Digital Autoradiography Research Articles

Tumor Microenvironment–Dependent 18F-FDG, 18F-Fluorothymidine, and 18F-Misonidazole Uptake: A Pilot Study in Mouse Models of Human Non–Small Cell Lung Cancer

(18)F-FDG, (18)F-fluorothymidine, and (18)F-misonidazole PET scans have emerged as important clinical tools in the management of cancer; however, none of them have demonstrated conclusive superiority. The aim of this study was to compare the intratumoral accumulation of (18)F-FDG, (18)F-fluorothymidine, and (18)F-misonidazole and relate this to specific components of the tumor microenvironment in mouse models of human non-small cell lung cancer (NSCLC). We used NSCLC A549 and HTB177 cells to generate subcutaneous and peritoneal xenografts in nude mice. Animals were coinjected with a PET radiotracer, pimonidazole (hypoxia marker), and bromodeoxyuridine (proliferation marker) intravenously 1 h before animal euthanasia. Tumor perfusion was assessed by Hoechst 33342 injection, given 1 min before sacrifice. The intratumoral distribution of PET radiotracers was visualized by digital autoradiography and related to microscopic visualization of proliferation, hypoxia, perfusion, stroma, and necrosis. NSCLC xenografts had complex structures with intermingled regions of viable cancer cells, stroma, and necrosis. Cancer cells were either well oxygenated (staining negatively for pimonidazole) and highly proliferative (staining positively for bromodeoxyuridine) or hypoxic (pimonidazole-positive) and noncycling (little bromodeoxyuridine). Hypoxic cancer cells with a low proliferation rate had high(18)F-FDG and (18)F-misonidazole uptake but low (18)F-fluorothymidine accumulation. Well-oxygenated cancer cells with a high proliferation rate accumulated a high level of (18)F-fluorothymidine but low (18)F-FDG and(18)F-misonidazole. Tumor stroma and necrotic zones were always associated with low (18)F-FDG, (18)F-misonidazole, and (18)F-fluorothymidine activity. In NSCLC A549 and HTB177 subcutaneously or intraperitoneally growing xenografts, (18)F-fluorothymidine accumulates in well-oxygenated and proliferative cancer cells, whereas (18)F-misonidazole and (18)F-FDG accumulate mostly in poorly proliferative and hypoxic cancer cells. (18)F-FDG and (18)F-misonidazole display similar intratumoral distribution patterns, and both mutually exclude (18)F-fluorothymidine.

Abstract 2439: Molecular imaging of spatial and temporal heterogeneity of tumor micro-environment in mouse models of non-small cell lung cancer macroscopic xenografts and micro-metastases

Abstract 18F-fluoro-2-deoxyglucose (18F-FDG, imaging glucose metabolism), 18F-fluorothymidine (18F-FLT, cell proliferation) and 18F-misonidazole (18F-FMISO, hypoxia) PET scans have emerged as important clinical tools for management of cancer. The objective of this study was to spatially and temporally visualize tumor microenvironment of tumor hypoxia, proliferation and glucose metabolism using 18F-FDG, 18F-FLT and 18F-FMISO digital autoradiography and microPET comparing with histological findings. Methods: We used human non-small cell lung cancer (NSCLC) A549 and HTB177 cells to generate subcutaneous and peritoneal metastases in nude mice. Animals were coinjected with the mixture of one PET radiotracer, pimonidazole (hypoxia marker) and bromodeoxyuridine (proliferation marker) intravenously 1 hour before animal euthanasia. The intratumoral distributions of radiotracers were visualized by digital autoradiography (DAR) and related to microscopic visualization of cellular proliferation, tumor hypoxia, stroma and necrosis. Serial microPET scans (day 1to day5) were also performed in the same animals to investigate change in glucose metabolism (using 18F-FDG), proliferation (18F-FLT) and hypoxia (18F-FMISO). Results: NSCLC microenvironment was complex and highly heterogeneous: xenografts had complex structures with intermingled regions of well oxygenated (negative pimonidazole) and highly proliferative (positive bromodeoxyuridine) cancer cells, hypoxic (positive pimonidazole) and low proliferation (little bromodeoxyuridine) cancer cells stroma and necrosis. Hypoxic cancer cells had high18F-FDG and 18F-FMISO but low 18F-FLT accumulation, indicating increased glucose metabolism is not a common feature of cancer cells but only hypoxic ones. Well oxygenated cancer cells with high proliferation rate accumulated high level of 18F-FLT, but low 18F-FDG and18F-FMISO. Stroma and necrotic zones always associated with low activity in all radiotracers we tested. MicroPET scans revealed apparent change in intratumor distribution of 18F-FLT, 18F-FDG as well as 18F-FMISO in as short as ∼48 hrs interval, indicating temporal heterogeneity of tumor microenvironment in term of proliferation, glucose metabolism and hypoxia. Conclusions: Both macroscopic xenografts and micrometastases of NSCLC have spatial heterogeneity of tumor microenvironment. Temporal change of tumor microenvironment did occur in a very short of interval of natural growth process in NSCLC. We have investigated to spatial and temporal behavior of heterogeneity of tumor microenvironment which is important for better understanding cancer biology and cancer management, if our findings in mice models are clinically applicable. Acknowledgement: This study was supported by Kentucky Lung Cancer Research Program Award to Dr. Xiao-Feng Li. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2439. doi:1538-7445.AM2012-2439

Targeting Free Prostate-Specific Antigen forIn VivoImaging of Prostate Cancer Using a Monoclonal Antibody Specific for Unique Epitopes Accessible on Free Prostate-Specific Antigen Alone

This study investigated the feasibility of targeting the free, unbound forms of prostate-specific antigen (fPSA) for in vivo imaging of prostate adenocarcinomas (PCa), as PSA is produced and secreted at abundance during every clinical stage and grade of PCa, including castration-resistant disease. We injected (125)I-labeled monoclonal antibody PSA30 (specific for an epitope uniquely accessible on fPSA alone) intravenously in male nude mice carrying subcutaneous xenografts of LNCaP tumors (n=36). Mice were sacrificed over a time course from 4 hours to 13 days after injecting (125)I-labeled PSA30. Tissue uptake of (125)I-PSA30 at 48 and 168 hours after intravenous injection was compared with two clinically used positron emission tomography radiopharmaceuticals, (18)F-fluoro-deoxy-glucose ((18)F-FDG) or (18)F-choline, in cryosections using Digital AutoRadiography (DAR) and also compared with immunohistochemical staining of PSA and histopathology. On DAR, the areas with high (125)I-PSA30 uptake corresponded mainly to morphologically intact and PSA-producing LNCaP cells, but did not associate with the areas of high uptake of either (18)F-FDG or (18)F-choline. Biodistribution of (125)I-PSA30 measured in dissected organs ex vivo during 4 to 312 hours after intravenous injection demonstrated maximum selective tumor uptake 24-48 hours after antibody injection. Our data showed selective uptake in vivo of a monoclonal antibody highly specific for fPSA in LNCaP cells. Hence, in vivo imaging of fPSA may be feasible with putative usefulness in disseminated PCa.

Micro-analytical characterisation of radioactive heterogeneities in samples from Central Asian TENORM sites

The present work focuses on the use of micro-analytical techniques to demonstrate the heterogeneous distribution of radionuclides and metals in soils collected at Former Soviet Union mining sites in Central Asia. Based on digital autoradiography, radionuclides were heterogeneously distributed in soil samples collected at the abandoned uranium mining sites Kurday, Kazakhstan, Kadji Sai, Kyrgyzstan and Taboshar, Tajikistan. Using electron microscopy interfaced with X-ray microanalysis submicron – mm-sized radioactive particles and rock fragments with U, As, Se and toxic metals on the surfaces were identified in Kurday and Kadji Sai samples. Employing scanning and tomographic (3D) synchrotron radiation based micro-X-ray fluorescence (μ-SRXRF) and synchrotron radiation based micro-X-ray diffraction (μ-SRXRD) allowed us to observe the inner structure of the particles without physical sectioning. The distribution of elements in virtual crosssections demonstrated that U and a series of toxic elements were rather heterogeneously distributed also within individual radioactive TENORM particles. Compared to archived data, U in Kadji Sai particles was present as uraninite (U4O9+y or UO2+x) or Na-zippeite ((Na4(UO2)6[(OH)10(SO4)3]·4H2O), i.e. U minerals with very low solubility. The results suggested that TENORM particles can carry substantial amount of radioactivity, which can be subject to re-suspension, atmospheric transport and water transport. Thus, the potential radioecological and radioanalytical impact of radioactive particles at NORM and TENORM sites worldwide should be taken into account. The present work also demonstrates that radioecological studies should benefit from the use of advanced methods such as synchrotron radiation based techniques.

Small-Animal PET Imaging of Amyloid-Beta Plaques with [11C]PiB and Its Multi-Modal Validation in an APP/PS1 Mouse Model of Alzheimer's Disease

In vivo imaging and quantification of amyloid-β plaque (Aβ) burden in small-animal models of Alzheimer's disease (AD) is a valuable tool for translational research such as developing specific imaging markers and monitoring new therapy approaches. Methodological constraints such as image resolution of positron emission tomography (PET) and lack of suitable AD models have limited the feasibility of PET in mice. In this study, we evaluated a feasible protocol for PET imaging of Aβ in mouse brain with [11C]PiB and specific activities commonly used in human studies. In vivo mouse brain MRI for anatomical reference was acquired with a clinical 1.5 T system. A recently characterized APP/PS1 mouse was employed to measure Aβ at different disease stages in homozygous and hemizygous animals. We performed multi-modal cross-validations for the PET results with ex vivo and in vitro methodologies, including regional brain biodistribution, multi-label digital autoradiography, protein quantification with ELISA, fluorescence microscopy, semi-automated histological quantification and radioligand binding assays. Specific [11C]PiB uptake in individual brain regions with Aβ deposition was demonstrated and validated in all animals of the study cohort including homozygous AD animals as young as nine months. Corresponding to the extent of Aβ pathology, old homozygous AD animals (21 months) showed the highest uptake followed by old hemizygous (23 months) and young homozygous mice (9 months). In all AD age groups the cerebellum was shown to be suitable as an intracerebral reference region. PET results were cross-validated and consistent with all applied ex vivo and in vitro methodologies. The results confirm that the experimental setup for non-invasive [11C]PiB imaging of Aβ in the APP/PS1 mice provides a feasible, reproducible and robust protocol for small-animal Aβ imaging. It allows longitudinal imaging studies with follow-up periods of approximately one and a half years and provides a foundation for translational Alzheimer neuroimaging in transgenic mice.

99mTc-Labeled Superparamagnetic Iron Oxide Nanoparticles for Multimodality SPECT/MRI of Sentinel Lymph Nodes

The purpose of this study was to develop multimodality SPECT/MRI contrast agents for sentinel lymph node (SLN) mapping in vivo. Nanoparticles with a solid iron oxide core and a polyethylene glycol coating were labeled with (99m)Tc. The labeling efficiency was determined with instant thin-layer chromatography and magnetic separation. The stability of the radiolabeled superparamagnetic iron oxide nanoparticles (SPIONs) was verified in both sterile water and human serum at room temperature 6 and 24 h after labeling. Five Wistar rats were injected subcutaneously in the right hind paw with (99m)Tc-SPIONs (25-50 MBq, ∼0.2 mg of Fe) and sacrificed 4 h after injection. Two animals were imaged with SPECT/MRI. All 5 rats were dissected; the lymph nodes, liver, kidneys, spleen, and hind paw containing the injection site were removed and weighed; and activity in the samples was measured. The microdistribution within the lymph nodes was studied with digital autoradiography. The efficiency of labeling of the SPIONs was 99% 6 h after labeling in both water and human serum. The labeling yield was 98% in water and 97% in human serum 24 h after labeling. The SLN could be identified in vivo with SPECT/MRI. The accumulation of (99m)Tc-SPIONs (as the percentage injected dose/g [%ID/g]) in the SLN was 100 %ID/g, whereas in the liver and spleen it was less than 2 %ID/g. Digital autoradiography images revealed a nonhomogeneous distribution of (99m)Tc-SPIONs within the lymph nodes; nanoparticles were found in the cortical, subcapsular, and medullary sinuses. This study revealed the feasibility of labeling SPIONs with (99m)Tc. The accumulation of (99m)Tc-SPIONs in lymph nodes after subcutaneous injection in animals, verified by SPECT/MRI, is encouraging for applications in breast cancer and malignant melanoma.

A Dual-radiolabel Marker Quantifies Decrease in HT29 Xenograft Hypoxia Induced by Mild Temperature Hyperthermia

Purpose: In this project, we developed novel methods to quantify changes in tumor hypoxia following a mild tempera-ture hyperthermia (MTH) treatment in rat HT29 human colon adenocarcinoma xenograft. Materials and Methods: An exogenous hypoxia marker (IAZGP) was labeled with two radioisotopes of iodine (131I and 123I, respectively) to form two distinct tracers. The two tracers were injected into HT29-bearing nude rats 4-hour before and immediately following 41.5℃, 45-minute mild hyperthermia treatment. The distributions of the two hypoxia tracers were obtained by performing digital autoradiography on tumor sections, and image processing resulted in quantitative information at 50 μm pixel size. Results: Following the hyperthermia treatment, there was a remarkable decrease in hypoxia tracer binding. The average whole tumor hypoxia tracer targeted fraction in five animals changed from 30.3% ± 9.7% to 13.0% ± 5.3% after the hyperthermia treatment (P = 0.001). Detailed pixelby-pixel analysis of the image data revealed a decline in hypoxia tracer uptake after hyperthermia in most regions. However, there was concomitant emergence of some new regions of hypoxia identified by increased tracer uptake. In the control group, the overall hypoxia tracer targeted fraction remained almost constant, with some hypoxic tracer redistribution (putative acute hypoxia) observed. Conclusion: Reoxygenation occurred in the rat HT29 xenograft following MTH treatment. This was evident with preponderance of decreased hypoxia specific tracer uptake on tumor sections. Our methodology might be a useful tool in hypoxia study.

Chemical element imaging for speleothem geochemistry: Application to a uranium-bearing corallite with aragonite diagenesis to opal (Eastern Siberia, Russia)

Two complementary image-based microanalysis methods, digital autoradiography of radioactive elements and X-ray fluorescence microspectroscopy (micro-XRF), were used to study the geochemistry of a speleothem from Eastern Siberia. Our objective was to show the interest of these micro-chemical methods to investigate the distribution of U and some other elements as Ca, Sr, and Si in speleothems. The sample studied was a corallite concretion that formed slowly by the precipitation of minerals from moisture that had condensed underground. Polished sections of the sample revealed alternating laminas of gray calcite and white aragonite. The concretion, which is older than 400Kyr (U/Th-TIMS), showed no signs of detrital contamination (230Th/232Th>10,000). Digital autoradiography and XRF analyses indicated exceptionally high uranium contents, ranging from 3–10 to more than 1300μg∙g−1 in different areas of the sample. Element maps for calcium (Ca), uranium (U), strontium (Sr) and silicon (Si) showed inverse correlations between Ca and U and between Ca and Si, but there is a strong correlation between Si and U. Under the optical microscope, the low Ca appears as corrosion voids, which were found to contain uranium-enriched amorphous silica replacing areas of aragonite and, in some cases, calcite. This postgenetic amorphous and uraniferous silica is composed of two kinds of opal: opal A formed by large microspheres of 30–40μm in diameter and a gray opal CT with irregular lepispheres. This late opal, dated around 385–412Kyr, is probably due to a special volcanic event formed by uraniferous glass ashes easily soluble by meteoric waters, as suggested by the presence of volcanic suite in the fine fraction of the opal. Opal precipitated after speleothem crystallization by evaporation or cryogenic supersaturation. The study of this exceptional concretion shows the value of direct chemical imaging methods, as they easily revealed the presence and distribution of U in the concretion, and allowed an unusual case of opal diagenesis in a carbonate speleothem to be described. Given the value of speleothems as geo-chronometers and recorders of environmental changes at different time scales, from season to isotopic stage, this methodology opens new perspectives in understanding the geochemical system and consequently the validity of radiometric datings, U/Th and U/Pb, especially in speleothem samples.

Detection, Quantification, and Microlocalisation of Targets of Pesticides Using Microchannel Plate Autoradiographic Imagers

Organophosphorus (OP) compounds are a diverse chemical group that includes nerve agents and pesticides. They share a common chemical signature that facilitates their binding and adduction of acetylcholinesterase (AChE) within nerve synapses to induce cholinergic toxicity. However, this group diversity results in non-uniform binding and inactivation of other secondary protein targets, some of which may be adducted and protein activity influenced, even when only a relatively minor portion of tissue AChE is inhibited. The determination of individual OP protein binding targets has been hampered by the sensitivity of methods of detection and quantification of protein-pesticide adducts. We have overcome this limitation by the employment of a microchannel plate (MCP) autoradiographic detector to monitor a radiolabelled OP tracer compound. We preincubated rat thymus tissue in vitro with the OP pesticides, azamethiphos-oxon, chlorfenvinphos-oxon, chlorpyrifos-oxon, diazinon-oxon, and malaoxon, and then subsequently radiolabelled the free OP binding sites remaining with 3H-diisopropylfluorophosphate (3H-DFP). Proteins adducted by OP pesticides were detected as a reduction in 3H-DFP radiolabelling after protein separation by one dimensional polyacrylamide gel electrophoresis and quantitative digital autoradiography using the MCP imager. Thymus tissue proteins of molecular weights ~28 kDa, 59 kDa, 66 kDa, and 82 kDa displayed responsiveness to adduction by this panel of pesticides. The 59 kDa protein target (previously putatively identified as carboxylesterase I) was only significantly adducted by chlorfenvinphos-oxon (p < 0.001), chlorpyrifos-oxon (p < 0.0001), and diazinon-oxon (p < 0.01), the 66 kDa protein target (previously identified as serum albumin) similarly only adducted by the same three pesticides (p < 0.0001), (p < 0.001), and (p < 0.01), and the 82 kDa protein target (previously identified as acyl peptide hydrolase) only adducted by chlorpyrifos-oxon (p < 0.0001) and diazinon-oxon (p < 0.001), when the average values of tissue AChE inhibition were 30%, 35%, and 32% respectively. The ~28 kDa protein target was shown to be heterogeneous in nature and was resolved to reveal nineteen 3H-DFP radiolabelled protein spots by two dimensional polyacrylamide gel electrophoresis and MCP autoradiography. Some of these 3H-DFP proteins spots were responsive to adduction by preincubation with chlorfenvinphos-oxon. In addition, we exploited the useful spatial resolution of the MCP imager (~70 μm) to determine pesticide micolocalisation in vivo, after animal dosing and autoradiography of brain tissue sections. Collectively, MCP autoradiographic imaging provided a means to detect targets of OP pesticides, quantify their sensitivity of adduction relative to tissue AChE inhibition, and highlighted that these common pesticides exhibit specific binding character to protein targets, and therefore their toxicity will need to be evaluated on an individual compound basis. In addition, MCP autoradiography afforded a useful method of visualisation of the localisation of a small radiolabelled tracer within brain tissue.

Tritium analysis in building dismantling process using digital autoradiography

The aim of our study was to develop a sensitive analytical technique to investigate tritium traces on concrete floor, ceiling and walls of a laboratory under dismantling process. In dismantling process, the behaviour of tritium must be followed very closely and in particular releases to atmosphere and exposure to public must be prevented. The investigations were carried out using liquid scintillation counting (LSC) and digital autoradiography techniques. The LSC investigations were carried out using wipe tests. It was concluded that even though LSC technique provided accurate information on the level of contamination, the loss of exact localisation of contamination spots was considered to be a major disadvantage in this application. The digital autoradiography was shown to provide both quantitative information and localisation of contamination spots. The exposure time on the digital autoradiography films were optimized, the matrix effect and the selectivity of the films were studied using polymethylmethacrylate standards. The studies showed that the digital autoradiography technique can give quantitative results, especially if standards of the investigated material (concrete) are used.

Development of digital gamma-activation autoradiography for analysis of samples of large area

Abstract Gamma-activation autoradiography is a prospective method for screening detection of inclusions of precious metals in geochemical samples. Its characteristics allow analysis of thin sections of large size (tens of cm 2 ), that favourably distinguishes it among the other methods for local analysis. At the same time, the activating field of the accelerator bremsstrahlung, displays a sharp intensity decrease relative to the distance along the axis. A method for activation dose “equalization” during irradiation of the large size thin sections has been developed. The method is based on the usage of a hardware-software system. This includes a device for moving the sample during the irradiation, a program for computer modelling of the acquired activating dose for the chosen kinematics of the sample movement and a program for pixel-by pixel correction of the autoradiographic images. For detection of inclusions of precious metals, a method for analysis of the acquired dose dynamics during sample decay has been developed. The method is based on the software processing pixel by pixel a time-series of coaxial autoradiographic images and generation of the secondary meta-images allowing interpretation regarding the presence of interesting inclusions based on half-lives. The method is tested for analysis of copper-nickel polymetallic ores. The developed solutions considerably expand the possible applications of digital gamma-activation autoradiography.

Enhancement of digital gamma activation autoradiography capabilities by means of computer analysis of the time series images

It has been demonstrated that pixel-by pixel processing of series of autoradiography images for revealing the dynamics of decay of the induced radionuclides is an efficient approach for mapping of radionuclides in the sample in activation autoradiography. Concepts of virtual scanner and corresponding software for linearization of dependence of optical density on scanner response (luminosity) have been introduced. The concept provides unification of the subsequent processing of autoradiograms, irrespective of the method as to how the digital image has been obtained. Algorithms and the software for estimation of decay parameters of a radionuclide mixture for each pixel using a series of coaxially positioned images have been developed. The software is able generate a set of the derivative meta-images allowing a conclusion to be made about the presence of the inclusions in question. To increase the reliability of radionuclide mapping it is suggested to use analysis of distribution of half-life values estimated for pixels of image zone(s) pointed by a special mask.

A proposal for phosphor imager acceptance testing procedure and routine quality controls in nuclear pharmacy practice

The digital autoradiography system is currently used in nuclear medicine for quantitative imaging of radioactivity distribution (thin layer chromatography samples, tissue sections, and cell cultures). The aim of this study was to define a set of tests for setting up a specific acceptance testing procedure and routine quality controls for this instrument. Over a 3-month period, we analyzed the active components of the instrument (phosphor screen and photometer) by using suitable self-manufactured equipment (phantoms, lead plate, lead cylinder, and photographic paper) required to realize, in a routine quality program, the following tests: integral uniformity (IU) and differential uniformity (DU) in a useful field of view (UFOV) and a central field of view (CFOV), resolution, geometric linearity, and sensitivity. Screen IU was 19.7 ± 2.3% (UFOV) and 11.1 ± 3.7% (CFOV). Screen DU ranged between 1.6 ± 1.1 and 1.8 ± 0.9% for UFOV and between 1.2 ± 0.4 and 1.4 ± 0.6% for CFOV. Screen resolution measured as full-width at half-maximum was 1.94 ± 0.08 mm. Screen sensitivity was 505.1 ± 10.4 digital light units and ranged between -3.15 and +3.49% with reference to the mean of measured values. Photometer IU was 17.4 ± 0.2% (UFOV) and 13.7 ± 1.1% (CFOV). Photometer DU ranged between 1.9 ± 0.9 and 2.3 ± 1.2% for UFOV and between 1.9 ± 0.8 and 2.1 ± 1.1% for CFOV. Photometer resolution was good (full-width at half-maximum =0.5 ± 0.076 mm). Our results suggest that the methodology we propose could be an easy, accurate, quick, and low-cost tool to guarantee the correct instrument basic function.

SU-F-BRA-01: Object-Based Analysis of Spatial Concordance Between Coregistered Images of Intratumoral Uptake of [18F]FLT and Distribution of Cell Proliferation Markers

Purpose: To propose a new objective method for deformable coregistration of multimodality images acquired with digital autoradiography (DAR) and microscopy in the context of PET tracer histopathological validation. To analyze the spatial concordance between the uptake pattern of 18F-fluorothymidine (FLT) as imaged with DAR and the distribution of cell proliferation as revealed by immunofluorescence microscopy imaging.Methods: Tumor-bearing mice were injected with FLT and other markers including bromodeoxyuridine (cell proliferation). After sacrifice, tumors were excised, frozen and sectioned. Multiple stacks of sequential 8μm sections were collected from each tumor. Selected sections were used for DAR to image FLT uptake distribution. Adjacent sections were used to acquire histopathological data. To correct for imperfections of the tissue cutting and collection, all images were deformably coregistered to the FLT DAR image based on biological images of tumor blood flow (Hoechst) that was acquired from each tissue section used. For each FLT DAR — cell proliferation microscopy image pair, object-based analysis was conducted, including overlap and relative operating characteristics (ROC) analysis. Results: Total registration error of proposed coregistration method was 44.86μm. This supersedes current rigid registration methods with reported errors of 100–200μm. In tumors with well-compartmentalized functional aspects, area under the ROC curve (AUCroc= 0.7) indicated FLT DAR image thresholding as an accurate method of detecting cell proliferation. For these tumors, Dice overlap index indicated maximum detection rates at thresholds between 20% and 40% of the maximum DAR intensity. For the tumors characterized by more heterogeneous distribution of cell proliferation across the tumor section, FLT DAR image thresholding could not predict cell proliferation beyond random chance. Conclusions: We developed a comprehensive method of obtaining and analyzing coregistered images of cell proliferation markers and intratumoral uptake of FLT. Tumor microenvironment heterogeneity is a significant factor affecting the utility of FLT for imaging cell proliferation.

Correlation of In Vivo and In Vitro Measures of Carbonic Anhydrase IX Antigen Expression in Renal Masses Using Antibody 124I-cG250

The goal of this study was to determine whether there is a potential correlation between quantification of radiolabeled macromolecular uptake in tumors determined in vivo using PET/CT and in vitro using autoradiography and γ-counting of tumor tissue. Twenty-six patients with renal masses scheduled for surgical resection received (124)I-labeled antibody cG250. Tumor specimens obtained from resection were studied. Fifteen of these patients had clear cell cancer demonstrated by positive findings on PET/CT images and histopathology. Radioactivity in tumors was measured on PET/CT images and expressed as percentage injected dose per gram. These values were then normalized to measurements of known serum radioactivity from a venous blood sample obtained at the time of PET/CT. Comparable measurements were obtained in vitro using γ-well counting and digital autoradiography of tumor tissue. There was a significant correlation between tumor radioactivity estimated in vivo and in vitro (Spearman correlation coefficient comparing normalized PET measurements with well counting of 0.84, P < 0.000001, and with autoradiography of 0.88, P < 0.000001). PET/CT measurements of tumor uptake were lower than measurements obtained with either of the in vitro methods, and digital autoradiography resulted in the highest measurements. PET/CT can be reliably used to quantify radiolabeled macromolecular uptake in vivo, suggesting important implications for quantitative pharmacokinetic estimates of macromolecular biodistribution.

14C autoradiography with an energy-sensitive silicon pixel detector

The first performance tests are presented of a carbon-14 (14C) beta-particle digital autoradiography system with an energy-sensitive hybrid silicon pixel detector based on the Timepix readout circuit. Timepix was developed by the Medipix2 Collaboration and it is similar to the photon-counting Medipix2 circuit, except for an added time-based synchronization logic which allows derivation of energy information from the time-over-threshold signal. This feature permits direct energy measurements in each pixel of the detector array. Timepix is bump-bonded to a 300 µm thick silicon detector with 256 × 256 pixels of 55 µm pitch. Since an energetic beta-particle could release its kinetic energy in more than one detector pixel as it slows down in the semiconductor detector, an off-line image analysis procedure was adopted in which the single-particle cluster of hit pixels is recognized; its total energy is calculated and the position of interaction on the detector surface is attributed to the centre of the charge cluster. Measurements reported are detector sensitivity, (4.11 ± 0.03) × 10−3 cps mm−2 kBq−1 g, background level, (3.59 ± 0.01) × 10−5 cps mm−2, and minimum detectable activity, 0.0077 Bq. The spatial resolution is 76.9 µm full-width at half-maximum. These figures are compared with several digital imaging detectors for 14C beta-particle digital autoradiography.

Energy sensitive Timepix silicon detector for electron imaging

We present the first measurements with the energy sensitive Timepix pixel detector for electron imaging. The hybrid pixel detector consists of a silicon detector, 300 μm thick, bump-bonded to the Timepix readout chip developed by the Medipix2 collaboration (256×256 pixels, 55 μm pitch, 14.08×14.08 mm 2 sensitive area). Each Timepix pixel can be independently operated in one of three modes: 1. counting of the detected particles; 2. measurement of the particle energy; 3. measurement of the time of particle interaction. The energy measurement in the second mode is performed via the determination of the “Time-Over-Threshold” (TOT). The ionization charge generated by the particle along its track is often registered by several adjacent pixels forming a cluster. The shape of the cluster is affected also by lateral charge spread. It is often possible to determine a particle type, its energy, entrance point and direction by online or offline analysis of shapes of recorded clusters. This way an influence of background or noise can be significantly reduced in measured data. The energy spectrum for 90 Sr/ 90 Y electrons was measured by Timepix detector and compared with the β − decay spectrum and the Monte Carlo simulated spectrum. In order to improve spatial resolution, we analyzed the tracks of all electrons and substituted each cluster with the position of its centroid. We measured the spatial resolution with a 90 Sr/ 90 Y source irradiating at 10 cm distance, a 100 μm thick steel edge slightly tilted with respect to the detector lines. The oversampled Line Spread Function shows an FWHM of 27.5±1.1 μm. The Timepix Si detector will be used for digital autoradiography with β − and β + tracers, and it could be used for electron microscopy . First tests were performed with a 14 C autoradiography sample.

Renal uptake of bismuth-213 and its contribution to kidney radiation dose following administration of actinium-225-labeled antibody

Clinical therapeutic studies using 225Ac-labeled antibodies have begun. Of major concern is renal toxicity that may result from the three alpha-emitting progeny generated following the decay of 225Ac. The purpose of this study was to determine the amount of 225Ac and non-equilibrium progeny in the mouse kidney after the injection of 225Ac-huM195 antibody and examine the dosimetric consequences. Groups of mice were sacrificed at 24, 96 and 144 h after injection with 225Ac-huM195 antibody and kidneys excised. One kidney was used for gamma ray spectroscopic measurements by a high-purity germanium (HPGe) detector. The second kidney was used to generate frozen tissue sections which were examined by digital autoradiography (DAR). Two measurements were performed on each kidney specimen: (1) immediately post-resection and (2) after sufficient time for any non-equilibrium excess 213Bi to decay completely. Comparison of these measurements enabled estimation of the amount of excess 213Bi reaching the kidney (γ-ray spectroscopy) and its sub-regional distribution (DAR). The average absorbed dose to whole kidney, determined by spectroscopy, was 0.77 (SD 0.21) Gy kBq−1, of which 0.46 (SD 0.16) Gy kBq−1 (i.e. 60%) was due to non-equilibrium excess 213Bi. The relative contributions to renal cortex and medulla were determined by DAR. The estimated dose to the cortex from non-equilibrium excess 213Bi (0.31 (SD 0.11) Gy kBq−1) represented ∼46% of the total. For the medulla the dose contribution from excess 213Bi (0.81 (SD 0.28) Gy kBq−1) was ∼80% of the total. Based on these estimates, for human patients we project a kidney-absorbed dose of 0.28 Gy MBq−1 following administration of 225Ac-huM195 with non-equilibrium excess 213Bi responsible for approximately 60% of the total. Methods to reduce renal accumulation of radioactive progeny appear to be necessary for the success of 225Ac radioimmunotherapy.

The α-Camera: A Quantitative Digital Autoradiography Technique Using a Charge-Coupled Device for Ex Vivo High-Resolution Bioimaging of α-Particles

Bioconjugates used in internal radiotherapy exhibit heterogeneous distributions in organs and tumors, implying a risk of nonuniform dose distribution in therapeutic applications using α-particle emitters. Tools are required that provide data on the activity distribution for estimation of absorbed dose on a suborgan level. The α-camera is a quantitative imaging technique developed to detect α-particles in tissues ex vivo. The aim of this study was to evaluate the characteristics of this imaging system and to exemplify its potential use in the development of α-radioimmunotherapy. The α-camera combines autoradiography with a scintillating technique and optical registration by a charge-coupled device (CCD). The imaging system characteristics were evaluated by measurements of linearity, uniformity, and spatial resolution. The technique was applied for quantitative imaging of (211)At activity distribution in cryosections of tumors, kidney, and whole body. Intratumoral activity distributions of tumor-specific (211)At-MX35-F(ab')(2) were studied at various times after injection. The postinjection activity distributions in the renal cortex and whole kidneys were compared for (211)At-F(ab')(2) and (211)At-IgG trastuzumab. Quantitative analysis of α-camera images demonstrated that the pixel intensity increased linearly with activity in the imaged specimen. The spatial resolution was 35 ± 11 μm (mean ± SD) and the uniformity better than 2%. Kidney cryosections revealed a higher cortex-to-whole kidney ratio for (211)At-F(ab')(2) than for (211)At-IgG (1.38 ± 0.03 and 0.77 ± 0.04, respectively) at 2 h after injection. Nonuniform intratumoral activity distributions were found for tumor-specific (211)At-MX35-F(ab')(2) at 10 min and 7 h after injection; after 21 h, the distribution was more uniform. The characteristics of the α-camera are promising, suggesting that this bioimaging system can assist the development, evaluation, and refinement of future targeted radiotherapy approaches using α-emitters. The α-camera provides quantitative data on the activity distribution in tissues on a near-cellular scale and can therefore be used for small-scale dosimetry, improving the prediction of biologic outcomes with α-particles with short path length and high linear energy transfer.

PET imaging of inflammation and adenocarcinoma xenografts using vascular adhesion protein 1 targeting peptide 68Ga-DOTAVAP-P1: comparison with 18F-FDG

The aim of this study was to evaluate inflammation and tumour imaging with a vascular adhesion protein 1 (VAP-1) targeting peptide (68)Ga-DOTAVAP-P1 in comparison with (18)F-FDG. Rats with both subcutaneous human pancreatic adenocarcinoma xenografts and turpentine oil-induced acute sterile inflammation were evaluated by dynamic positron emission tomography (PET) and by digital autoradiography of tissue cryosections. Subsequently, the autoradiographs were combined with histological and immunohistological analysis of the sections. (68)Ga-DOTAVAP-P1 delineated acute, sterile inflammation comparable with (18)F-FDG. However, the tumour uptake of (68)Ga-DOTAVAP-P1 was low in contrast to prominent (18)F-FDG uptake. The standardised uptake values of inflammation and tumours by PET were 1.1 +/- 0.4 (mean +/- SEM) and 0.4 +/- 0.1 for (68)Ga-DOTAVAP-P1 and 2.0 +/- 0.5 and 1.6 +/- 0.8 for (18)F-FDG, respectively. In addition, PET studies showed inflammation to muscle and tumour to muscle ratios of 5.1 +/- 3.1 and 1.7 +/- 0.3 for (68)Ga-DOTAVAP-P1 and 6.2 +/- 0.7 and 4.6 +/- 2.2 for (18)F-FDG, respectively. Immunohistochemistry revealed increased expression of luminal VAP-1 on the endothelium at the site of inflammation and low expression in the tumour The (68)Ga-DOTAVAP-P1 PET was able to visualise inflammation better than tumour, which was in accordance with the luminal expression of VAP-1 on vasculature in these experimental models.