Investigation of imaging the somatostatin receptor by opening the blood-brain barrier with melittin - A feasibility study using positron emission tomography and [64Cu]Cu-DOTATATE.

DOTATATE is a somatostatin peptide analog used in the clinic to detect somatostatin receptors which are highly expressed on neuroendocrine tumors. Somatostatin receptors are found naturally in the intestines, pancreas, lungs, and brain (mainly cortex). In vivo measurement of the somatostatin receptors in the cortex has been challenging because available tracers cannot cross the blood-brain barrier (BBB) due to their intrinsic polarity. A peptide called melittin, a main component of honeybee venom, has been shown to disrupt plasma membranes and increase the permeability of biological membranes. In this study, we assessed the feasibility of using melittin to facilitate the passage of [64Cu]Cu-DOTATATE through the BBB and its binding to somatostatin receptors in the cortex. Evaluation included in vitro autoradiography on Long Evans rat brains to estimate the binding affinity of [64Cu]Cu-DOTATATE to the somatostatin receptors in the cortex and an in vivo evaluation of [64Cu]Cu-DOTATATE binding in NMRI mice after injection of melittin. This study found an in vitro Bmax = 89 ± 4 nM and KD = 4.5 ± 0.6 nM in the cortex, resulting in a theoretical binding potential (BP) calculated as Bmax/KD ≈ 20, which is believed suitable for in vivo brain PET imaging. However, the in vivo results showed no significant difference between the control and melittin injected mice, indicating that the honeybee venom failed to open the BBB. Additional experiments, potentially involving faster injection rates are required to verify that melittin can increase brain uptake of non-BBB permeable PET tracers. Furthermore, an evaluation of whether a venom with a narrow therapeutic range can be used for clinical purposes needs to be considered.

Targeted imaging of uPAR expression in vivo with cyclic AE105 variants.

A comprehensive literature reports on the correlation between elevated levels of urokinase-type plasminogen activator receptor (uPAR) and the severity of diseases with chronic inflammation including solid cancers. Molecular imaging is widely used as a non-invasive method to locate disease dissemination via full body scans and to stratify patients for targeted treatment. To date, the only imaging probe targeting uPAR that has reached clinical phase-II testing relies on a high-affinity 9-mer peptide (AE105), and several studies by positron emission tomography (PET) scanning or near-infra red (NIR) fluorescence imaging have validated its utility and specificity in vivo. While our previous studies focused on applying various reporter groups, the current study aims to improve uPAR-targeting properties of AE105. We successfully stabilized the small uPAR-targeting core of AE105 by constraining its conformational landscape by disulfide-mediated cyclization. Importantly, this modification mitigated the penalty on uPAR-affinity typically observed after conjugation to macrocyclic chelators. Cyclization did not impair tumor targeting efficiency of AE105 in vivo as assessed by PET imaging and a trend towards increased tracer uptake was observed. In future studies, we predict that this knowledge will aid development of new fluorescent AE105 derivatives with a view to optical imaging of uPAR to assist precision guided cancer surgery.

A nanobody recognizes a unique conserved epitope and potently neutralizes SARS-CoV-2 omicron variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) Omicron variant sub-lineages spread rapidly worldwide, mostly due to their immune-evasive properties. This has put a significant part of the population at risk for severe disease and underscores the need for effective anti-SARS-CoV-2 agents against emergent strains in vulnerable patients. Camelid nanobodies are attractive therapeutic candidates due to their high stability, ease of large-scale production, and potential for delivery via inhalation. Here, we characterize the receptor binding domain (RBD)-specific nanobody W25 and show superior neutralization activity toward Omicron sub-lineages in comparison to all other SARS-CoV2 variants. Structure analysis of W25 in complex with the SARS-CoV2 spike glycoprotein shows that W25 engages an RBD epitope not covered by any of the antibodies previously approved for emergency use. In vivo evaluation of W25 prophylactic and therapeutic treatments across multiple SARS-CoV-2 variant infection models, together with W25 biodistribution analysis in mice, demonstrates favorable pre-clinical properties. Together, these data endorse W25 for further clinical development.

Neoadjuvant Gold Nanoshell-Based Photothermal Therapy Combined with Liposomal Doxorubicin in a Mouse Model of Colorectal Cancer.

Introduction: Traditional cancer treatments, such as chemotherapy, are often incapable of achieving complete responses as standalone therapies. Hence, current treatment strategies typically rely on a combination of several approaches. Nanoparticle-based photothermal therapy (PTT) is a technique used to kill cancer cells through localized, severe hyperthermia that has shown promise as an add-on treatment to multiple cancer therapies. Here, we evaluated whether the combination of gold nanoshell (NS)-based PTT and liposomal doxorubicin could improve outcome in a mouse model of colorectal cancer. Methods: First, NS-based PTT was performed on tumor-bearing mice. Radiolabeled liposomes were then injected at different timepoints to follow their accumulation in the tumor and determine the ideal injection time after PTT. In addition, fluorescent liposomes were used to observe the liposomal distribution in the tumor after PTT. Finally, we combined PTT and doxorubicin-loaded liposomes and studied the effect of the treatment strategy on the mice by following tumor growth and survival. Results: PTT significantly improved liposomal accumulation in the tumor, but only when the liposomes were injected immediately after the therapy. The liposomes accumulated mostly in regions adjacent to the ablated areas. When PTT was combined with liposomal doxorubicin, the mice experienced a slowdown in tumor growth and an improvement in survival. Conclusion: According to our preclinical study, NS-based PTT seems promising as an add-on treatment for liposomal chemotherapy and potentially other systemic therapies, and could be relevant for future application in a clinical setting.

Synthesis and radiolabeling of a polar [125 I]I-1,2,4,5-tetrazine.

Pretargeting imaging has gained a lot of prominence, due to its excellent bioorthogonality and improved imaging contrast compared to conventional imaging. A new iodo tetrazine (Tz) derivative has been synthesized and further developed into the corresponding iodine-125 (125 I) analog (12), via the trimethylstannane precursor. Radiolabeling with either N-chlorosuccinimide or chloramine-T, in either MeCN or MeOH proceeded with a radiochemical conversion (RCC) of >80%. Subsequent deprotection only proved successful, among the tested conditions, when the radiolabeled Tz was stirred in 6-M HCl(aq.) at 60°C for 2.5 h. To the best of our knowledge, this is the first H-tetrazine labeled with iodine. In vivo investigations on the pretargeting ability of 12 are currently under way.

Combination of [177Lu]Lu-DOTA-TATE Targeted Radionuclide Therapy and Photothermal Therapy as a Promising Approach for Cancer Treatment: In Vivo Studies in a Human Xenograft Mouse Model.

Peptide receptor radionuclide therapy (PRRT) relies on α- and ß-emitting radionuclides bound to a peptide that commonly targets somatostatin receptors (SSTRs) for the localized killing of tumors through ionizing radiation. A Lutetium-177 (177Lu)-based probe linked to the somatostatin analog octreotate ([177Lu]Lu-DOTA-TATE) is approved for the treatment of certain SSTR-expressing tumors and has been shown to improve survival. However, a limiting factor of PRRT is the potential toxicity derived from the high doses needed to kill the tumor. This could be circumvented by combining PRRT with other treatments for an enhanced anti-tumor effect. Photothermal therapy (PTT) relies on nanoparticle-induced hyperthermia for cancer treatment and could be a useful add-on to PRRT. Here, we investigate a strategy combining [177Lu]Lu-DOTA-TATE PRRT and nanoshell (NS)-based PTT for the treatment of SSTR-expressing small-cell lung tumors in mice. Our results showed that the combination treatment improved survival compared to PRRT alone, but only when PTT was performed one day after [177Lu]Lu-DOTA-TATE injection (one of the timepoints examined), showcasing the effect of treatment timing in relation to outcome. Furthermore, the combination treatment was well-tolerated in the mice. This indicates that strategies involving NS-based PTT as an add-on to PRRT could be promising and should be investigated further.

Development of 18F-Labeled Bispyridyl Tetrazines for In Vivo Pretargeted PET Imaging.

Pretargeted PET imaging is an emerging and fast-developing method to monitor immuno-oncology strategies. Currently, tetrazine ligation is considered the most promising bioorthogonal reaction for pretargeting in vivo. Recently, we have developed a method to 18F-label ultrareactive tetrazines by copper-mediated fluorinations. However, bispyridyl tetrazines-one of the most promising structures for in vivo pretargeted applications-were inaccessible using this strategy. We believed that our successful efforts to 18F-label H-tetrazines using low basic labeling conditions could also be used to label bispyridyl tetrazines via aliphatic nucleophilic substitution. Here, we report the first direct 18F-labeling of bispyridyl tetrazines, their optimization for in vivo use, as well as their successful application in pretargeted PET imaging. This strategy resulted in the design of [18F]45, which could be labeled in a satisfactorily radiochemical yield (RCY = 16%), molar activity (Am = 57 GBq/µmol), and high radiochemical purity (RCP > 98%). The [18F]45 displayed a target-to-background ratio comparable to previously successfully applied tracers for pretargeted imaging. This study showed that bispyridyl tetrazines can be developed into pretargeted imaging agents. These structures allow an easy chemical modification of 18F-labeled tetrazines, paving the road toward highly functionalized pretargeting tools. Moreover, bispyridyl tetrazines led to near-instant drug release of iTCO-tetrazine-based 'click-to-release' reactions. Consequently, 18F-labeled bispyridyl tetrazines bear the possibility to quantify such release in vivo in the future.

Photothermal Therapy as Adjuvant to Surgery in an Orthotopic Mouse Model of Human Fibrosarcoma.

Surgery is still the first-line treatment for multiple solid cancers. However, recurrence is a common issue, especially when dealing with aggressive tumors or tumors that are difficult to completely remove due to their location. Getting clear surgical margins can be challenging, but treatment strategies combining surgery with other anti-cancer therapies can potentially improve the outcome. Photothermal therapy (PTT) is a technique that relies on photoabsorbing agents, such as gold nanoparticles, to transform light into local hyperthermia. This technique can be used to ablate tumor tissue where the photoabsorbing agent accumulates, sparing healthy surrounding tissue. In this study, we examined the potential of gold nanoparticle-based PTT as an adjuvant treatment to surgery in a mouse model of human fibrosarcoma. For this we performed subtotal tumor resection to mimic a clinical situation where total tumor removal is not achieved, and subsequent PTT was applied on the surgical field. Our results showed that animals undergoing adjuvant PTT after surgery presented sustained delayed tumor growth and improved survival when compared to animals that only underwent surgery. We believe that these findings show the potential of PTT as an adjuvant method to traditional tumor surgery and could pave way to more personalized treatment options.

The use of a uPAR-targeted probe for photothermal cancer therapy prolongs survival in a xenograft mouse model of glioblastoma.

The development of tumor-targeted probes that can efficiently reach cancerous tissue is an important focus of preclinical research. Photothermal cancer therapy (PTT) relies on light-absorbing molecules, which are directed towards tumor tissue and irradiated with an external source of light. This light is transformed into heat, causing localized hyperthermia and tumor death. The fluorescent probe indocyanine green (ICG) is already used as an imaging agent both preclinically and in clinical settings, but its use for PTT is yet to be fully exploited due to its short retention time and non-specific tumor targeting. Therefore, increasing ICG tumor uptake is necessary to improve treatment outcome. The urokinase-type plasminogen activator receptor, uPAR, is overexpressed in multiple tumor types. ICG-Glu-Glu-AE105, consisting of the uPAR-targeting peptide AE105 conjugated to ICG, has shown great potential for fluorescence-guided surgery. In this study, ICG-Glu-Glu-AE105 was evaluated as photothermal agent in a subcutaneous mouse model of human glioblastoma. We observed that the photothermal abilities of ICG-Glu-Glu-AE105 triggered high temperatures in the tumor during PTT, leading to tumor death and prolonged survival. This confirms the potential of ICG-Glu-Glu-AE105 as photothermal agent and indicates that it could be used as an add-on to the application of the probe for fluorescence-guided surgery.

18F-FDG positron emission tomography and diffusion-weighted magnetic resonance imaging for response evaluation of nanoparticle-mediated photothermal therapy.

Nanoparticle-mediated photothermal cancer therapy (PTT) is a treatment which creates localized damage to tumors via nanoparticles that generate heat when irradiated with near infrared light. Substantial work has been dedicated to developing efficient heat-transducing nanoparticles that can be delivered systemically to the tumor. However, less attention has been given to clinically relevant assessment methods of treatment outcome that could be used for personalizing the therapy. Here, we compare 18F-FDG positron emission tomography combined with computed tomography (PET/CT) and diffusion-weighted imaging (DWI) for early evaluation and prognosis of PTT in tumor-bearing mice using silica-gold nanoshells (NS). The NS-treated mice experienced inhibited tumor growth and significantly prolonged survival compared to control mice. One day after PTT, PET/CT and DWI scans showed a decrease in tumor 18F-FDG uptake of ~90% and an increase of ~50% in apparent diffusion coefficient (ADC) values respectively, compared to baseline. No significant changes were observed for control groups. Additionally, the changes in 18F-FDG uptake and ADC values correlated significantly with survival, demonstrating that both methods can be used for early evaluation of PTT although 18F-FDG PET/CT showed the strongest prognostic value. Based on these results, both modalities should be considered for therapy monitoring of PTT when clinically translated.

Fractionated photothermal therapy in a murine tumor model: comparison with single dose.

Purpose: Photothermal therapy (PTT) exploits the light-absorbing properties of nanomaterials such as silica-gold nanoshells (NS) to inflict tumor death through local hyperthermia. However, in in vivo studies of PTT, the heat distribution is often found to be heterogeneous throughout the tumor volume, which leaves parts of the tumor untreated and impairs the overall treatment outcome. As this challenges PTT as a one-dose therapy, this study here investigates if giving the treatment repeatedly, ie, fractionated PTT, increases the efficacy in mice bearing subcutaneous tumors. Methods: The NS heating properties were first optimized in vitro and in vivo. Two fractionated PTT protocols, consisting of two and four laser treatments, respectively, were developed and applied in a murine subcutaneous colorectal tumor model. The efficacy of the two fractionated protocols was evaluated both by longitudinal monitoring of tumor growth and, at an early time point, by positron emission tomography (PET) imaging of 18F-labeled glucose analog 18F-FDG. Results: Overall, there were no significant differences in tumor growth and survival between groups of mice receiving single-dose PTT and fractionated PTT in our study. Nonetheless, some animals did experience inhibited tumor growth or even complete tumor disappearance due to fractionated PTT, and these animals also showed a significant decrease in tumor uptake of 18F-FDG after therapy. Conclusion: This study only found an effect of giving PTT to tumors in fractions compared to a single-dose approach in a few animals. However, many factors can affect the outcome of PTT, and reliable tools for optimization of treatment protocol are needed. Despite the modest treatment effect, our results indicate that 18F-FDG PET/CT imaging can be useful to guide the number of treatment sessions necessary.

Non-invasive Early Response Monitoring of Nanoparticle-assisted Photothermal Cancer Therapy Using 18F-FDG, 18F-FLT, and 18F-FET PET/CT Imaging.

Rationale: Since its first implementation nanoparticle-assisted photothermal cancer therapy has been studied extensively, although mainly with focus on optimal nanoparticle design. However, development of efficient treatment protocols, as well as reliable and early evaluation tools in vivo, are needed to push the therapy towards clinical translation. Positron emission tomography (PET) is a non-invasive imaging technique that is currently finding extensive use for early evaluation of cancer therapies; an approach that has become of increasing interest due to its great potential for personalized medicine. Methods: In this study, we performed PET imaging to evaluate the treatment response two days after nanoparticle-assisted photothermal cancer therapy in tumor-bearing mice. We used three different tracers; 2'-deoxy-2'-18F-fluoro-D-glucose (18F-FDG), 3'-deoxy-3'-18F-fluorothymidine (18F-FLT), and O-(2'-18F-fluoroethyl)-L-tyrosine (18F-FET) to image and measure treatment induced changes in glucose uptake, cell proliferation, and amino acid transport, respectively. After therapy, tumor growth was monitored longitudinally until endpoint was reached. Results: We found that nanoparticle-assisted photothermal therapy overall inhibited tumor growth and prolonged survival. All three PET tracers had a significant decrease in tumor uptake two days after therapy and these changes correlated with future tumor growth, with 18F-FDG having the most predictive value in this tumor model. Conclusion: This study shows that 18F-FDG, 18F-FLT, and 18F-FET are all robust markers for the treatment response of photothermal therapy, and demonstrate that PET imaging can be used for stratification and optimization of the therapy. Furthermore, having a selection of PET tracers that can reliably measure treatment response is highly valuable as the individual tracer might be excluded in certain applications where physiological processes limit their contrast to background.

Liposome accumulation in irradiated tumors display important tumor and dose dependent differences.

Radiation therapy may affect several important parameters in the tumor microenvironment and thereby influence the accumulation of liposomes by the enhanced permeability and retention (EPR)-effect. Here we investigate the effect of single dose radiation therapy on liposome tumor accumulation by PET/CT imaging using radiolabeled liposomes. Head and neck cancer xenografts (FaDu) and syngenic colorectal (CT26) cancer models were investigated. Radiotherapy displayed opposite effects in the two models. FaDu tumors displayed increased mean accumulation of liposomes for radiation doses up to 10 Gy, whereas CT26 tumors displayed a tendency for decreased accumulation. Tumor hypoxia was found negatively correlated to microregional distribution of liposomes. However, liposome distribution in relation to hypoxia was improved at lower radiation doses. The study reveals that the heterogeneity in liposome tumor accumulation between tumors and different radiation protocols are important factors that need to be taken into consideration to achieve optimal effect of liposome based radio-sensitizer therapy.

Single Particle and PET-based Platform for Identifying Optimal Plasmonic Nano-Heaters for Photothermal Cancer Therapy.

Plasmonic nanoparticle-based photothermal cancer therapy is a promising new tool to inflict localized and irreversible damage to tumor tissue by hyperthermia, without harming surrounding healthy tissue. We developed a single particle and positron emission tomography (PET)-based platform to quantitatively correlate the heat generation of plasmonic nanoparticles with their potential as cancer killing agents. In vitro, the heat generation and absorption cross-section of single irradiated nanoparticles were quantified using a temperature sensitive lipid-based assay and compared to their theoretically predicted photo-absorption. In vivo, the heat generation of irradiated nanoparticles was evaluated in human tumor xenografts in mice using 2-deoxy-2-[F-18]fluoro-D-glucose ((18)F-FDG) PET imaging. To validate the use of this platform, we quantified the photothermal efficiency of near infrared resonant silica-gold nanoshells (AuNSs) and benchmarked this against the heating of colloidal spherical, solid gold nanoparticles (AuNPs). As expected, both in vitro and in vivo the heat generation of the resonant AuNSs performed superior compared to the non-resonant AuNPs. Furthermore, the results showed that PET imaging could be reliably used to monitor early treatment response of photothermal treatment. This multidisciplinary approach provides a much needed platform to benchmark the emerging plethora of novel plasmonic nanoparticles for their potential for photothermal cancer therapy.

Pharmacokinetic Analysis of (64)Cu-ATSM Dynamic PET in Human Xenograft Tumors in Mice.

UNLABELLED: The aim of this study was to evaluate the feasibility to perform voxel-wise kinetic modeling on datasets obtained from tumor-bearing mice that underwent dynamic PET scans with (64)Cu-ATSM and extract useful physiological parameters. METHODS: Tumor-bearing mice underwent 90-min dynamic PET scans with (64)Cu-ATSM and CT scans with contrast. Irreversible and reversible two-tissue compartment models were fitted to time activity curves (TACs) obtained from whole tumor volumes and compared using the Akaike information criterion (AIC). Based on voxel-wise pharmacokinetic analysis, parametric maps of model rate constants k1, k3 and Ki were generated and compared to (64)Cu-ATSM uptake. RESULTS: Based on the AIC, an irreversible two-tissue compartment model was selected for voxel-wise pharmacokinetic analysis. Of the extracted parameters, k1 (~perfusion) showed a strong correlation with early tracer uptake (mean spearman R = 0.88) 5 min post injection (pi). Moreover, positive relationships were found between late tracer uptake (90 min pi) and both k3 and the net influx rate constant, Ki (mean spearman R = 0.56 and R = 0.86; respectively). CONCLUSION: This study shows the feasibility to extract relevant parameters from voxel-wise pharmacokinetic analysis to be used for preclinical validation of (64)Cu-ATSM as a hypoxia-specific PET tracer.

High tumor uptake of (64)Cu: implications for molecular imaging of tumor characteristics with copper-based PET tracers.

INTRODUCTION: The use of copper-based positron emission tomography (PET) tracers in cancer studies is increasing. However, as copper has previously been found in high concentrations in human tumor tissue in vivo, instability of PET tracers could result in tumor accumulation of non-tracer-bound radioactive copper that may influence PET measurements. Here we determine the degree of (64)Cu uptake in five commonly used human cancer xenograft models in mice. Additionally, we compare copper accumulation in tumor tissue to gene expression of human copper transporter 1 (CTR1). METHODS: Small animal PET scans were performed on five different human cancer xenograft mice models 1h and 22h post injection (p.i.) of (64)CuCl2. Regions of interest (ROIs) were drawn on tumor tissue and sections of various organs on all images. Quantitative real-time PCR (qPCR) gene expression measurements of CTR1 were performed on tumor samples obtained after the 22h scan. RESULTS: A relatively high tumor uptake of (64)Cu was seen in four out of five tumor types and an increase in (64)Cu accumulation was seen in three out of five tumor types between 1h and 22h p.i. No relationship was found between tumor uptake of (64)Cu and gene expression of CTR1. CONCLUSIONS: The relatively high, time- and tumor type dependent (64)Cu uptake demonstrated here in five different human cancer xenograft models in mice, emphasizes the importance of validating tracer uptake and indicates that high in vivo stability of copper-based PET tracers is of particular importance because non-tracer-bound copper can accumulate in tumor tissue to a level that could potentially lead to misinterpretation of PET data.

(18)F-FDG PET imaging of murine atherosclerosis: association with gene expression of key molecular markers.

AIM: To study whether (18)F-FDG can be used for in vivo imaging of atherogenesis by examining the correlation between (18)F-FDG uptake and gene expression of key molecular markers of atherosclerosis in apoE(-/-) mice. METHODS: Nine groups of apoE(-/-) mice were given normal chow or high-fat diet. At different time-points, (18)F-FDG PET/contrast-enhanced CT scans were performed on dedicated animal scanners. After scans, animals were euthanized, aortas removed, gamma counted, RNA extracted from the tissue, and gene expression of chemo (C-X-C motif) ligand 1 (CXCL-1), monocyte chemoattractant protein (MCP)-1, vascular cell adhesion molecule (VCAM)-1, cluster of differentiation molecule (CD)-68, osteopontin (OPN), lectin-like oxidized LDL-receptor (LOX)-1, hypoxia-inducible factor (HIF)-1α, HIF-2α, vascular endothelial growth factor A (VEGF), and tissue factor (TF) was measured by means of qPCR. RESULTS: The uptake of (18)F-FDG increased over time in the groups of mice receiving high-fat diet measured by PET and ex vivo gamma counting. The gene expression of all examined markers of atherosclerosis correlated significantly with (18)F-FDG uptake. The strongest correlation was seen with TF and CD68 (p<0.001). A multivariate analysis showed CD68, OPN, TF, and VCAM-1 to be the most important contributors to the uptake of (18)F-FDG. Together they could explain 60% of the (18)F-FDG uptake. CONCLUSION: We have demonstrated that (18)F-FDG can be used to follow the progression of atherosclerosis in apoE(-/-) mice. The gene expression of ten molecular markers representing different molecular processes important for atherosclerosis was shown to correlate with the uptake of (18)F-FDG. Especially, the gene expressions of CD68, OPN, TF, and VCAM-1 were strong predictors for the uptake.

Quantitative PET of human urokinase-type plasminogen activator receptor with 64Cu-DOTA-AE105: implications for visualizing cancer invasion.

UNLABELLED: Expression levels of the urokinase-type plasminogen activator receptor (uPAR) represent an established biomarker for poor prognosis in a variety of human cancers. The objective of the present study was to explore whether noninvasive PET can be used to perform a quantitative assessment of expression levels of uPAR across different human cancer xenograft models in mice and to illustrate the clinical potential of uPAR PET in future settings for individualized therapy. METHODS: To accomplish our objective, a linear, high-affinity uPAR peptide antagonist, AE105, was conjugated with DOTA and labeled with (64)Cu ((64)Cu-DOTA-AE105). Small-animal PET was performed in 3 human cancer xenograft mice models, expressing different levels of human uPAR, and the tumor uptake was correlated with the uPAR expression level determined by uPAR enzyme-linked immunosorbent assay. The tumor uptake pattern of this tracer was furthermore compared with (18)F-FDG uptake, and finally the correlation between sensitivity toward 5-fluorouracil therapy and uPAR expression level was investigated. RESULTS: The uPAR-targeting PET tracer was produced in high purity and with high specific radioactivity. A significant correlation between tumor uptake of (64)Cu-DOTA-AE105 and uPAR expression was found (R(2) = 0.73; P < 0.0001) across 3 cancer xenografts, thus providing a strong argument for specificity. A significantly different uptake pattern of (64)Cu-DOTA-AE105, compared with that of (18)F-FDG, was observed, thus emphasizing the additional information that can be obtained on tumor biology using (64)Cu-DOTA-AE105 PET. Furthermore, a significant correlation between baseline uPAR expression and sensitivity toward 5-fluorouracil was revealed, thus illustrating the possible potentials of uPAR PET in a clinical setting. CONCLUSION: Our results clearly demonstrate that the peptide-based PET tracer (64)Cu-DOTA-AE105 enables the noninvasive quantification of uPAR expression in tumors in vivo, thus emphasizing its potential use in a clinical setting to detect invasive cancer foci and for individualized cancer therapy.

Importance of Attenuation Correction (AC) for Small Animal PET Imaging.

UNLABELLED: The purpose of this study was to investigate whether a correction for annihilation photon attenuation in small objects such as mice is necessary. The attenuation recovery for specific organs and subcutaneous tumors was investigated. A comparison between different attenuation correction methods was performed. METHODS: Ten NMRI nude mice with subcutaneous implantation of human breast cancer cells (MCF-7) were scanned consecutively in small animal PET and CT scanners (MicroPET(TM) Focus 120 and ImTek's MicroCAT(TM) II). CT-based AC, PET-based AC and uniform AC methods were compared. RESULTS: The activity concentration in the same organ with and without AC revealed an overall attenuation recovery of 9-21% for MAP reconstructed images, i.e., SUV without AC could underestimate the true activity at this level. For subcutaneous tumors, the attenuation was 13 ± 4% (9-17%), for kidneys 20 ± 1% (19-21%), and for bladder 18 ± 3% (15-21%). The FBP reconstructed images showed almost the same attenuation levels as the MAP reconstructed images for all organs. CONCLUSIONS: The annihilation photons are suffering attenuation even in small subjects. Both PET-based and CT-based are adequate as AC methods. The amplitude of the AC recovery could be overestimated using the uniform map. Therefore, application of a global attenuation factor on PET data might not be accurate for attenuation correction.

Tumor volume in subcutaneous mouse xenografts measured by microCT is more accurate and reproducible than determined by 18F-FDG-microPET or external caliper.

BACKGROUND: In animal studies tumor size is used to assess responses to anticancer therapy. Current standard for volumetric measurement of xenografted tumors is by external caliper, a method often affected by error. The aim of the present study was to evaluate if microCT gives more accurate and reproducible measures of tumor size in mice compared with caliper measurements. Furthermore, we evaluated the accuracy of tumor volume determined from 18F-fluorodeoxyglucose (18F-FDG) PET. METHODS: Subcutaneously implanted human breast adenocarcinoma cells in NMRI nude mice served as tumor model. Tumor volume (n = 20) was determined in vivo by external caliper, microCT and 18F-FDG-PET and subsequently reference volume was determined ex vivo. Intra-observer reproducibility of the microCT and caliper methods were determined by acquiring 10 repeated volume measurements. Volumes of a group of tumors (n = 10) were determined independently by two observers to assess inter-observer variation. RESULTS: Tumor volume measured by microCT, PET and caliper all correlated with reference volume. No significant bias of microCT measurements compared with the reference was found, whereas both PET and caliper had systematic bias compared to reference volume. Coefficients of variation for intra-observer variation were 7% and 14% for microCT and caliper measurements, respectively. Regression coefficients between observers were 0.97 for microCT and 0.91 for caliper measurements. CONCLUSION: MicroCT was more accurate than both caliper and 18F-FDG-PET for in vivo volumetric measurements of subcutaneous tumors in mice.18F-FDG-PET was considered unsuitable for determination of tumor size. External caliper were inaccurate and encumbered with a significant and size dependent bias. MicroCT was also the most reproducible of the methods.