In vivo detection of antigen-specific CD8+ T cells by immuno-positron emission tomography.

The immune system's ability to recognize peptides on major histocompatibility molecules contributes to the eradication of cancers and pathogens. Tracking these responses in vivo could help evaluate the efficacy of immune interventions and improve mechanistic understanding of immune responses. For this purpose, we employ synTacs, which are dimeric major histocompatibility molecule scaffolds of defined composition. SynTacs, when labeled with positron-emitting isotopes, can noninvasively image antigen-specific CD8+ T cells in vivo. Using radiolabeled synTacs loaded with the appropriate peptides, we imaged human papillomavirus-specific CD8+ T cells by positron emission tomography in mice bearing human papillomavirus-positive tumors, as well as influenza A virus-specific CD8+ T cells in the lungs of influenza A virus-infected mice. It is thus possible to visualize antigen-specific CD8+ T-cell populations in vivo, which may serve prognostic and diagnostic roles.

The ionic charge of copper-64 complexes conjugated to an engineered antibody affects biodistribution.

The development of biomolecules as imaging probes requires radiolabeling methods that do not significantly influence their biodistribution. Sarcophagine (Sar) chelators form extremely stable complexes with copper and are therefore a promising option for labeling proteins with (64)Cu. However, initial studies using the first-generation sarcophagine bifunctional chelator SarAr to label the engineered antibody fragment ch14.18-ΔCH2 (MW 120 kDa) with (64)Cu showed high tracer retention in the kidneys, presumably because the high local positive charge on the Cu(II)-SarAr moiety resulted in increased binding of the labeled protein to the negatively charged basal cells of the glomerulus. To test this hypothesis, ch14.18-ΔCH2 was conjugated with a series of Sar derivatives of decreasing positive charge and three commonly used macrocyclic polyaza polycarboxylate (PAC) bifunctional chelators (BFC). The immunoconjugates were labeled with (64)Cu and injected into mice, and PET/CT images were obtained at 24 and 48 h postinjection (p.i.). At 48 h p.i., ex vivo biodistribution was assessed. In addition, to demonstrate the potential of metastasis detection using (64)Cu-labeled ch14.18-ΔCH2, a preclinical imaging study of intrahepatic neuroblastoma tumors was performed. Reducing the positive charge on the Sar chelators decreased kidney uptake of Cu-labeled ch14.18-ΔCH2 by more than 6-fold, from >45 to <6% ID/g, whereas the uptake in most other tissues, including liver, was relatively unchanged. However, despite this dramatic decrease, the renal uptake of the PAC BFCs was generally lower than that of the Sar derivatives, as was the liver uptake. Uptake of (64)Cu-labeled ch14.18-ΔCH2 in neuroblastoma hepatic metastases was detected using PET.

Mitochondrial transplantation: Effects on chemotherapy in prostate and ovarian cancer cells in vitro and in vivo.

Prostate and ovarian cancers affect the male and female reproductive organs and are among the most common cancers in developing countries. Previous studies have demonstrated that cancer cells have a high rate of aerobic glycolysis that is present in nearly all invasive human cancers and persists even under normoxic conditions. Aerobic glycolysis has been correlated with chemotherapeutic resistance and tumor aggressiveness. These data suggest that mitochondrial dysfunction may confer a significant proliferative advantage during the somatic evolution of cancer. In this study we investigated the effect of direct mitochondria transplantation on cancer cell proliferation and chemotherapeutic sensitivity in prostate and ovarian cancer models, both in vitro and in vivo. Our results show that the transplantation of viable, respiration competent mitochondria has no effect on cancer cell proliferation but significantly decreases migration and alters cell cycle checkpoints. Our results further demonstrate that mitochondrial transplantation significantly increases chemotherapeutic sensitivity, providing similar apoptotic levels with low-dose chemotherapy as that achieved with high-dose chemotherapy. These results suggest that mitochondria transplantation provides a novel approach for early prostate and ovarian cancer therapy, significantly increasing chemotherapeutic sensitivity in in vitro and in vivo murine models.

Low-Fouling Zwitterionic Polymeric Colloids as Resuscitation Fluids for Hemorrhagic Shock.

Colloids, known as volume expanders, have been used as resuscitation fluids for hypovolemic shock for decades, as they increase plasma oncotic pressure and expand intravascular volume. However, recent studies show that commonly used synthetic colloids have adverse interactions with human biological systems. In this work, a low-fouling amine(N)-oxide-based zwitterionic polymer as an alternative volume expander with improved biocompatibility and efficacy is designed. It is demonstrated that the polymer possesses antifouling ability, resisting cell interaction and deposition in major organs, and is rapidly cleared via renal filtration and hepatic circulation, reducing the risk of long-term side effects. Furthermore, in vitro and in vivo studies show an absence of adverse effects on hemostasis or any acute safety risks. Finally, it is shown that, in a head-to-head comparison with existing colloids and plasma, the zwitterionic polymer serves as a more potent oncotic agent for restoring intravascular volume in a hemorrhagic shock model. The design of N-oxide-based zwitterionic polymers may lead to the development of alternative fluid therapies to treat hypovolemic shock and to improve fluid management in general.

Detection and therapy of neuroblastoma minimal residual disease using [64/67Cu]Cu-SARTATE in a preclinical model of hepatic metastases.

BACKGROUND: A major challenge to the long-term success of neuroblastoma therapy is widespread metastases that survive initial therapy as minimal residual disease (MRD). The SSTR2 receptor is expressed by most neuroblastoma tumors making it an attractive target for molecularly targeted radionuclide therapy. SARTATE consists of octreotate, which targets the SSTR2 receptor, conjugated to MeCOSar, a bifunctional chelator with high affinity for copper. Cu-SARTATE offers the potential to both detect and treat neuroblastoma MRD by using [64Cu]Cu-SARTATE to detect and monitor the disease and [67Cu]Cu-SARTATE as the companion therapeutic agent. In the present study, we tested this theranostic pair in a preclinical model of neuroblastoma MRD. An intrahepatic model of metastatic neuroblastoma was established using IMR32 cells in nude mice. The biodistribution of [64Cu]Cu-SARTATE was measured using small-animal PET and ex vivo tissue analysis. Survival studies were carried out using the same model: mice (6-8 mice/group) were given single doses of saline, or 9.25 MBq (250 µCi), or 18.5 MBq (500 µCi) of [67Cu]Cu-SARTATE at either 2 or 4 weeks after tumor cell inoculation. RESULTS: PET imaging and ex vivo biodistribution confirmed tumor uptake of [64Cu]Cu-SARTATE and rapid clearance from other tissues. The major clearance tissues were the kidneys (15.6 ± 5.8% IA/g at 24 h post-injection, 11.5 ± 2.8% IA/g at 48 h, n = 3/4). Autoradiography and histological analysis confirmed [64Cu]Cu-SARTATE uptake in viable, SSTR2-positive tumor regions with mean tumor uptakes of 14.1-25.0% IA/g at 24 h. [67Cu]Cu-SARTATE therapy was effective when started 2 weeks after tumor cell inoculation, extending survival by an average of 13 days (30%) compared with the untreated group (mean survival of control group 43.0 ± 8.1 days vs. 55.6 ± 9.1 days for the treated group; p = 0.012). No significant therapeutic effect was observed when [67Cu]Cu-SARTATE was started 4 weeks after tumor cell inoculation, when the tumors would have been larger (control group 14.6 ± 8.5 days; 9.25 MBq group 9.5 ± 1.6 days; 18.5 MBq group 15.6 ± 4.1 days; p = 0.064). CONCLUSIONS: Clinical experiences of peptide-receptor radionuclide therapy for metastatic disease have been encouraging. This study demonstrates the potential for a theranostic approach using [64/67Cu]Cu-SARTATE for the detection and treatment of SSTR2-positive neuroblastoma MRD.

Conformation-sensitive targeting of lipid nanoparticles for RNA therapeutics.

The successful in vivo implementation of gene expression modulation strategies relies on effective, non-immunogenic delivery vehicles. Lipid nanoparticles are one of the most advanced non-viral clinically approved nucleic-acid delivery systems. Yet lipid nanoparticles accumulate naturally in liver cells upon intravenous administration, and hence, there is an urgent need to enhance uptake by other cell types. Here we use a conformation-sensitive targeting strategy to achieve in vivo gene silencing in a selective subset of leukocytes and show potential therapeutic applications in a murine model of colitis. In particular, by targeting the high-affinity conformation of α4ß7 integrin, which is a hallmark of inflammatory gut-homing leukocytes, we silenced interferon-γ in the gut, resulting in an improved therapeutic outcome in experimental colitis. The lipid nanoparticles did not induce adverse immune activation or liver toxicity. These results suggest that our lipid nanoparticle targeting strategy might be applied for selective delivery of payloads to other conformation-sensitive targets.

Microdistribution of targeted, fluorescently labeled anti-carcinoembryonic antigen antibody in metastatic colorectal cancer: implications for radioimmunotherapy.

PURPOSE: Most radioimmunotherapy studies on radiolabeled antibody distribution are based on autoradiographic and radioluminographic data, which provide a lack of detailed information due to low resolution. We used fluorescently labeled anti-carcinoembryonic antigen (CEA) antibody (A5B7) to investigate quantitatively the kinetics and microdistribution of antibody in a clinically relevant orthotopic colorectal cancer model (LS174T) using high-resolution digital microscopy. EXPERIMENTAL DESIGN: Nude mice bearing LS174T liver orthotopic tumors received a single i.v. injection of fluorescently labeled A5B7 and were sacrificed at 10 minutes, 1 hour, or 24 hours postinjection. Before sacrifice, mice were injected with the perfusion marker Hoechst 33342. An anti-CD31 antibody was used to detect blood vessel distribution. Cryostat sections were processed with immunofluorescence procedures and analyzed with fluorescence microscopy and image analysis techniques. The fluorescence images were related to morphologic images of the same or adjacent tumor sections. RESULTS: Fluorescently labeled antibody showed rapid, selective uptake into tumor deposits, with a strong negative correlation with tumor size at 10 minutes and 1 hour (P < or = 0.01). By 24 hours, the correlation was no longer significant. The study showed movement of antibody across the tumor with time and a tendency to localize more uniformly by later time points (24 hours). The rate of antibody motility was similar in small and large tumor metastases, but small deposits showed more rapid antibody localization. Intratumoral vessels were positively related to tumor size (P < or = 0.001). CONCLUSION: The obtained data suggest that radioimmunotherapy can be highly efficient in an adjuvant or minimal residual disease setting.

Positron Emission Tomography Detects In Vivo Expression of Disialoganglioside GD2 in Mouse Models of Primary and Metastatic Osteosarcoma.

The cell membrane glycolipid GD2 is expressed by multiple solid tumors, including 88% of osteosarcomas and 98% of neuroblastomas. However, osteosarcomas are highly heterogeneous, with many tumors exhibiting GD2 expression on <50% of the individual cells, while some tumors are essentially GD2-negative. Anti-GD2 immunotherapy is the current standard of care for high-risk neuroblastoma, but its application to recurrent osteosarcomas, for which no effective therapies exist, has been extremely limited. This is, in part, because the standard assays to measure GD2 expression in these heterogeneous tumors are not quantitative and are subject to tissue availability and sampling bias. To address these limitations, we evaluated a novel, sensitive radiotracer [64Cu]Cu-Bn-NOTA-hu14.18K322A to detect GD2 expression in osteosarcomas (six patient-derived xenografts and one cell line) in vivo using positron emission tomography (PET). Tumor uptake of the radiolabeled, humanized anti-GD2 antibody [64Cu]Cu-Bn-NOTA-hu14.18K322A was 7-fold higher in modestly GD2-expressing osteosarcomas (32% GD2-positive cells) than in a GD2-negative tumor (9.8% vs. 1.3% of the injected dose per cc, respectively). This radiotracer also identified lesions as small as 29 mm3 in a 34% GD2-positive model of metastatic osteosarcoma of the lung. Radiolabeled antibody accumulation in patient-derived xenografts correlated with GD2 expression as measured by flow cytometry (Pearson r = 0.88, P = 0.01), distinguishing moderately GD2-expressing osteosarcomas (32%-69% GD2-positive cells) from high GD2 expressors (>99%, P < 0.05). These results support the utility of GD2 imaging with PET to measure GD2 expression in osteosarcoma and thus maximize the clinical impact of anti-GD2 immunotherapy. SIGNIFICANCE: In situ assessment of all GD2-positive osteosarcoma sites with a novel PET radiotracer could significantly impact anti-GD2 immunotherapy patient selection and enable noninvasive probing of correlations between target expression and therapeutic response.

Combining radioimmunotherapy with antihypoxia therapy 2-deoxy-D-glucose results in reduction of therapeutic efficacy.

PURPOSE: The efficacy of solid tumor radioimmunotherapy is reduced by heterogeneous tumor distribution of the radionuclide, with dose mainly deposited in the normoxic region and by the relative radioresistance of hypoxic tumor cells. In an attempt to overcome these challenges, radioimmunotherapy was combined with 2-deoxy-d-glucose (2DG), a hypoxia-selective cytotoxic inhibitor of glucose metabolism. EXPERIMENTAL DESIGN: In vitro toxicity of 2DG in LS174T cultures was tested using a colony-forming assay. The effect of combining 2DG with radioimmunotherapy in vivo was tested by administering radiolabeled anti-carcinoembryonic antigen antibody ([(131)I]A5B7 IgG1 whole monoclonal) to nude mice bearing s.c. LS174T tumors, followed by 10 daily injections of 2DG (2.0 g/kg). Tumors were measured to assess therapeutic efficacy. RESULTS: Data from in vitro studies confirmed 2DG cytotoxicity in this cell line. Greater toxicity was observed under standard laboratory conditions and in hypoxic cultures than at intermediate, physiologically relevant levels of glucose and oxygen. Alone, 2DG had no effect on in vivo tumor growth (P = 0.377 compared with saline-treated controls). Combination of radioimmunotherapy with 2DG reduced the therapeutic effect of radioimmunotherapy (e.g., 150 microCi (131)I alone mean survival time, 48.33 +/- 16.83 days; combined with 2DG, 30.67 +/- 5.62 days, P = 0.038). CONCLUSIONS: The combination investigated had a detrimental effect on survival. It is suggested that a cellular metabolic response to more aggressive therapy, previously reported in vitro, caused this. The results of this study have implications for the clinical application of combined cancer therapies with an antimetabolic modality component.

Combretastatin A-4-phosphate effectively increases tumor retention of the therapeutic antibody, 131I-A5B7, even at doses that are sub-optimal for vascular shut-down.

Radioimmunotherapy using 131I-A5B7, an anti-CEA antibody, in combination with the vascular disrupting agent, combretastatin A4-phosphate (CA-4-P, 200 mg/kg), has produced tumor cures in SW1222 colorectal xenografts. CA-4-P causes acute tumor blood vessel shutdown, which can be monitored in clinical trials using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI). The purpose of this study was to determine the magnitude of the anti-vascular effect of CA-4-P in the SW1222 tumor, at 200 mg/kg and at lower, more clinically relevant doses, using conventional assays; relate effects to changes in DCE-MRI parameters and determine the corresponding effects on tumor retention of 131I-A5B7. The tumor vascular effects of 30, 100 and 200 mg/kg CA-4-P were determined, at 4- and 24-h post-treatment, using DCE-MRI, uptake of Hoechst 33342 for tumor vascular volume and conventional histology for necrosis. The effect of CA-4-P on tumor and normal tissue 131I-A5B7 retention was also determined. A significant reduction in tumor DCE-MRI kinetic parameters, the initial area under the contrast agent concentration time curve (IAUGC) and the transfer constant (Ktrans), was demonstrated at 4 h after CA-4-P, for all dose levels. These effects persisted for at least 24 h for the 200 mg/kg group but not for lower doses. A similar pattern was seen for vascular volume and necrosis. Despite this dose response, all three dose levels increased tumor retention of radio labeled antibody to a similar degree. These results demonstrate that moderate tumor blood flow reduction following antibody administration is sufficient to improve tumor antibody retention. This is encouraging for the combination of CA-4-P and 131I-A5B7 in clinical trials.

Molecular imaging in nanomedicine - A developmental tool and a clinical necessity.

The development of nanomedicines presents the potential to deliver more potent drugs targeted more specifically to the site(s) of disease than is currently achievable. While encouraging results have been achieved, including at the clinical level, significant challenges and opportunities for development remain, both in terms of further developing the technology and in understanding the underlying biology. Given the lessons learned regarding variations in nanomedicine delivery to different tumor types and between different patients with the same tumor type, this is an area of drug development that, rather than simply benefiting from a patient-specific approach, actually demands it. The only way that this distribution information can be obtained is through imaging, and this requires labeling of the nanomedicine to enable detection outside the body. In this review, we describe recent advances in the labeling of nanomedicines, how imaging studies are guiding nanomedicine development, and the role of imaging in the future development of nanomedicines.

A Sensitive Method for the Measurement of Copper at Trace Levels Using an HPLC-Based Assay.

BACKGROUND: Measurement of trace metal contamination is critical in the production of radiometals, such as 64Cu, for protein labeling. ICP-MS provides these data with high sensitivity and high specificity, but at high (instrument) cost. TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11- tetraacetic acid) titration provides high sensitivity at low cost but with low specificity. A method that allowed the measurement of trace metals with high sensitivity but also at relatively low cost would, therefore, be very useful in the development of new radiometal production methods. OBJECTIVE: The goal of this project was to develop an analytical method for copper that uses readily available laboratory equipment while minimally achieving low ppm sensitivity. METHOD: The metal-chelating macrocycle 2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetraacetic acid (DOTA) was coupled to fluorescein to produce a molecule that combines high UV absorbance and high quantum yield with the ability to chelate a wide range of transition metals. The fluorescein-DOTA was mixed with Cu(II) samples at low ppm concentrations, and the samples were analyzed by reversed-phase HPLC. RESULTS: Copper chelation by the DOTA moiety decreased its overall charge, leading to a delayed elution from a C18 HPLC column. The absorbance signal of the fluorescein-DOTA-Cu(II) peak (453 nm) linearly correlated with the copper concentration allowing measurement of Cu(II) down to 1.25 ppm. Furthermore, using fluorescence detection (521 nm) the detection limit was reduced by almost three orders of magnitude, to 2.5 ppb (p<0.05). CONCLUSION: Using a fluorescent dye (fluorescein) coupled to a macrocyclic chelator (DOTA) and an HPLC equipped with a standard UV detector is it possible to measure Cu at ppm concentrations, the Cu concentration observed in typical samples of 64Cu.

Colitis ImmunoPET: Defining Target Cell Populations and Optimizing Pharmacokinetics.

BACKGROUND: Positron emission tomography combined with a specific probe presents the ability to noninvasively assess inflammatory bowel disease. We previously reported increased intestinal uptake of a Cu-labeled anti-ß7 integrin antibody (clone FIB504.64) in colitic mice. Here, we evaluated an anti-α4ß7 integrin antibody (clone DATK32), and the F(ab')2 and Fab fragments of the anti-ß7 antibody, which should have faster blood clearance than the intact antibody, as imaging probes for the detection of colitis in a mouse model. METHODS: The immunoproteins were labeled with Cu, injected into mice with dextran sodium sulphate-induced colitis. Positron emission tomography data were collected between 1 and 48 hours postinjection. RESULTS: Focal uptake of the anti-ß7 fragments was observed in the gut as early as 1 hour postinjection, and they cleared more rapidly from normal tissues than the whole antibody. For example, the blood concentrations at 24 hours postinjection were 23.3 ± 3.0% ID/g for Cu-labeled DATK32, 12.9 ± 2.1% ID/g for FIB504.64, 4.1 ± 0.4% ID/g for FIB504.64-F(ab')2, and 0.62 ± 0.2% ID/g for FIB504.64-Fab (P < 0.0001, analysis of variance). The ratio of uptake of DATK32 between the colitis and control groups in the large intestine (1.38) was lower than for the FIB504.64 fragments (3.15 for F(ab')2, 1.84 for Fab) or intact FIB504.64 (1.78). CONCLUSIONS: The lower intestinal uptake ratio of the Cu-labeled anti-α4ß7 antibody (DATK32) compared with the anti-ß7 immunoproteins suggests that targeting all ß7-expressing lymphocytes, not just those expressing α4ß7, is a more promising route to the development of an inflammatory bowel disease imaging agent. The FIB504.64-F(ab')2 fragment demonstrated the greatest differential between colitis and control groups, and is therefore the most promising lead molecule for the development of an inflammatory bowel disease-specific imaging agent.

Tumour parameters affected by combretastatin A-4 phosphate therapy in a human colorectal xenograft model in nude mice.

Combretastatin A-4 phosphate (CA4-P) is an antivascular agent which inhibits tumour blood flow. The effects of CA4-P were studied at 1 and 24h in colorectal xenografts by the concomitant imaging of multiple physiological parameters (hypoxia, blood vessels and perfusion), selected to demonstrate changes related to vascular shut-down. Untreated tumours were viable, with perfused blood vessels throughout and only small areas of hypoxia. At 1h post-treatment, although blood vessels remained throughout the tumour, perfused vessels were mainly restricted to the rim. However, hypoxia was widespread in both peripheral and central parts of the tumour. Quantitative analysis also revealed a significant decrease in perfusion and a maximum increase in hypoxia at this time-point. Conversely, at 24h after treatment, when most of the tumour was necrotic, pathophysiological conditions in the surviving viable rim were already returning to normal: perfusion was increasing, and hypoxia was greatly reduced and restricted to regions bordering central necrosis. In conclusion, these data provide an insight into the actions by which CA4-P may exert its effects on solid tumours.

Use of [18F]FDG Positron Emission Tomography to Monitor the Development of Cardiac Allograft Rejection.

BACKGROUND: Positron emission tomography (PET) has the potential to be a specific, sensitive and quantitative diagnostic test for transplant rejection. To test this hypothesis, we evaluated F-labeled fluorodeoxyglucose ([F]FDG) and N-labeled ammonia ([N]NH3) small animal PET imaging in a well-established murine cardiac rejection model. METHODS: Heterotopic transplants were performed using minor major histocompatibility complex-mismatched B6.C-H2 donor hearts in C57BL/6(H-2) recipients. C57BL/6 donor hearts into C57BL/6 recipients served as isograft controls. [F]FDG PET imaging was performed weekly between posttransplant days 7 and 42, and the percent injected dose was computed for each graft. [N]NH3 imaging was performed to evaluate myocardial perfusion. RESULTS: There was a significant increase in [F]FDG uptake in allografts from day 14 to day 21 (1.6% to 5.2%; P < 0.001) and uptake in allografts was significantly increased on posttransplant days 21 (5.2% vs 0.9%; P = 0.005) and 28 (4.8% vs 0.9%; P = 0.006) compared to isograft controls. Furthermore, [F]FDG uptake correlated with an increase in rejection grade within allografts between days 14 and 28 after transplantation. Finally, the uptake of [N]NH3 was significantly lower relative to the native heart in allografts with chronic vasculopathy compared to isograft controls on day 28 (P = 0.01). CONCLUSIONS: PET imaging with [F]FDG can be used after transplantation to monitor the evolution of rejection. Decreased uptake of [N]NH3 in rejecting allografts may be reflective of decreased myocardial blood flow. These data suggest that combined [F]FDG and [N]NH3 PET imaging could be used as a noninvasive, quantitative technique for serial monitoring of allograft rejection and has potential application in human transplant recipients.

Synergy between vascular targeting agents and antibody-directed therapy.

PURPOSE: Tumor heterogeneity necessitates the use of combined therapies. We have shown that combining antibody-directed therapy with antivascular agents converts a subcurative to a curative treatment. The purpose of this study was to investigate, by radioluminographic and microscopic techniques, the regional effects of the two complementary therapies. METHODS AND MATERIALS: Nude mice bearing colorectal tumors were injected with 125I-labeled anti-carcinoembryonic antigen antibody, and images were obtained for antibody distribution and modeling studies using radioluminography. For therapy studies, the mice were given radioimmunotherapy alone (131I-A5B7 anti-carcinoembryonic antigen antibody), the antivascular agent combretastatin A-4 3-0-phosphate (200 mg/kg), or both. Extra mice were used to study the regional tumor effects of these therapies over time: relevant histochemical procedures were performed on tissue sections to obtain composite digital microscopic images of apoptosis, blood vessels, perfusion, hypoxia, and morphology. RESULTS: Antibody distribution, modeling, and immunohistochemistry showed how radioimmunotherapy (7.4 MBq/40 microg antibody) effectively treated the outer, well-oxygenated tumor region only. Combretastatin A-4 3-0-phosphate treated the more hypoxic center, and in doing so altered the relationship between tumor parameters. CONCLUSION: The combined complementary therapies produced cures by destroying tumor regions with different pathophysiologies. Relating these regional therapeutic effects to the relevant tumor parameters microscopically allows optimization of therapy and improved translation to clinical trials.

Analysis of the regional uptake of radiolabeled deoxyglucose analogs in human tumor xenografts.

UNLABELLED: It has been shown in vitro that the cell uptake of (18)F-FDG, a tracer of glucose metabolism, increases under hypoxia. This is consistent with increased glycolytic metabolism. We have previously shown that in ischemic heart ex vivo the rates of uptake of (18)F-FDG and 2-(14)C-deoxy-D-glucose ((14)C-2DG) are both reduced. In this study, we investigated this effect in tumors by comparing the microdistribution of (18)F-FDG and (14)C-2DG in hypoxic and normoxic regions. METHODS: Mice (MF1) bearing LS174T human tumor xenografts were injected with premixed (18)F-FDG (100 MBq), (14)C-2DG (0.37 MBq), and pimonidazole hydrochloride (60 mg/kg). After 30, 60, and 120 min, tissues (n = 4) were taken and counted for whole-body biodistribution. Tumors were frozen, sectioned, and exposed to phosphor image plates to obtain a quantitative digital image of radionuclide distribution. Sections were then stained to reveal tumor pathophysiology: Hematoxylin and eosin staining demonstrated viable and necrotic regions, and immunohistochemical staining detected pimonidazole metabolism in hypoxic cells. The images of radionuclide microdistribution and histology were then coregistered and analyzed to assess radionuclide trapping throughout the tumor on a pixel-by-pixel basis. The Pearson correlation coefficients between the 2 radionuclides were calculated. The relative amounts of nuclide were then analyzed in viable and necrotic regions and in normoxic and hypoxic regions. RESULTS: Whole-body biodistributions for the 2 radiotracers were similar. A high Pearson correlation coefficient was obtained for the 2 radionuclides throughout the tumors (r = 0.85 +/- 0.10, P < 0.0001), indicating a highly similar microdistribution. When the tumors were divided into viable and necrotic regions, the ratio of mean counts per pixel was 1.96 (P < 0.0001), whereas for hypoxic versus normoxic regions it was 1.26 (P < 0.0001). There was no significant difference in selectivity for hypoxia between the 2 radiotracers (P = 0.86). CONCLUSION: The tumor microdistribution of deoxyglucose in viable, hypoxic, and necrotic regions show that there was little change in the microdistribution of deoxyglucose throughout this time course. This study extends previous in vitro work and confirms the selectivity of deoxyglucose for viable cells over necrotic regions and for hypoxic cells over normoxic regions in vivo.

The nonuniformity of antibody distribution in the kidney and its influence on dosimetry.

The therapeutic efficacy of radiolabeled antibody fragments can be limited by nephrotoxicity, particularly when the kidney is the major route of extraction from the circulation. Conventional dose estimates in kidney assume uniform dose deposition, but we have shown increased antibody localization in the cortex after glomerular filtration. The purpose of this study was to measure the radioactivity in cortex relative to medulla for a range of antibodies and to assess the validity of the assumption of uniformity of dose deposition in the whole kidney and in the cortex for these antibodies with a range of radionuclides. Storage phosphor plate technology (radioluminography) was used to acquire images of the distributions of a range of antibodies of various sizes, labeled with 125I, in kidney sections. This allowed the calculation of the antibody concentration in the cortex relative to the medulla. Beta-particle point dose kernels were then used to generate the dose-rate distributions from 14C, 131I, 186Re, 32P and 90Y. The correlation between the actual dose-rate distribution and the corresponding distribution calculated assuming uniform antibody distribution throughout the kidney was used to test the validity of estimating dose by assuming uniformity in the kidney and in the cortex. There was a strong inverse relationship between the ratio of the radioactivity in the cortex relative to that in the medulla and the antibody size. The nonuniformity of dose deposition was greatest with the smallest antibody fragments but became more uniform as the range of the emissions from the radionuclide increased. Furthermore, there was a strong correlation between the actual dose-rate distribution and the distribution when assuming a uniform source in the kidney for intact antibodies along with medium- to long-range radionuclides, but there was no correlation for small antibody fragments with any radioisotope or for short-range radionuclides with any antibody. However, when the cortex was separated from the whole kidney, the correlation between the actual dose-rate distribution and the assumed dose-rate distribution, if the source was uniform, increased significantly. During radioimmunotherapy, the extent of nonuniformity of dose deposition in the kidney depends on the properties of the antibody and radionuclide. For dosimetry estimates, the cortex should be taken as a separate source region when the radiopharmaceutical is small enough to be filtered by the glomerulus.

Nonuniform absorbed dose distribution in the kidney: the influence of organ architecture.

The development of novel, systemically administered radionuclide therapies (such as radioimmunotherapy) relies on the ability to predict dose-limiting toxicity to normal tissue. Where the kidney is the principal route of excretion of the radionuclide preparation and/or breakdown of products, nephrotoxicity may be the dose-limiting factor. Until recently, conventional (MIRD) dosimetry assumed the distribution in the kidney to be uniform. A new MIRD phantom of the kidney models it as a set of uniform suborgans. In the work described here, the assumption of uniformity of distribution and of heterogeneity of dose rate (and, thus, absorbed dose) was tested in the mouse model. In this paper, we examine the nonuniformity of distribution and the subsequent dose rate for 4 antibody preparations (IgG (150 kD), F(ab)'(2) (100 kD), Fab (50 kD) and sFv (27 kD)) labeled with 4 radionuclides ((125)I, (131)I, (186)Re, and (90)Y) of interest in radioimmunotherapy (RIT). The kidney was considered as a whole and as two suborgans (cortex and medulla), and the nonuniformity of the dose-rate distribution was measured by a correlation of modeled dose-rate distribution with the dose-rate distribution obtained for an equivalent uniform radionuclide distribution. The heterogeneity of distribution, the inter- and intra-suborgan, was seen to increase as the molecular weight of the antibodies decreased. The assumption of uniform activity distribution for the whole kidney gives a poor estimation of the distribution of the dose rate. In the cortex, the longer-range emitters smooth out the effect of heterogeneous distribution and, in mice, an assumption of uniform cortex self-dose distribution may be sufficient for simple calculations. It is unclear how much this smoothing would be relevant in the human kidney.

Targeted imaging of Ewing sarcoma in preclinical models using a 64Cu-labeled anti-CD99 antibody.

PURPOSE: Ewing sarcoma is a tumor of the bone and soft tissue characterized by diffuse cell membrane expression of CD99 (MIC2). Single-site, surgically resectable disease is associated with an excellent 5-year event-free survival; conversely, patients with distant metastases have a poor prognosis. Noninvasive imaging is the standard approach to identifying sites of metastatic disease. We sought to develop a CD99-targeted imaging agent for staging Ewing sarcoma and other CD99-expressing tumors. EXPERIMENTAL DESIGN: We identified a CD99 antibody with highly specific binding in vitro and labeled this antibody with (64)Cu. Mice with either subcutaneous Ewing sarcoma xenograft tumors or micrometastases were imaged with the (64)Cu-labeled anti-CD99 antibody and these results were compared with conventional MRI and 2[18F]fluoro-2-deoxy-D-glucose-positron emission tomography (FDG-PET) imaging. RESULTS: (64)Cu-labeled anti-CD99 antibody demonstrated high avidity for the CD99-positive subcutaneous tumors, with a high tumor-to-background ratio, greater than that demonstrated with FDG-PET. Micrometastases, measuring 1 to 2 mm on MRI, were not detected with FDG-PET but were readily visualized with the (64)Cu-labeled anti-CD99 antibody. Probe biodistribution studies demonstrated high specificity of the probe for CD99-positive tumors. CONCLUSIONS: (64)Cu-labeled anti-CD99 antibody can detect subcutaneous Ewing sarcoma tumors and metastatic sites with high sensitivity, outperforming FDG-PET in preclinical studies. This targeted radiotracer may have important implications for the diagnosis, surveillance, and treatment of Ewing sarcoma. Similarly, it may impact the management of other CD99 positive tumors.