A novel method for tracking nitrogen kinetics in vivo under hyperbaric conditions using radioactive nitrogen-13 gas and positron emission tomography.

Decompression sickness (DCS) is caused by gaseous nitrogen dissolved in tissues forming bubbles during decompression. To date, no method exists to identify nitrogen within tissues, but with advances in positron-emission tomography (PET) technology, it may be possible to track gaseous radionuclides into tissues. We aimed to develop a method to track nitrogen movement in vivo and under hyperbaric pressure that could then be used to further our understanding of DCS using nitrogen-13 (13N2). A single anesthetized female Sprague-Dawley rat was exposed to 625 kPa, composed of air, isoflurane, and 13N2 for 10 min. The PET scanner recorded 13N2 during the hyperbaric exposure with energy windows of 250-750 keV. The PET showed an increase in 13N2 concentration in the lung, heart, and abdominal regions, which all reached a plateau after ∼4 min. This showed that it is possible to gain noninvasive in vivo measurements of nitrogen kinetics through the body while at hyperbaric pressures. Tissue samples showed radioactivity above background levels in the blood, brain, liver, femur, and thigh muscle when assessed using a γ counter. The method can be used to evaluate an array of challenges to our understanding of decompression physiology by quantifying nitrogen load through γ counts of 13N2, and signal intensity of the PET. Further development of the method will improve the specificity of the measured outcomes, and enable it to be used with larger mammals, including humans.NEW & NOTEWORTHY This article describes a method for the in vivo quantification and tracking of nitrogen through the mammalian body whilst exposed to hyperbaric pressure. The method has the potential to further our understanding of decompression sickness, and quantitatively evaluate the effectiveness of both the treatment and prevention of decompression sickness.

Fluorescent Guided Sentinel Lymph Mapping of the Oral Cavity with Fluorescent-Labeled Tilmanocept.

OBJECTIVE: With the shift toward utilization of sentinel lymph node biopsy (SLNB) in oral cavity cancer, improved techniques for intraoperative sentinel node identification are needed. This study investigates the feasibility of fluorescently labeled tilmanoscept in SLNB in an oral cancer rabbit model. METHODS: An animal study was designed using 21 healthy male New Zealand rabbits. Gallium-68-labeled tilmanocept labeled with IRDye800CW was injected submucosally into the buccal mucosa (n = 6) or lateral tongue (n = 7) followed by PET imaging. One hour after injection, SLNB was performed using fluorescence imaging followed by a bilateral neck dissection and sampling of non-nodal surrounding tissue. All tissues were measured for radioactivity and fluorescence. In addition, eight rabbits were injected with delayed SLNB performed 48 h after injection. RESULTS: Buccal injections all had ipsilateral SLN drainage and tongue injections exhibited 18.2% contralateral drainage. An average of 1.9 ± 1.0 SLN (range 1-5) were identified. In addition, an average of 16.9 ± 3.3 non-sentinel lymph nodes were removed per animal. SLNs had an average of 0.69 ± 0.60 percent-of-injected dose (%ID) compared with non-sentinel nodes with 0.012 ± 0.025 %ID and surrounding tissue with 0.0067 ± 0.015 %ID. There was 98.0% agreement between sentinel lymph nodes identified using fluorescence compared to radioactivity with Cohen's kappa coefficient of 0.879. In 48-h delayed SLNB, results were consistent with 97.8% agreement with radioactivity and Cohen's Kappa coefficient of 0.884. Fluorescence identified additional lymph nodes that were not identified by radioactivity, and with one false negative. CONCLUSION: Fluorescent-labeled Tc-99 m-tilmanocept represents a highly accurate adjunct to enhance SLNB for oral cavity cancer. LEVEL OF EVIDENCE: N/A Laryngoscope, 134:1299-1307, 2024.

Neuronal Ndst1 depletion accelerates prion protein clearance and slows neurodegeneration in prion infection.

Select prion diseases are characterized by widespread cerebral plaque-like deposits of amyloid fibrils enriched in heparan sulfate (HS), a abundant extracellular matrix component. HS facilitates fibril formation in vitro, yet how HS impacts fibrillar plaque growth within the brain is unclear. Here we found that prion-bound HS chains are highly sulfated, and that the sulfation is essential for accelerating prion conversion in vitro. Using conditional knockout mice to deplete the HS sulfation enzyme, Ndst1 (N-deacetylase / N-sulfotransferase) from neurons or astrocytes, we investigated how reducing HS sulfation impacts survival and prion aggregate distribution during a prion infection. Neuronal Ndst1-depleted mice survived longer and showed fewer and smaller parenchymal plaques, shorter fibrils, and increased vascular amyloid, consistent with enhanced aggregate transit toward perivascular drainage channels. The prolonged survival was strain-dependent, affecting mice infected with extracellular, plaque-forming, but not membrane bound, prions. Live PET imaging revealed rapid clearance of recombinant prion protein monomers into the CSF of neuronal Ndst1- deficient mice, neuronal, further suggesting that HS sulfate groups hinder transit of extracellular prion protein monomers. Our results directly show how a host cofactor slows the spread of prion protein through the extracellular space and identify an enzyme to target to facilitate aggregate clearance.

The kidney drug transporter OAT1 regulates gut microbiome-dependent host metabolism.

Organic anion transporter 1 (OAT1/SLC22A6, NKT) is a multispecific drug transporter in the kidney with numerous substrates, including pharmaceuticals, endogenous metabolites, natural products, and uremic toxins. Here, we show that OAT1 regulates levels of gut microbiome-derived metabolites. We depleted the gut microbiome of Oat1-KO and WT mice and performed metabolomics to analyze the effects of genotype (KO versus WT) and microbiome depletion. OAT1 is an in vivo intermediary between the host and the microbes, with 40 of the 162 metabolites dependent on the gut microbiome also impacted by loss of Oat1. Chemoinformatic analysis revealed that the altered metabolites (e.g., indoxyl sulfate, p-cresol sulfate, deoxycholate) had more ring structures and sulfate groups. This indicates a pathway from gut microbes to liver phase II metabolism, to renal OAT1-mediated transport. The idea that multiple gut-derived metabolites directly interact with OAT1 was confirmed by in vitro transport and magnetic bead binding assays. We show that gut microbiome-derived metabolites dependent on OAT1 are impacted in a chronic kidney disease (CKD) model and human drug-metabolite interactions. Consistent with the Remote Sensing and Signaling Theory, our results support the view that drug transporters (e.g., OAT1, OAT3, OATP1B1, OATP1B3, MRP2, MRP4, ABCG2) play a central role in regulating gut microbe-dependent metabolism, as well as interorganismal communication between the host and microbiome.

Role of sentinel lymph node biopsy for oral squamous cell carcinoma: Current evidence and future challenges.

Sentinel lymph node biopsy (SLNB) has been used across oncological specialties for prognostication, staging, and identification of occult nodal metastasis. Recent studies demonstrated the potential clinical utility of SLNB in oral cavity squamous cell carcinoma (OCSCC). Elective neck dissection is the current standard of care in early management of OCSCC with depth of invasion greater than 2-4 mm; however, majority of patients ultimately do not have nodal disease on final pathology. SLNB is an alternative procedure widely adopted in early cancer management in many oncological subspecialities. Several considerations such as depth of invasion, nodal mapping, histopathology methods, operator variability, postoperative complications, and advancement in preoperative and intraoperative imaging technology can guide the appropriate application to SLNB in OCSCC. The aim of this review is to discuss the current evidence for SLNB in the treatment of early stage OCSCC, imaging technologies that support SLNB procedures, and studies that are currently underway.

MicroPET evidence for a hypersensitive neuroinflammatory profile of gp120 mouse model of HIV.

Despite increased survivability for people living with HIV (PLWH), HIV-related cognitive deficits persist. Determining biological mechanism(s) underlying abnormalities is critical to minimize the long-term impact of HIV. Positron emission tomography (PET) studies reveal that PLWH exhibit elevated neuroinflammation, potentially contributing to these problems. PLWH are hypersensitive to environmental insults that drive elevated inflammatory profiles. Gp120 is an envelope glycoprotein exposed on the surface of the HIV envelope which enables HIV entry into a cell contributing to HIV-related neurotoxicity. In vivo evidence for mice overexpressing gp120 (transgenic) mice exhibiting neuroinflammation remains unclear. Here, we conducted microPET imaging in gp120 transgenic and wildtype mice, using the radiotracer [(18)F]FEPPA (binds to the translocator protein expressed by activated microglial serving as a neuroinflammatory marker). Imaging was performed at baseline and 24 h after lipopolysaccharide (LPS; 5 mg/kg) treatment (endotoxin that triggers an immune response). Gp120 transgenic mice exhibited elevated [(18F)]FEPPA in response to LPS vs. wildtype mice throughout the brain including dorsal and ventral striata, hypothalamus, and hippocampus. Gp120 transgenic mice are hypersensitive to environmental inflammatory insults, consistent with PLWH, measurable in vivo. It remains to-be-determined whether this heightened sensitivity is connected to the behavioral abnormalities of these mice or sensitive to any treatments.

A receptor-binding radiopharmaceutical for imaging of traumatic brain injury in a rodent model: [99mTc]Tc-tilmanocept.

INTRODUCTION: Blood-brain barrier (BBB) disruption and subsequent neuro-inflammation occur following traumatic brain injury (TBI), resulting in a spectrum of human nervous system disorders. [99mTc]Tc-tilmanocept is a receptor-binding radiopharmaceutical FDA-approved for sentinel lymph node mapping. We hypothesize that after an intravenous (i.v.) injection, [99mTc]Tc-tilmanocept, will traverse a disrupted BBB and bind to CD206-bearing microglial cells. METHODS: Age-matched mice were divided into three groups: 5-days post TBI (n = 4), and 5-days post sham (n = 4), and naïve controls (n = 4). IRDye800CW-labeled [99mTc]Tc-tilmanocept (0.15 nmol per gram body weight) and FITC-labeled bovine serum albumin (FITC-BSA) were injected (i.v.) into each mouse. Mice were imaged with a high-resolution gamma camera for 45 min. Immediately after imaging, the brains were perfused with fixative, excised, imaged with a fluorescence scanner, assayed for radioactivity, and prepared for histology. RESULTS: In vivo nuclear imaging, ex vivo fluorescence imaging, ex vivo gamma well counting, and histo-microscopy demonstrated enhanced tilmanocept uptake in the TBI region. The normalized [99mTc]Tc-tilmanocept uptake value from nuclear imaging and the maximum pixel intensity from fluorescence imaging of the TBI group (1.12 ±â€¯0.12 and 2288 ±â€¯278 a.u., respectively) were significantly (P < 0.04) higher than the sham group (0.64 ±â€¯0.28 and 1708 ±â€¯101 a.u., respectively) and the naive group (0.76 ±â€¯0.24 and 1643 ±â€¯391 a.u., respectively). The mean [99mTc]Tc-tilmanocept scaled uptake in the TBI brains (0.058 ±â€¯0.013%/g) was significantly (P < 0.010) higher than the scaled brain uptake of the sham group (0.031 ±â€¯0.011%/g) and higher (P = 0.04) than the uptake of the naïve group (0.020 ±â€¯0.002%/g). Fluorescence microscopy demonstrated increased uptake of the IRDye800CW-tilmanocept and FITC-BSA in the TBI brain regions. CONCLUSION: [99mTc]Tc-tilmanocept traverses disrupted blood-brain barrier and localizes within the injured region. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: [99mTc]Tc-tilmanocept could serve as an imaging biomarker for TBI-associated neuroinflammation and any disease process that involves a disruption of the blood-brain barrier.

Concepts for design and analysis of receptor radiopharmaceuticals: The Receptor-Binding Radiotracers series of meetings provided the foundation.

A symposium at George Washington University on Receptor-Binding Radiotracers in 1980 and three follow-up meetings held at University of California, San Diego provided a forum for debating the critical concepts involved in the new field of designing and evaluating radiotracers for imaging receptors and transporters. This review is intended to educate young investigators who may be relatively new to receptor radiopharmaceutical development. Our anticipated audience includes researchers in basic pharmacology, radiochemistry, imaging technology and kinetic data analysis and how these disciplines have worked together to build our understanding of the human biology of transporters and receptor signaling in health and disease. We have chosen to focus on radiochemical design of a useful imaging agent and how design is coupled to analysis of data collected from dynamic imaging with that agent. Some pharmacology may be required for designing the imaging agent and some imaging physics may be important in optimizing the quality of data that is collected. However, the key to a successful imaging agent is matching the radiotracer to the target receptor and to analysis of the time-course data that is used to parse delivery from specific binding and subsequent metabolism or degradation. Properly designed imaging agents are providing critical information about human biology in health and disease as well as pharmacodynamic response to drug interventions. The review emphasizes some of the ideas that were controversial at the 1980 conference and chronicles with literature examples how they have resolved over the four decades of using radiotracers to study transporters and receptors in human subjects. These examples show that there are situations where a very small KD, i.e. high affinity, has the potential to yield an image that reflects blood flow more than receptor density. The examples also show that by combining two studies, one with high specific activity and a second with low specific activity injections one can unravel the pseudo-first order rate B'max into the true second-order rate constant, k3, and the unoccupied receptor density. The final section describes how mathematical methods first presented to the receptor-imaging community in 1980 are now being used to provide confidence in the analysis of kinetic biodistribution studies. Our hope is that by bringing these concepts together in a single review, the next generation of scientists developing receptor imaging agents can be much more efficient than their pioneers in developing useful imaging methods.

Molecular Imaging of the Glomerulus via Mesangial Cell Uptake of Radiolabeled Tilmanocept.

An unmet need for the clinical management of chronic kidney disease is a predictive tool of kidney function during the first decade of the disease, when there is silent loss of glomerular function. The objective of this study was to demonstrate receptor-mediated binding of tilmanocept to CD206 within the kidney and provide evidence of kinetic sensitivity of this binding to renal function. Methods: Rats were positioned in a PET scanner with the liver and kidneys within the field of view. After an intravenous injection of 68Ga-IRDye800-tilmanocept, using 1 of 2 scaled molar doses (0.02 nmol/g, n = 5; or 0.10 nmol/g, n = 5), or coinjection (n = 3) of 68Ga-IRDye800-tilmanocept (0.10 nmol/g) and unlabeled tilmanocept (5.0 nmol/g), or a negative control, 68Ga-IRDye800-DTPA-galactosyl-dextran (0.02 nmol/g, n = 5), each animal was imaged for 20 min followed by a whole-body scan. Frozen kidney sections were stained for podocytes and CD206 using immunofluorescence. Molecular imaging of diabetic db/db mice (4.9 wk, n = 6; 7.3 wk, n = 4; 13.3 wk, n = 6) and nondiabetic db/m mice (n = 6) was performed with fluorescence-labeled 99mTc-tilmanocept (18.5 MBq, 2.6 nmol). Thirty minutes after injection, blood, liver, kidneys, and urine were assayed for radioactivity. Renal time-activity curves were generated. Results: Rat PET whole-body images and time-activity curves of 68Ga-IRDye800-tilmanocept demonstrated receptor-mediated renal accumulation with evidence of glomerular uptake. Activity within the renal cortex persisted during the 40-min study. Histologic examination demonstrated colocalization of CD206 and IRDye800-tilmanocept within the glomerulus. The glomerular accumulation of the coinjection and the negative control studies were significantly less than the CD206-targeted agent. The db/db mice displayed a multiphasic renal time-activity curve with high urinary bladder accumulation; the nondiabetic mice exhibited renal uptake curves dominated by a single phase with low bladder accumulation. Conclusion: This study demonstrated receptor-mediated binding to the glomerular mesangial cells and kinetic sensitivity of tilmanocept to chronic renal disease. Given the role of mesangial cells during the progression of diabetic nephropathy, PET or SPECT renal imaging with radiolabeled tilmanocept may provide a noninvasive quantitative assessment of glomerular function.

Molecular Imaging of endometrial sentinel lymph nodes utilizing fluorescent-labeled Tilmanocept during robotic-assisted surgery in a porcine model.

Molecular imaging with a fluorescent version of Tilmanocept may permit an accurate and facile detection of sentinel nodes of endometrial cancer. Tilmanocept accumulates in sentinel lymph nodes (SLN) by binding to a cell surface receptor unique to macrophages and dendritic cells. Four female Yorkshire pigs underwent cervical stromal injection of IRDye800-Tilmanocept, a molecular imaging agent tagged with near-infrared fluorescent dye and radiolabeled with gallium-68 and technetium-99m. PET/CT scans 1.5 hours post-injection provided pre-operative SLN mapping. Robotic-assisted lymphadenectomy was performed two days after injection, using the FireFly imaging system to identify nodes demonstrating fluorescent signal. After removal of fluorescent nodes, pelvic and periaortic node dissections were performed. Nodes were assayed for technetium-99m activity, and SLNs were established using the "10%-rule", requiring that the radioactivity of additional SLNs be greater than 10% of the "hottest" SLN. Thirty-four nodal samples were assayed ex vivo for radioactivity. All the SLNs satisfying the "10%-rule" were detected using the FireFly system. Five fluorescent nodes were detected, corresponding with preoperative PET/CT scan. Three pigs had one SLN and one pig had two SLNs, with 100% concordance between fluorescence and radioactivity. Fluorescent-labeled Tilmanocept permits real-time intraoperative detection of SLNs during robotic-assisted lymphadenectomy for endometrial cancer in a porcine model. When radiolabeled with gallium-68, Tilmanocept allows for preoperative localization of SLNs using PET/CT, and shows specificity to SLNs with persistent fluorescent signal, detectable using the FireFly system, for two days post-injection. In conclusion, these findings suggest that a phase I trial in human subjects is warranted, and that a long-term goal of an intra-operative administration of non-radioactive fluorescent-labeled Tilmanocept is possible.

Targeting mannose receptor expression on macrophages in atherosclerotic plaques of apolipoprotein E-knockout mice using 111In-tilmanocept.

BACKGROUND: Atherosclerotic plaque phenotypes are classified based on the extent of macrophage infiltration into the lesions, and the presence of certain macrophage subsets might be a sign for plaque vulnerability. The mannose receptor (MR) is over-expressed in activated macrophages. Tilmanocept is a tracer that targets MR and is approved in Europe and the USA for the detection of sentinel lymph nodes. In this study, our aim was to evaluate the potential of 111In-labelled tilmanocept for the detection of MR-positive macrophages in atherosclerotic plaques of apolipoprotein E-knockout (ApoE-KO) mouse model. METHODS: Tilmanocept was labelled with 111In. The labelling stability and biodistribution of the tracer was first evaluated in control mice (n = 10) 1 h post injection (p.i.). For in vivo imaging studies, 111In-tilmanocept was injected into ApoE-KO (n = 8) and control (n = 8) mice intravenously (i.v.). The mice were scanned 90 min p.i. using a dedicated animal SPECT/CT. For testing the specificity of 111In-tilmanocept uptake in plaques, a group of ApoE-KO mice was co-injected with excess amount of non-labelled tilmanocept. For ex vivo imaging studies, the whole aortas (n = 9 from ApoE-KO and n = 4 from control mice) were harvested free from adventitial tissue for Sudan IV staining and autoradiography. Cryosections were prepared for immunohistochemistry (IHC). RESULTS: 111In radiolabelling of tilmanocept provided a yield of greater than 99%. After i.v. injection, 111In-tilmanocept accumulated in vivo in MR-expressing organs (i.e. liver and spleen) and showed only low residual blood signal 1 h p.i. MR-binding specificity in receptor-positive organs was demonstrated by a 1.5- to 3-fold reduced uptake of 111In-tilmanocept after co-injection of a blocking dose of non-labelled tilmanocept. Focal signal was detected in atherosclerotic plaques of ApoE-KO mice, whereas no signal was detected in the aortas of control mice. 111In-tilmanocept uptake was detected in atherosclerotic plaques on autoradiography correlating well with Sudan IV-positive areas and associating with subendothelial accumulations of MR-positive macrophages as demonstrated by IHC. CONCLUSIONS: After i.v. injection, 111In-tilmanocept accumulated in MR-expressing organs and was associated with only low residual blood signal. In addition, 111In-tilmanocept uptake was detected in atherosclerotic plaques of mice containing MR-expressing macrophages suggesting that tilmanocept represents a promising tracer for the non-invasive detection of macrophages in atherosclerotic plaques.

Fluorescence-Based Molecular Imaging of Porcine Urinary Bladder Sentinel Lymph Nodes.

The primary objective was to test the ability of a laparoscopic camera system to detect the fluorescent signal emanating from sentinel lymph nodes (SLNs) approximately 2 d after injection and imaging of a positron-emitting molecular imaging agent into the submucosa of the porcine urinary bladder. Methods: Three female pigs underwent a submucosal injection of the bladder with fluorescent-tagged tilmanocept, radiolabeled with both 68Ga and 99mTc. One hour after injection, a pelvic PET/CT scan was acquired for preoperative SLN mapping. Approximately 36 h later, robotic SLN mapping was performed using a fluorescence-capable camera system. After identification of the fluorescent lymph nodes, a pelvic lymph node dissection was completed with robotic assistance. All excised nodal packets (n = 36) were assayed for 99mTc activity, which established a lymph node as an SLN. 99mTc activity was also used to calculate the amount of dye within each lymph node. Results: All of the SLNs defined by the ex vivo γ-well assay of 99mTc activity were detected by fluorescence mode imaging. The time between injection and robotic SLN mapping ranged from 32 to 38 h. A total of 5 fluorescent lymph nodes were detected; 2 pigs had 2 fluorescent lymph nodes and 1 pig exhibited a single lymph node. Four of the 5 SLNs exhibited increased SUVs of 12.4-139.0 obtained from PET/CT. The dye content of the injection sites ranged from 371 to 1,441 pmol, which represented 16.5%-64.1% of the injected dose; the amount of dye within the SLNs ranged from 8.5 to 88 pmol, which was equivalent to 0.38%-3.91% of the administered dose. Conclusion: Fluorescent-labeled 68Ga-tilmanocept allows for PET imaging and real-time intraoperative detection of SLNs during robotic surgery.

New Dioxaborolane Chemistry Enables [(18)F]-Positron-Emitting, Fluorescent [(18)F]-Multimodality Biomolecule Generation from the Solid Phase.

New protecting group chemistry is used to greatly simplify imaging probe production. Temperature and organic solvent-sensitive biomolecules are covalently attached to a biotin-bearing dioxaborolane, which facilitates antibody immobilization on a streptavidin-agarose solid-phase support. Treatment with aqueous fluoride triggers fluoride-labeled antibody release from the solid phase, separated from unlabeled antibody, and creates [(18)F]-trifluoroborate-antibody for positron emission tomography and near-infrared fluorescent (PET/NIRF) multimodality imaging. This dioxaborolane-fluoride reaction is bioorthogonal, does not inhibit antigen binding, and increases [(18)F]-specific activity relative to solution-based radiosyntheses. Two applications are investigated: an anti-epithelial cell adhesion molecule (EpCAM) monoclonal antibody (mAb) that labels prostate tumors and Cetuximab, an anti-epidermal growth factor receptor (EGFR) mAb (FDA approved) that labels lung adenocarcinoma tumors. Colocalized, tumor-specific NIRF and PET imaging confirm utility of the new technology. The described chemistry should allow labeling of many commercial systems, diabodies, nanoparticles, and small molecules for dual modality imaging of many diseases.

Staging of fibrosis in experimental non-alcoholic steatohepatitis by quantitative molecular imaging in rat models.

OBJECTIVES: The aim of this study was to test the ability of hepatocyte-specific functional imaging to stage fibrosis in experimental rat models of liver fibrosis and progressive NASH. Using ROC analysis we tested the ability of a functional imaging metric to discriminate early (F1) from moderate (F2) fibrosis in the absence and presence of non-alcoholic steatohepatitis, which has not been achieved by any modality other than biopsy. METHODS: Galactosyl Human Serum Albumin (GSA) was radiolabeled with the positron-emitter, (68)Ga, and injected (i.v., 45-95 µCi, 1.5 pmol/g TBW) into 44 healthy, 19 DEN-, and 22 CDAA-treated male rats. Quantification of liver function was achieved by calculating T90, defined as the time for the liver to accumulate 90 percent of the [(68)Ga]GSA plateau value. All livers were excised immediately after imaging and prepared for a "blinded" histologic examination, which included fibrosis and fat content scores. Two sets of fibrosis scores were recorded for all of animals. The dominant fibrosis stage was recorded as the "Dominant Pattern" score and the "Maximum Pattern" score was assigned if a smaller distinct region with a higher fibrosis score was observed. RESULTS: Animals with Dominant Pattern F0-F1 liver fibrosis (D(-)=39) demonstrated significantly (P<0.0001) faster accumulation of [(68)Ga]GSA (2.40 ± 0.52 min) than those with moderate to advanced Dominant Pattern fibrosis F2 and F4 (D(+)=26) (3.48 ± 1.01 min). ROC analysis (F0-F1 vs F2-F4) produced an area under the binormal curve (AUC) of 0.867 ± 0.045. Twenty-seven of the 65 rats had small regions with higher fibrosis scores. Six of these Maximum Pattern scores reclassified the animals from D(-) to D(+). ROC analysis of F0-F1 versus F2-F4 rats without liver fat produced AUCs of 0.881 ± 0.053 for the Dominant Pattern Score and 0.944 ± 0.035 for the Maximum Pattern Score. CONCLUSIONS: PET Functional Imaging of [(68)Ga]GSA accurately discriminates early from moderate experimental fibrosis independent of steatosis grade. If validated in human studies, molecular imaging may emerge as a potential alternative to invasive liver biopsy.

A tri-modal molecular imaging agent for sentinel lymph node mapping.

INTRODUCTION: We report an "instant kit" method to radiolabel fluorescent-tilmanocept with (68)Ga and (99m)Tc for tri-modal molecular imaging of sentinel lymph nodes (SLNs). METHODS: Solutions of sodium acetate, (68)GaCl(3) and Na(99m)TcO(4) were added successively to a "kit vial" containing lyophilized 800CW-tilmanocept, SnCl(2), trehalose and ascorbic acid. After a 30-min incubation, the pH was neutralized with PBS. No purification was required. Radiochemical and fluorescence purity was measured by HPLC and ITLC techniques. In vitro stability was measured by standing gel chromatography (SGC) and ITLC by a 100-fold dilution 0.25 h after radiolabeling. In vivo stability was measured by SGC and ITLC after an 11h incubation in human plasma. A dose (0.1 nmol, ~1MBq (68)Ga, ~25M Bq (99m)Tc) was injected to the footpad of 4 mice. Popliteal SLNs were imaged by PET and fluorescence imaging systems at 0.5, 24, 48, 72 h, then excised and assayed for (99m)Tc. RESULTS: Radiochemical and fluorescent purity exceeded 98%. The in vitro stability assay demonstrated high irreversibility of both radiolabels and the fluorescent label, and in vivo stability assay demonstrated high stability of the technetium and fluorescent labels to plasma metabolism. Popliteal SLNs were identified by PET and fluorescence imaging within 0.5 h of injection. SLN fluorescence intensity remained constant for 72 h, when ~1% of the injected dose resided in the SLN. CONCLUSIONS: Fluorescent-labeled tilmanocept can be radiolabeled with (68)Ga and (99m)Tc by the sequential addition of each generator eluate to a lyophilized kit. The resulting tri-modal agent provides: PET images for pre-operative SLN mapping, fluorescence imaging up to 72 hours after injection, and quantitative radiometric measurement of SLN accumulation after excision.

Comparison of Post-injection Site Pain Between Technetium Sulfur Colloid and Technetium Tilmanocept in Breast Cancer Patients Undergoing Sentinel Lymph Node Biopsy.

BACKGROUND: No prior studies have examined injection pain associated with Technetium-99m Tilmanocept (TcTM). METHODS: This was a randomized, double-blinded study comparing postinjection site pain between filtered Technetium Sulfur Colloid (fTcSC) and TcTM in breast cancer lymphoscintigraphy. Pain was evaluated with a visual analogue scale (VAS) (0-100 mm) and the short-form McGill Pain Questionnaire (SF-MPQ). The primary endpoint was mean difference in VAS scores at 1-min postinjection between fTcSC and TcTM. Secondary endpoints included a comparison of SF-MPQ scores between the groups at 5 min postinjection and construction of a linear mixed effects model to evaluate the changes in pain during the 5-min postinjection period. RESULTS: Fifty-two patients underwent injection (27-fTcSC, 25-TcTM). At 1-min postinjection, patients who received fTcSC experienced a mean change in pain of 16.8 mm (standard deviation (SD) 19.5) compared with 0.2 mm (SD 7.3) in TcTM (p = 0.0002). At 5 min postinjection, the mean total score on the SF-MPQ was 2.8 (SD 3.0) for fTcSC versus 2.1 (SD 2.5) for TcTM (p = 0.36). In the mixed effects model, injection agent (p < 0.001), time (p < 0.001) and their interaction (p < 0.001) were associated with change in pain during the 5-min postinjection period. The model found fTcSC resulted in significantly more pain of 15.2 mm (p < 0.001), 11.3 mm (p = 0.001), and 7.5 mm (p = 0.013) at 1, 2, and 3 min postinjection, respectively. CONCLUSIONS: Injection with fTcSC causes significantly more pain during the first 3 min postinjection compared with TcTM in women undergoing lymphoscintigraphy for breast cancer.

γ-Tilmanocept, a New Radiopharmaceutical Tracer for Cancer Sentinel Lymph Nodes, Binds to the Mannose Receptor (CD206).

γ-Tilmanocept ((99m)Tc-labeled-tilmanocept or [(99m)Tc]-tilmanocept) is the first mannose-containing, receptor-directed, radiolabeled tracer for the highly sensitive imaging of sentinel lymph nodes in solid tumor staging. To elucidate the mannose-binding receptor that retains tilmanocept in this microenvironment, human macrophages were used that have high expression of the C-type lectin mannose receptor (MR; CD206). Cy3-labeled tilmanocept exhibited high specificity binding to macrophages that was nearly abolished in competitive inhibition experiments. Furthermore, Cy3-tilmanocept binding was markedly reduced on macrophages deficient in the MR by small interfering RNA treatment and was increased on MR-transfected HEK 293 cells. Finally, confocal microscopy revealed colocalization of Cy3-tilmanocept with the macrophage membrane MR and binding of labeled tilmanocept to MR(+) cells (macrophages and/or dendritic cells) in human sentinel lymph node tissues. Together these data provide strong evidence that CD206 is a major binding receptor for γ-tilmanocept. Identification of CD206 as the γ-tilmanocept-binding receptor enables opportunities for designing receptor-targeted advanced imaging agents and therapeutics for cancer and other diseases.

Sentinel lymph node biopsy in bladder cancer: Systematic review and technology update.

A sentinel lymph node (SLN) is the first lymph node to drain a solid tumor and likely the first place metastasis will travel. SLN biopsy has been well established as a staging tool for melanoma and breast cancer to guide lymph node dissection (LND); its utility in bladder cancer is debated. We performed a systematic search of PubMed for both human and animal studies that looked at SLN detection in cases of urothelial carcinoma of the bladder. We identified a total of nine studies that assessed a variety of imaging techniques to identify SLNs in patients with urothelial carcinoma of the bladder. Eight studies investigated human patients while one looked at animal (dog) models. Seven studies representing 156 patients noted the negative predictive value of the SLN to predict a metastasis free state was 92% (92/100). The SLN biopsy was less accurate in metastatic patients with a positive predictive value of only 77% (43/56) with a false negative range of in individual studies of 0-19%. Clinically, positive nodes routinely do not take up the pharmaceutical agent for SLN. Therefore, SLN biopsy is a promising concept with a 92% negative predictive value; however, the false negative rates are high which may be improved by standardizing populations and indications. Novel technologies are improving the detection of SLN and may provide the surgeon with an improved ability to detect micrometastasis, guide surgery, and reduce patient morbidity.

Comparison of [(99m)Tc]tilmanocept and filtered [(99m)Tc]sulfur colloid for identification of SLNs in breast cancer patients.

BACKGROUND: The efficacy of sentinel lymph node (SLN) surgery requires targeted removal of first-draining nodes; however, frequently more nodes are removed than necessary. [(99m)Tc]tilmanocept (TcTM) is a molecular-targeted radiopharmaceutical specifically designed for SLN mapping. We evaluated technical outcomes of SLN biopsy in breast cancer patients mapped with TcTM + vital blue dye (VBD) versus filtered [(99m)Tc]sulfur colloid (fTcSC) + VBD. METHODS: There were 84 versus 115 patients in the TcTM versus fTcSC cohorts, respectively. Main measures were the number of SLNs removed per patient and factors influencing number of nodes removed. We also evaluated whether the radiotracer injected affected the proportion of positive nodes removed in node-positive patients. RESULTS: Fewer nodes were removed among patients mapped with TcTM compared to fTcSC (mean TcTM: 1.85 vs. fTcSC: 3.24, p < 0.001). Logistic regression analysis adjusted for tumor characteristics showed that injection of fTcSC (p < 0.001) independently predicted removal of greater than 3 nodes. A similar proportion of patients was identified as node-positive, whether mapped with TcTM or with fTcSC (TcTM: 24 % vs. fTcSC: 17 %, p = 0.3); however, TcTM detected a greater proportion of positive nodes among node-positive patients compared with fTcSC (0.73 vs. 0.43, p = 0.001). CONCLUSIONS: Patients undergoing SLN biopsy with TcTM required fewer SLNs to identify the same rate of node-positive patients compared with fTcSC in breast cancer patients with similar risk of axillary metastatic disease. These data suggest that a molecularly targeted mechanism of SLN identification may reduce the total number of nodes necessary for accurate axillary staging.

Robotic-assisted fluorescence sentinel lymph node mapping using multimodal image guidance in an animal model.

OBJECTIVE: To investigate positron emission tomography/computed tomography (PET/CT) preoperative imaging and intraoperative detection of a fluorescent-labeled receptor-targeted radiopharmaceutical in a prostate cancer animal model. MATERIALS AND METHODS: Three male beagle dogs underwent an intraprostatic injection of fluorescent-tagged tilmanocept, radiolabeled with both gallium Ga-68 and technetium Tc-99m. One hour after injection, a pelvic PET/CT scan was performed for preoperative sentinel lymph node (SLN) mapping. The definition of SLN was a standardized uptake value that exceeded 5% of the lymph node with the highest standardized uptake value. Thirty-six hours later, we performed robotic-assisted SLN dissection using a fluorescence-capable camera system. Fluorescent lymph nodes were clipped, the abdomen was opened, and the pelvic and retroperitoneal nodes were excised. All excised nodal packets were assayed by in vitro nuclear counting and reported as the percentage of injected dose. RESULTS: Preoperative PET/CT imaging identified a median of 3 SLNs per animal. All SLNs (100%) identified by the PET/CT were fluorescent during robotic-assisted lymph node dissection. Of all fluorescent nodes visualized by the camera system, 9 of 12 nodes (75%) satisfied the 5% rule defined by the PET/CT scan. The 2 lymph nodes that did not qualify accumulated <0.002% of the injected dose. CONCLUSION: Fluorescent-labeled tilmanocept has optimal logistic properties to obtain preoperative PET/CT and subsequent real-time intraoperative confirmation during robotic-assisted SLN dissection.