Alessandro Ruggiero

Cerenkov Luminescence Imaging of Medical Isotopes

The development of novel multimodality imaging agents and techniques represents the current frontier of research in the field of medical imaging science. However, the combination of nuclear tomography with optical techniques has yet to be established. Here, we report the use of the inherent optical emissions from the decay of radiopharmaceuticals for Cerenkov luminescence imaging (CLI) of tumors in vivo and correlate the results with those obtained from concordant immuno-PET studies. Methods: In vitro phantom studies were used to validate the visible light emission observed from a range of radionuclides including the positron emitters 18F, 64Cu, 89Zr, and 124I; β-emitter 131I; and α-particle emitter 225Ac for potential use in CLI. The novel radiolabeled monoclonal antibody 89Zr-desferrioxamine B [DFO]-J591 for immuno-PET of prostate-specific membrane antigen (PSMA) expression was used to coregister and correlate the CLI signal observed with the immuno-PET images and biodistribution studies. Results: Phantom studies confirmed that Cerenkov radiation can be observed from a range of positron-, β-, and α-emitting radionuclides using standard optical imaging devices. The change in light emission intensity versus time was concordant with radionuclide decay and was also found to correlate linearly with both the activity concentration and the measured PET signal (percentage injected dose per gram). In vivo studies conducted in male severe combined immune deficient mice bearing PSMA-positive, subcutaneous LNCaP tumors demonstrated that tumor-specific uptake of 89Zr-DFO-J591 could be visualized by both immuno-PET and CLI. Optical and immuno-PET signal intensities were found to increase over time from 24 to 96 h, and biodistribution studies were found to correlate well with both imaging modalities. Conclusion: These studies represent the first, to our knowledge, quantitative assessment of CLI for measuring radiotracer uptake in vivo. Many radionuclides common to both nuclear tomographic imaging and radiotherapy have the potential to be used in CLI. The value of CLI lies in its ability to image radionuclides that do not emit either positrons or γ-rays and are, thus, unsuitable for use with current nuclear imaging modalities. Optical imaging of Cerenkov radiation emission shows excellent promise as a potential new imaging modality for the rapid, high-throughput screening of radiopharmaceuticals.

Paradoxical glomerular filtration of carbon nanotubes

The molecular weight cutoff for glomerular filtration is thought to be 30–50 kDa. Here we report rapid and efficient filtration of molecules 10–20 times that mass and a model for the mechanism of this filtration. We conducted multimodal imaging studies in mice to investigate renal clearance of a single-walled carbon nanotube (SWCNT) construct covalently appended with ligands allowing simultaneous dynamic positron emission tomography, near-infrared fluorescence imaging, and microscopy. These SWCNTs have a length distribution ranging from 100 to 500 nm. The average length was determined to be 200–300 nm, which would yield a functionalized construct with a molecular weight of ∼350–500 kDa. The construct was rapidly (t1/2 ∼ 6 min) renally cleared intact by glomerular filtration, with partial tubular reabsorption and transient translocation into the proximal tubular cell nuclei. Directional absorption was confirmed in vitro using polarized renal cells. Active secretion via transporters was not involved. Mathematical modeling of the rotational diffusivity showed the tendency of flow to orient SWCNTs of this size to allow clearance via the glomerular pores. Surprisingly, these results raise questions about the rules for renal filtration, given that these large molecules (with aspect ratios ranging from 100:1 to 500:1) were cleared similarly to small molecules. SWCNTs and other novel nanomaterials are being actively investigated for potential biomedical applications, and these observations—that high aspect ratio as well as large molecular size have an impact on glomerular filtration—will allow the design of novel nanoscale-based therapeutics with unusual pharmacologic characteristics.

Receptor Activator of NF-kB (RANK) Expression in Primary Tumors Associates with Bone Metastasis Occurrence in Breast Cancer Patients

Background Receptor activator of NFkB (RANK), its ligand (RANKL) and the decoy receptor of RANKL (osteoprotegerin, OPG) play a pivotal role in bone remodeling by regulating osteoclasts formation and activity. RANKL stimulates migration of RANK-expressing tumor cells in vitro, conversely inhibited by OPG. Materials and Methods We examined mRNA expression levels of RANKL/RANK/OPG in a publicly available microarray dataset of 295 primary breast cancer patients. We next analyzed RANK expression by immunohistochemistry in an independent series of 93 primary breast cancer specimens and investigated a possible association with clinicopathological parameters, bone recurrence and survival. Results Microarray analysis showed that lower RANK and high OPG mRNA levels correlate with longer overall survival (P = 0.0078 and 0.0335, respectively) and disease-free survival (P = 0.059 and 0.0402, respectively). Immunohistochemical analysis of RANK showed a positive correlation with the development of bone metastases (P = 0.023) and a shorter skeletal disease-free survival (SDFS, P = 0.037). Specifically, univariate analysis of survival showed that “RANK-negative” and “RANK-positive” patients had a SDFS of 105.7 months (95% CI: 73.9–124.4) and 58.9 months (95% CI: 34.7–68.5), respectively. RANK protein expression was also associated with accelerated bone metastasis formation in a multivariate analysis (P = 0.029). Conclusions This is the first demonstration of the role of RANK expression in primary tumors as a predictive marker of bone metastasis occurrence and SDFS in a large population of breast cancer patients.

Intraoperative Imaging of Positron Emission Tomographic Radiotracers Using Cerenkov Luminescence Emissions

Imaging the location and extent of cancer provides invaluable information before, during, and after surgery. The majority of “image-guided” methods that use, for example, positron emission tomography (PET) involve preoperative imaging and do not provide real-time information during surgery. It is now well established that the inherent optical emissions (Cerenkov radiation) from various β-emitting radionuclides can be visualized by Cerenkov luminescence imaging (CLI). Here we report the full characterization of CLI using the positron-emitting radiotracer 89Zr-DFO-trastuzumab for target-specific, quantitative imaging of HER2/neu-positive tumors in vivo. We also provide the first demonstration of the feasibility of using CLI for true image-guided, intraoperative surgical resection of tumors. Analysis of optical CLIs provided accurate, quantitative information on radiotracer biodistribution and tissue uptake that correlated well with the concordant PET images. CLI, PET, and biodistribution studies revealed target-specific uptake of 89Zr-DFO-trastuzumab in BT-474 (HER2/neu positive) versus MDA-MB-468 (HER2/neu negative) xenografts in the same mice. Competitive inhibition (blocking) studies followed by CLI also confirmed the in vivo immunoreactivity and specificity of 89Zr-DFO-trastuzumab for HER2/neu. Overall, these results strongly support the continued development of CLI as a preclinical and possible clinical tool for use in molecular imaging and surgical procedures for accurately defining tumor margins.

Targeting the Internal Epitope of Prostate-Specific Membrane Antigen with 89Zr-7E11 Immuno-PET

The potential of the positron-emitting 89Zr has been recently investigated for the design of radioimmunoconjugates for immuno-PET. In this study, we report the preparation and in vivo evaluation of 89Zr-desferrioxamine B (DFO)-7E11, a novel 89Zr-labeled monoclonal antibody (mAb) construct for targeted imaging of prostate-specific membrane antigen (PSMA), a prototypical cell surface marker highly overexpressed in prostate cancer. The ability of 89Zr-DFO-7E11 to delineate tumor response to therapy was also investigated, because it binds to the intracellular epitope of PSMA, which becomes available only on membrane disruption in dead or dying cells. Methods: 7E11 as a marker of dying cells was studied by flow cytometry and microscopy of cells after antiandrogen-, radio-, and chemotherapy in LNCaP and PC3 PSMA–positive cells. The in vivo behavior of 89Zr-DFO-7E11 was characterized in mice bearing subcutaneous LNCaP (PSMA-positive) tumors by biodistribution studies and immuno-PET. The potential of assessing tumor response was evaluated in vivo after radiotherapy. Results: In vitro studies correlated 7E11 binding with markers of apoptosis (7-amino-actinomycin-D and caspase-3). In vivo biodistribution experiments revealed high, target-specific uptake of 89Zr-DFO-7E11 in LNCaP tumors after 24 h (20.35 ± 7.50 percentage injected dose per gram [%ID/g]), 48 h (22.82 ± 3.58 %ID/g), 96 h (36.94 ± 6.27 %ID/g), and 120 h (25.23 ± 4.82 %ID/g). Excellent image contrast was observed with immuno-PET. 7E11 uptake was statistically increased in irradiated versus control tumor as measured by immuno-PET and biodistribution studies. Binding specificity was assessed by effective blocking studies at 48 h. Conclusion: These findings suggest that 89Zr-DFO-7E11 displays high tumor–to–background tissue contrast in immuno-PET and can be used as a tool to monitor and quantify, with high specificity, tumor response in PSMA-positive prostate cancer.

In vivo quantitative assessment of cell viability of gadolinium or iron-labeled cells using MRI and bioluminescence imaging

In cell therapy, noninvasive monitoring of in vivo cell fate is challenging. In this study we investigated possible differences in R₁, R₂ or R₂* relaxation rate as a measure of overall cell viability for mesenchymal stem cells labeled with Gd-liposomes (Gd-MSCs) or iron oxide nanoparticles (SPIO-MSCs). Cells were also transduced with a luciferase vector, facilitating a correlation between MRI findings and cell viability using bioluminescence imaging (BLI). Viable Gd-MSCs were clearly distinguishable from nonviable Gd-MSCs under both in vitro and in vivo conditions, clearly differing quantitatively (ΔR₁ and ΔR₂) as well as by visual appearance (hypo- or hyperintense contrast). Immediately post-injection,viable Gd-MSCs caused a substantially larger ΔR₂ and lower ΔR₁ effect compared with nonviable MSCs. With time, the ΔR₁ and ΔR₂ relaxation rate showed a good negative correlation with increasing cell number following proliferation. Upon injection, no substantial quantitative or visual differences between viable and nonviable SPIO-MSCs were detected. Moreover, nonviable SPIO-MSCs caused a persisting signal void in vivo, compromising the specificity of this contrast agent. In vivo persistence of SPIO particles was confirmed by histological staining. A large difference was found between SPIO- and Gd-labeled cells in the accuracy of MR relaxometry in assessing the cell viability status. Gd-liposomes provide a more accurate and specific assessment of cell viability than SPIO particles. Viable Gd cells can be differentiated from nonviable Gd cells even by visual interpretation. These findings clearly indicate Gd to be the favourable contrast agent in qualitative and quantitative evaluation of labeled cell fate in future cell therapy experiments.

Prevalence of interstitial lung involvement in patients with connective tissue diseases assessed with high-resolution computed tomography

Objectives: To assess the prevalence of interstitial lung disease (ILD) in patients with different forms of connective tissue disease (CTD) using non‐invasive procedures including high‐resolution computed tomography (HRCT) and to evaluate the relationship between the imaging and functional status of the patients. Methods: Eighty‐one subjects with CTD (47 inpatients and 34 outpatients) were evaluated with pulmonary function tests (PFT) and radiological investigations. The extent and severity of lung disease was quantified with an HRCT scoring system previously used in patients with systemic sclerosis (SSc). Interstitial lung involvement was defined as predominantly fibrotic or inflammatory based on HRCT abnormalities. Results: HRCT abnormalities suggestive of ILD were observed in 69 patients (85.1%), whereas PFT and plain radiograph alterations occurred less frequently (40.7%). The most frequent HRCT abnormalities were septal/subpleural lines and ground‐glass appearance whereas lesions consistent with advanced fibrosis were observed in a minority of patients. The HRCT score was higher in patients with abnormal PFT (p<0.001). Thirty‐five patients had predominant fibrosis and 34 patients predominantly inflammatory abnormalities. A score of 10 points represented the best compromise between sensitivity and specificity in predicting functional impairment. Conclusions: A high prevalence of ILD was found based on HRCT abnormalities. However, HRCT scans characterized by minor abnormalities have poor specificity for clinically significant disease and functional findings should also be considered. The large number of patients with predominantly inflammatory HRCT abnormalities suggests that many cases of ILD may be diagnosed in a relatively early stage of the disease.

Tumor Volume as an Alternative Response Measurement for Imatinib Treated GIST Patients

Background Assessment of tumor size changes is crucial in clinical trials and patient care. We compared imatinib-induced volume changes of liver metastases (LM) from gastro-intestinal stromal tumors (GIST) to RECIST and Choi criteria and their association with overall survival (OS). Methods LM from 84 GIST patients (training and validation set) were evaluated using manual and semi-automated Computed Tomography measurements at baseline, after 3, 6 and 12 months of imatinib. The ability of uni-dimensional (1D) and three-dimensional (3D) measurements to detect size changes (increase/decrease) ≥20% was evaluated. Volumetric response cut-offs were derived from minimally relevant changes (+20/−30%) by RECIST, considering lesions as spherical or ellipsoidal. Results 3D measurements detected size changes ≥20% more frequently than 1D at every time-point (P≤0.008). 3D and Choi criteria registered more responses than RECIST at 3 and 6 months for 3D-spheres (P≤0.03) and at all time-points for 3D-ellipsoids and Choi criteria (P<0.001). Progressive disease by 3D criteria seems to better correlate to OS at late time-points than other criteria. Conclusion Volume criteria (especially ellipsoids) classify a higher number of patients as imatinib-responders than RECIST. Volume discriminates size changes better than diameter in GIST and constitutes a feasible and robust method to evaluate response and predict patient benefit.

Deconvoluting hepatic processing of carbon nanotubes.

Single-wall carbon nanotubes present unique opportunities for drug delivery, but have not advanced into the clinic. Differential nanotube accretion and clearance from critical organs have been observed, but the mechanism not fully elucidated. The liver has a complex cellular composition that regulates a range of metabolic functions and coincidently accumulates most particulate drugs. Here we provide the unexpected details of hepatic processing of covalently functionalized nanotubes including receptor-mediated endocytosis, cellular trafficking and biliary elimination. Ammonium-functionalized fibrillar nanocarbon is found to preferentially localize in the fenestrated sinusoidal endothelium of the liver but not resident macrophages. Stabilin receptors mediate the endocytic clearance of nanotubes. Biocompatibility is evidenced by the absence of cell death and no immune cell infiltration. Towards clinical application of this platform, nanotubes were evaluated for the first time in non-human primates. The pharmacologic profile in cynomolgus monkeys is equivalent to what was reported in mice and suggests that nanotubes should behave similarly in humans.

Different Strategies for Reducing Intestinal Background Radioactivity Associated with Imaging HSV1- tk Expression Using Established Radionucleoside Probes

One limitation of HSV1-tk reporter positron emission tomography (PET) with nucleoside analogues is the high background radioactivity in the intestine. We hypothesized that endogenous expression of thymidine kinase in bacterial flora could phosphorylate and trap such radiotracers, contributing to the high radioactivity levels in the bowel, and therefore explored different strategies to increase fecal elimination of radiotracer. Intestinal radioactivity was assessed by in vivo microPET imaging and ex vivo tissue sampling following intravenous injection of 18F-FEAU, 124I-FIAU, or 18F-FHBG in a germ-free mouse strain. We also explored the use of an osmotic laxative agent and/or a 100% enzymatically hydrolyzed liquid diet. No significant differences in intestinal radioactivity were observed between germ-free and normal mice. 18F-FHBG-derived intestinal radioactivity levels were higher than those of 18F-FEAU and 124I-FIAU; the intestine to blood ratio was more than 20-fold higher for 18F-FHBG than for 18F-FEAU and 124I-FIAU. The combination of Peptamen and Nulytely lowered intestinal radioactivity levels and increased (2.2-fold) the HSV1-tk transduced xenograft to intestine ratio for 18F-FEAU. Intestinal bacteria in germ-free mice do not contribute to the high intestinal levels of radioactivity following injection of radionucleoside analogues. The combination of Peptamen and Nulytely increased radiotracer elimination by increasing bowel motility without inducing dehydration.