plasma at 37°C and capable of stimulating T cells to an extent similar to native IL2. A biodistribution study showed highest tracer uptake in the kidneys and bladder due to rapid renal clearance of the tracer. Small-animal PET images showed binding of (18)F-FB-IL2 to activated hPBMc proportional to the number of injected cells. CONCLUSION: We report the successful labeling of IL2 with (18)F for PET of activated T lymphocytes. (18)F-FB-IL2 is stable, is biologically active, and allows in vivo detection of activated T lymphocytes.

Investigation of 99mTc-labelling of recombinant human interleukin-2 via hydrazinonicotinamide.

INTRODUCTION: Interleukin-2 (IL-2) when radiolabelled with (99m)Tc has been proved useful in imaging the side of lymphocytic infiltration in patients with autoimmune disorders and plays a significant role as a T-cell imaging agent. However, the labelling procedures used so far appeared to be rather complex and laborious. The aim of present study was to develop an efficient procedure of (99m)Tc-labelling of recombinant human interleukin-2 (rhIL-2) via hydrazinonicotinamide (HYNIC) to develop a dry kit formulation. METHODS: Various molar ratios of rhIL-2/HYNIC (from 1:2 to 1:12) were used at the conjugation step. The conjugates were purified on a PD-10 column to remove the excess of unbound HYNIC, as well as of any aggregates. The final peptide concentration was quantified by the BCA method, and the number of HYNIC molecules incorporated into a rhIL-2 molecule was determined based on the reaction with 2-sulfobenzaldehyde. The (99m)Tc-labelling was optimized using various amounts of HYNIC-rhIL-2, (99m)Tc, SnCl(2), tricine and nicotinic acid (NA). Quality control included GF-HPLC, ITLC, SDS-PAGE and biological assay. Biodistribution studies were performed in Swiss mice and Wistar rats. RESULTS: Generally, the highest radiolabelling yields were achieved when the HYNIC-rhIL-2 conjugates of ca. 2-4 HYNIC molecule substitution ratios were used. The optimal pH of the reaction medium was found to be in the range of 6.5 to 7.0. GF-HPLC analysis indicated that monomer and aggregates of (99m)Tc-HYNIC-rhIL-2 are formed during radiolabelling. At optimized conditions of wet radiolabelling, the (99m)Tc-HYNIC-rhIL-2 monomer was obtained with radiochemical purity >99%, specific activity of ca. 4 GBq/mg rhIL-2 and overall yield of ca. 65%. The two-vial freeze-dried kit was prepared: the first vial contained 30 µg HYNIC-rhIL-2, co-ligands, buffer and antioxidant; the second vial contained tricine and SnCl(2). The monomer of (99m)Tc-HYNIC-rhIL-2 was obtained by gel chromatography on a PD-10 column. No differences between labelled and unlabelled IL2 in terms of biological activity were observed. CONCLUSIONS: Our study shows that rhIL-2 can be efficiently radiolabelled with (99m)Tc via HYNIC, with tricine and NA as co-ligands using a two-vial freeze-dried kit. This enables the preparation of sterile and ready-to-use (99m)Tc-HYNIC(tricine,NA)-rhIL-2 within 1 h.

Synthesis and optimization of the labeling procedure of 99mTc-HYNIC-interleukin-2 for in vivo imaging of activated T lymphocytes.

INTRODUCTION: We have previously described the labeling of interleukin-2 (IL2) with (123)I and (99m)Tc-N(3)S. Both radiopharmaceuticals were successfully applied in humans to image several inflammatory lesions and autoimmune diseases characterized by tissue infiltrating lymphocytes expressing the IL2 receptor (CD25). However, both radiopharmaceuticals had some specific disadvantages, such as cost and time of synthesis. MATERIALS AND METHODS: Here, we describe a new improved method for labeling interleukin-2 with (99m)Tc using HYNIC-NHS and tricine as coligand. Several optimizations of reagent concentrations and labeling conditions were performed in order to standardize the procedure. After labeling, IL2 was purified by tC2 reverse-phase chromatography and tested in vitro and in vivo, in mice and in a normal volunteer. Results showed that this labeling procedure is cheap, fast, reliable, and reproducible, leading to a product with high specific activity (153 µCi/µg), high stability and capable of binding in vitro to CD25 positive cells. In vivo biodistribution in mice and human volunteer did not show any significant different from (99m)Tc-N(3)S-IL2. CONCLUSION: In conclusion, the optimization of (99m)Tc-HYNIC-IL2 has a great advantage in terms of cost and time of production and a simple kit formulation can be considered for routine application to study patients affected by autoimmune diseases, graft rejection, or other chronic inflammatory disorders.

ATM-ATR-dependent up-regulation of DNAM-1 and NKG2D ligands on multiple myeloma cells by therapeutic agents results in enhanced NK-cell susceptibility and is associated with a senescent phenotype.

There is much evidence to support a role for natural killer (NK) cells in controlling the progression of multiple myeloma (MM), a malignancy characterized by an abnormal plasma cell proliferation in the bone marrow (BM). Induction of DNA damage response has been recently shown capable of enhancing NKG2D ligand (NKG2DL) expression, but nothing is known about DNAM-1 ligand (DNAM-1L) regulation. In this study, we show that myeloma cells treated with low doses of therapeutic agents commonly used in the management of patients with MM, such as doxorubicin, melphalan, and bortezomib, up-regulate DNAM-1 and NKG2D ligands. Accordingly, therapeutic drug treatment of MM cells increases NK-cell degranulation, the NKG2D and DNAM-1 receptors being the major triggering molecules. Similar data were also obtained using ex vivo primary plasma cells derived from MM patients. Drug-induced DNAM-1 and NKG2D ligand expression was abolished after treatment with the ATM (ataxia telangiectasia mutated) and ATR (ATM- and RAD3-related) pharmacologic inhibitors caffeine and KU-55933, and was preferentially associated with senescent cells arrested in the G2 phase of the cell cycle. Altogether, our findings have identified a common pathway that can trigger the up-regulation of different NK cell-activating ligands and suggest that NK cells represent an immunosurveillance mechanism toward cells undergoing stress-induced senescent programs.