Valerie A. Longo

Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma

Nanoparticle-based materials, such as drug delivery vehicles and diagnostic probes, currently under evaluation in oncology clinical trials are largely not tumor selective. To be clinically successful, the next generation of nanoparticle agents should be tumor selective, nontoxic, and exhibit favorable targeting and clearance profiles. Developing probes meeting these criteria is challenging, requiring comprehensive in vivo evaluations. Here, we describe our full characterization of an approximately 7-nm diameter multimodal silica nanoparticle, exhibiting what we believe to be a unique combination of structural, optical, and biological properties. This ultrasmall cancer-selective silica particle was recently approved for a first-in-human clinical trial. Optimized for efficient renal clearance, it concurrently achieved specific tumor targeting. Dye-encapsulating particles, surface functionalized with cyclic arginine-glycine-aspartic acid peptide ligands and radioiodine, exhibited high-affinity/avidity binding, favorable tumor-to-blood residence time ratios, and enhanced tumor-selective accumulation in αvβ3 integrin-expressing melanoma xenografts in mice. Further, the sensitive, real-time detection and imaging of lymphatic drainage patterns, particle clearance rates, nodal metastases, and differential tumor burden in a large-animal model of melanoma highlighted the distinct potential advantage of this multimodal platform for staging metastatic disease in the clinical setting.

Measuring the Pharmacodynamic Effects of a Novel Hsp90 Inhibitor on HER2/neu Expression in Mice Using 89Zr-DFO-Trastuzumab

Background The positron-emitting radionuclide 89Zr (t 1/2 = 3.17 days) was used to prepare 89Zr-radiolabeled trastuzumab for use as a radiotracer for characterizing HER2/neu-positive breast tumors. In addition, pharmacodynamic studies on HER2/neu expression levels in response to therapeutic doses of PU-H71 (a specific inhibitor of heat-shock protein 90 [Hsp90]) were conducted. Methodology/Principal Findings Trastuzumab was functionalized with desferrioxamine B (DFO) and radiolabeled with [89Zr]Zr-oxalate at room temperature using modified literature methods. ImmunoPET and biodistribution experiments in female, athymic nu/nu mice bearing sub-cutaneous BT-474 (HER2/neu positive) and/or MDA-MB-468 (HER2/neu negative) tumor xenografts were conducted. The change in 89Zr-DFO-trastuzumab tissue uptake in response to high- and low-specific-activity formulations and co-administration of PU-H71 was evaluated by biodistribution studies, Western blot analysis and immunoPET. 89Zr-DFO-trastuzumab radiolabeling proceeded in high radiochemical yield and specific-activity 104.3±2.1 MBq/mg (2.82±0.05 mCi/mg of mAb). In vitro assays demonstrated >99% radiochemical purity with an immunoreactive fraction of 0.87±0.07. In vivo biodistribution experiments revealed high specific BT-474 uptake after 24, 48 and 72 h (64.68±13.06%ID/g; 71.71±10.35%ID/g and 85.18±11.10%ID/g, respectively) with retention of activity for over 120 h. Pre-treatment with PU-H71 was followed by biodistribution studies and immunoPET of 89Zr-DFO-trastuzumab. Expression levels of HER2/neu were modulated during the first 24 and 48 h post-administration (29.75±4.43%ID/g and 41.42±3.64%ID/g, respectively). By 72 h radiotracer uptake (73.64±12.17%ID/g) and Western blot analysis demonstrated that HER2/neu expression recovered to baseline levels. Conclusions/Significance The results indicate that 89Zr-DFO-trastuzumab provides quantitative and highly-specific delineation of HER2/neu positive tumors, and has potential to be used to measure the efficacy of long-term treatment with Hsp90 inhibitors, like PU-H71, which display extended pharmacodynamic profiles.

Endogenous expression of HrasG12V induces developmental defects and neoplasms with copy number imbalances of the oncogene

We developed mice with germline endogenous expression of oncogenic Hras to study effects on development and mechanisms of tumor initiation. They had high perinatal mortality, abnormal cranial dimensions, defective dental ameloblasts, and nasal septal deviation, consistent with some of the features of human Costello syndrome. These mice developed papillomas and angiosarcomas, which were associated with HrasG12V allelic imbalance and augmented Hras signaling. Endogenous expression of HrasG12V was also associated with a higher mutation rate in vivo. Tumor initiation by HrasG12V likely requires augmentation of signal output, which in papillomas and angiosarcomas is achieved via increased Hras-gene copy number, which may be favored by a higher mutation frequency in cells expressing the oncoprotein.

18F-Labeled-Bioorthogonal Liposomes for In Vivo Targeting

Liposomes are attractive vehicles for the controlled release of drugs and cytotoxins and have a long-standing history in medical research and clinical practice. In addition to established therapeutic indications, liposomes have several favorable properties for molecular imaging, including high stability and the ability to be labeled with radioisotopes, as well as paramagnetic and fluorescent contrast agents. However, long circulation times and difficulties in creating targeted liposomes have proven challenges for imaging. In this study, we have addressed these limitations using a recently developed strategy for bioorthogonal conjugation, the reaction between tetrazines and trans-cyclooctenes. By coating radiolabeled liposomes with trans-cyclooctene and pretargeting with a tetrazine coupled to a targeted peptide, we were able to selectively enhance the retention of liposomes and bind them to tumor tissue in live animals. The rapid reaction between tetrazines and trans-cyclooctenes allowed imaging to be performed with the short-lived PET tracer (18)F, yielding signal-to-background activity ratios of 7:1. The covalent, bioorthogonally driven tumor-targeting of liposomes by in vivo click chemistry is promising and should be explored for more selective and rapid delivery of radiodiagnostics and radiotherapeutics, two classes of drugs which particularly benefit from fast clearance, low nonspecific binding, and the associated reduced toxicity to kidneys and bone marrow.

A Modular Labeling Strategy for In Vivo PET and Near-Infrared Fluorescence Imaging of Nanoparticle Tumor Targeting

Advances in preclinical molecular imaging have generated new opportunities to noninvasively visualize the biodistribution and tumor targeting of nanoparticle therapeutics. Capitalizing on recent achievements in this area, we sought to develop an 89Zr-based labeling strategy for liposomal nanoparticles that accumulate in tumors via passive targeting mechanisms. Methods: 89Zr-labeled liposomes were prepared using 2 different approaches: click labeling and surface chelation. Pharmacokinetic and biodistribution studies, as well as PET/CT imaging of the radiolabeled nanoparticles, were performed on a mouse model of breast cancer. In addition, a dual PET/optical probe was prepared by incorporation of a near-infrared fluorophore and tested in vivo by PET and near-infrared fluorescence imaging. Results: The surface chelation approach proved to be superior in terms of radiochemical yield and stability, as well as in vivo performance. Accumulation of these liposomes in tumor peaked at 24 h after injection and was measured to be 13.7 ± 1.8 percentage injected dose per gram. The in vivo performance of this probe was not essentially perturbed by the incorporation of a near-infrared fluorophore. Conclusion: We have developed a highly modular and efficient strategy for the labeling of liposomal nanoparticles with 89Zr. In xenograft and orthotopic mouse models of breast cancer, we demonstrated that the biodistribution of these nanoparticles can be visualized by PET imaging. In combination with a near-infrared dye, these liposomal nanoparticles can serve as bimodal PET/optical imaging agents. The liposomes target malignant growth, and their bimodal features may be useful for simultaneous PET and intraoperative imaging.

Dynamic Small-Animal PET Imaging of Tumor Proliferation with 3′-Deoxy-3′-18F-Fluorothymidine in a Genetically Engineered Mouse Model of High-Grade Gliomas

3′-Deoxy-3′-18F-fluorothymidine (18F-FLT), a partially metabolized thymidine analog, has been used in preclinical and clinical settings for the diagnostic evaluation and therapeutic monitoring of tumor proliferation status. We investigated the use of 18F-FLT for detecting and characterizing genetically engineered mouse (GEM) high-grade gliomas and evaluating the pharmacokinetics in GEM gliomas and normal brain tissue. Our goal was to develop a robust and reproducible method of kinetic analysis for the quantitative evaluation of tumor proliferation. Methods: Dynamic 18F-FLT PET imaging was performed for 60 min in glioma-bearing mice (n = 10) and in non–tumor-bearing control mice (n = 4) by use of a dedicated small-animal PET scanner. A 3-compartment, 4-parameter model was used to characterize 18F-FLT kinetics in vivo. For compartmental analysis, the arterial input was measured by placing a region of interest over the left ventricular blood pool and was corrected for partial-volume averaging. The 18F-FLT “trapping” and tissue flux model parameters were correlated with measured uptake (percentage injected dose per gram [%ID/g]) values at 60 min. Results: 18F-FLT uptake values (%ID/g) at 1 h in brain tumors were significantly greater than those in control brains (mean ± SD: 4.33 ± 0.58 and 0.86 ± 0.22, respectively; P < 0.0004). Kinetic analyses of the measured time–activity curves yielded independent, robust estimates of tracer transport and metabolism, with compartmental model–derived time–activity data closely fitting the measured data. Except for tracer transport, statistically significant differences were found between the applicable model parameters for tumors and normal brains. The tracer retention rate constant strongly correlated with measured 18F-FLT uptake values (r = 0.85, P < 0.0025), whereas a more moderate correlation was found between net 18F-FLT flux and 18F-FLT uptake values (r = 0.61, P < 0.02). Conclusion: A clinically relevant mouse glioma model was characterized by both static and dynamic small-animal PET imaging of 18F-FLT uptake. Time–activity curves were kinetically modeled to distinguish early transport from a subsequent tracer retention phase. Estimated 18F-FLT rate constants correlated positively with %ID/g measurements. Dynamic evaluation of 18F-FLT uptake offers a promising approach for noninvasively assessing cellular proliferation in vivo and for quantitatively monitoring new antiproliferation therapies.

Gold Nanoparticles Provide Bright Long-Lasting Vascular Contrast for CT Imaging

OBJECTIVE Iodinated contrast agent for CT has a short half-life in the vasculature. As the field of interventional procedures expands, a more durable contrast agent would be highly useful. Our study investigated whether gold nanoparticles are feasible as a long-lasting vascular contrast agent for CT. MATERIALS AND METHODS Gold nanoparticles were synthesized by a modified Turkevich method, coated with methoxy-polyethylene glycol-thiol chains, and compared with an iodine-based contrast agent used in mice. Contrast agents were imaged in tubes by CT at 40, 60, and 140 kVp and then were tested in vivo by tail vein injection. Nine mice received gold nanoparticles, two received iodine-based contrast agent, and one received saline. CT of mice was performed at 60 kVp immediately, 6 hours, and 24 hours after injection. RESULTS In an isolated form in tubes, gold nanoparticles had greater radiographic density than did iodine-based contrast agent at 40 kVp and were comparable at the other CT voltages. In mice, gold nanoparticles provided bright contrast enhancement that enabled clear visualization of the abdominal aorta and renal arteries, which could not be distinguished without contrast agent. This persisted up to 24 hours, which was the last time point assessed. Contrast enhancement of the vasculature by iodine-based contrast agent was present immediately after injection but had disappeared by 6 hours. CONCLUSION Gold nanoparticles can provide clear and durable contrast enhancement of the vasculature even at 24 hours. These findings merit further study of gold nanoparticles for their potential as a contrast agent in CT and CT-guided interventional procedures.

A Vaccinia Virus Encoding the Human Sodium Iodide Symporter Facilitates Long-Term Image Monitoring of Virotherapy and Targeted Radiotherapy of Pancreatic Cancer

To assess therapeutic response and potential toxicity of oncolytic virotherapy, a noninvasive, deep-tissue imaging modality is needed. This study aimed to assess the feasibility, parameters, and determining factors of serial imaging and long-term monitoring of virotherapy and the therapeutic response of pancreatic cancer xenografts treated with a vaccinia virus carrying the human sodium iodide symporter GLV-1h153. Methods: Pancreatic cancer xenografts (PANC-1) in nude mice were treated systemically or intratumorally with GLV-1h153 and serially imaged using 124I PET at 1, 2, 3, and 5 wk after viral injection. Signal intensity was compared with tumor therapeutic response and optical imaging, and tumors were histologically analyzed for morphology and the presence of virus. Autoradiography was performed using technetium-pertechnetate and γ-scintigraphy to assess determining factors for radiouptake in tumors. Finally, the enhanced therapeutic effect of combination therapy with GLV-1h153 and systemic radioiodine was assessed. Results: GLV-1h153 successfully facilitated serial long-term imaging of virotherapy, with PET signal intensity correlating to tumor response. GLV-1h153 colonization of tumors mediated radioiodine uptake at potentially therapeutic doses. Successful radiouptake required the presence of virus, adequate blood flow, and viable tissue, whereas loss of signal intensity was linked to tumor death and necrosis. Finally, combining systemically administered GLV-1h153 and 131I led to enhanced tumor kill when compared with virus or 131I alone (P < 0.01). Conclusion: GLV-1h153 is a promising oncolytic agent for the treatment, long-term imaging, and monitoring of therapeutic response in a xenograft model of pancreatic cancer. GLV-1h153 provided insight into tumor biologic activity and facilitated enhanced tumor kill when combined with systemic targeted radiotherapy. These results warrant further investigation into parameters and potential synergistic effects of combination therapy.

Vaccinia virus GLV-1h153 in combination with 131I shows increased efficiency in treating triple-negative breast cancer

We investigated the therapeutic efficacy of a replication‐competent oncolytic vaccinia virus, GLV‐1h153, carrying human sodium iodide symporter (hNIS), in combination with radioiodine in an orthotopic triple‐negative breast cancer (TNBC) murine model. In vitro viral infection was confirmed by immunoblotting and radioiodine uptake assays. Orthotopic xenografts (MDA‐MB‐231 cells) received intratumoral injection of GLV‐1h153 or PBS. One week after viral injection, xenografts were randomized into 4 treatment groups: GLV‐1h153 alone, GLV‐1h153 and 131I (~5 mCi), 131I alone, or PBS, and followed for tumor growth. Kruskal‐Wallis and Wilcoxon tests were performed for statistical analysis. Radiouptake assay showed a 178‐fold increase of radioiodine uptake in hNIS‐expressing infected cells compared with PBS control. Systemic 131I‐iodide in combination with GLV‐1h153 resulted in a 6‐fold increase in tumor regression (24 compared to 146 mm3 for the virus‐only treatment group; P<0.05; d 40). We demonstrated that a novel vaccinia virus, GLV‐1h153, expresses hNIS, increases the expression of the symporter in TNBC cells, and serves both as a gene marker for noninvasive imaging of virus and as a vehicle for targeted radionuclide therapy with 131I.—Gholami, S., Chen, C‐H., Lou, E., Belin, L. J., Fujisawa, S., Longo, V. A. Chen, N. G., Gönen, M., Zanzonico, P. B., Szalay, A. A., Fong, Y. Vaccinia virus GLV‐1h153 in combination with 131I shows increased efficiency in treating triple‐negative breast cancer. FASEB J. 28, 676–682 (2014). www.fasebj.org

Registration of planar bioluminescence to magnetic resonance and x-ray computed tomography images as a platform for the development of bioluminescence tomography reconstruction algorithms

The procedures we propose make possible the mapping of two-dimensional (2-D) bioluminescence image (BLI) data onto a skin surface derived from a three-dimensional (3-D) anatomical modality [magnetic resonance (MR) or computed tomography (CT)] dataset. This mapping allows anatomical information to be incorporated into bioluminescence tomography (BLT) reconstruction procedures and, when applied using sources visible to both optical and anatomical modalities, can be used to evaluate the accuracy of those reconstructions. Our procedures, based on immobilization of the animal and a priori determined fixed projective transforms, should be more robust and accurate than previously described efforts, which rely on a poorly constrained retrospectively determined warping of the 3-D anatomical information. Experiments conducted to measure the accuracy of the proposed registration procedure found it to have a mean error of 0.36+/-0.23 mm. Additional experiments highlight some of the confounds that are often overlooked in the BLT reconstruction process, and for two of these confounds, simple corrections are proposed.