Ligand-targeted theranostic nanomedicines against cancer

AbstractNanomedicines have significant potential for cancer treatment. Although the majority of nanomedicines currently tested in clinical trials utilize simple, biocompatible liposome-based nanocarriers, their widespread use is limited by non-specificity and low target site concentration and thus, do not provide a substantial clinical advantage over conventional, systemic chemotherapy. In the past 20years, we have identified specific receptors expressed on the surfaces of tumor endothelial and perivascular cells, tumor cells, the extracellular matrix and stromal cells using combinatorial peptide libraries displayed on bacteriophage. These studies corroborate the notion that unique receptor proteins such as IL-11Rα, GRP78, EphA5, among others, are differentially overexpressed in tumors and present opportunities to deliver tumor-specific therapeutic drugs. By using peptides that bind to tumor-specific cell-surface receptors, therapeutic agents such as apoptotic peptides, suicide genes, imaging dyes or chemotherapeutics can be precisely and systemically delivered to reduce tumor growth in vivo, without harming healthy cells. Given the clinical applicability of peptide-based therapeutics, targeted delivery of nanocarriers loaded with therapeutic cargos seems plausible. We propose a modular design of a functionalized protocell in which a tumor-targeting moiety, such as a peptide or recombinant human antibody single chain variable fragment (scFv), is conjugated to a lipid bilayer surrounding a silica-based nanocarrier core containing a protected therapeutic cargo. The functionalized protocell can be tailored to a specific cancer subtype and treatment regimen by exchanging the tumor-targeting moiety and/or therapeutic cargo or used in combination to create unique, theranostic agents. In this review, we summarize the identification of tumor-specific receptors through combinatorial phage display technology and the use of antibody display selection to identify recombinant human scFvs against these tumor-specific receptors. We compare the characteristics of different types of simple and complex nanocarriers, and discuss potential types of therapeutic cargos and conjugation strategies. The modular design of functionalized protocells may improve the efficacy and safety of nanomedicines for future cancer therapy

Cerenkov Radiation Energy Transfer (CRET) Imaging: A Novel Method for Optical Imaging of PET Isotopes in Biological Systems

Positron emission tomography (PET) allows sensitive, non-invasive analysis of the distribution of radiopharmaceutical tracers labeled with positron (β(+))-emitting radionuclides in small animals and humans. Upon β(+) decay, the initial velocity of high-energy β(+) particles can momentarily exceed the speed of light in tissue, producing Cerenkov radiation that is detectable by optical imaging, but is highly absorbed in living organisms.To improve optical imaging of Cerenkov radiation in biological systems, we demonstrate that Cerenkov radiation from decay of the PET isotopes (64)Cu and (18)F can be spectrally coupled by energy transfer to high Stokes-shift quantum nanoparticles (Qtracker705) to produce highly red-shifted photonic emissions. Efficient energy transfer was not detected with (99m)Tc, a predominantly γ-emitting isotope. Similar to bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET), herein we define the Cerenkov radiation energy transfer (CRET) ratio as the normalized quotient of light detected within a spectral window centered on the fluorophore emission divided by light detected within a spectral window of the Cerenkov radiation emission to quantify imaging signals. Optical images of solutions containing Qtracker705 nanoparticles and [(18)F]FDG showed CRET ratios in vitro as high as 8.8±1.1, while images of mice with subcutaneous pseudotumors impregnated with Qtracker705 following intravenous injection of [(18)F]FDG showed CRET ratios in vivo as high as 3.5±0.3.Quantitative CRET imaging may afford a variety of novel optical imaging applications and activation strategies for PET radiopharmaceuticals and other isotopes in biomaterials, tissues and live animals

Quantitative Modeling of Cerenkov Light Production Efficiency from Medical Radionuclides

There has been recent and growing interest in applying Cerenkov radiation (CR) for biological applications. Knowledge of the production efficiency and other characteristics of the CR produced by various radionuclides would help in accessing the feasibility of proposed applications and guide the choice of radionuclides. To generate this information we developed models of CR production efficiency based on the Frank-Tamm equation and models of CR distribution based on Monte-Carlo simulations of photon and β particle transport. All models were validated against direct measurements using multiple radionuclides and then applied to a number of radionuclides commonly used in biomedical applications. We show that two radionuclides, Ac-225 and In-111, which have been reported to produce CR in water, do not in fact produce CR directly. We also propose a simple means of using this information to calibrate high sensitivity luminescence imaging systems and show evidence suggesting that this calibration may be more accurate than methods in routine current use

Targeted molecular-genetic imaging and ligand-directed therapy in aggressive variant prostate cancer

Aggressive variant prostate cancers (AVPC) are a clinically defined group of tumors of heterogeneous morphologies, characterized by poor patient survival and for which limited diagnostic and treatment options are currently available. We show that the cell surface 78-kDa glucose-regulated protein (GRP78), a receptor that binds to phage-display-selected ligands, such as the SNTRVAP motif, is a candidate target in AVPC. We report the presence and accessibility of this receptor in clinical specimens from index patients. We also demonstrate that human AVPC cells displaying GRP78 on their surface could be effectively targeted both in vitro and in vivo by SNTRVAP, which also enabled specific delivery of siRNA species to tumor xenografts in mice. Finally, we evaluated ligand-directed strategies based on SNTRVAP-displaying adeno-associated virus/phage (AAVP) particles in mice bearing MDA-PCa-118b, a patient-derived xenograft (PDX) of castration-resistant prostate cancer bone metastasis that we exploited as a model of AVPC. For theranostic (a merging of the terms therapeutic and diagnostic) studies, GRP78-targeting AAVP particles served to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) gene, which has a dual function as a molecular-genetic sensor/reporter and a cell suicide-inducing transgene. We observed specific and simultaneous PET imaging and treatment of tumors in this preclinical model of AVPC. Our findings demonstrate the feasibility of GPR78-targeting, ligand-directed theranostics for translational applications in AVPC

Prognostic Value of [18F]-Fluoro-Deoxy-Glucose PET/CT, S100 or MIA for Assessment of Cancer-Associated Mortality in Patients with High Risk Melanoma

PURPOSE: To assess the prognostic value of FDG PET/CT compared to the tumor markers S100B and melanoma inhibitory activity (MIA) in patients with high risk melanoma. METHODS: Retrospective study in 125 consecutive patients with high risk melanoma that underwent FDG PET/CT for re-staging. Diagnostic accuracy and prognostic value was determined for FDG PET/CT as well as for S100B and MIA. As standard of reference, cytological, histological, PET/CT or MRI follow-up findings as well as clinical follow-up were used. RESULTS: Of 125 patients, FDG PET/CT was positive in 62 patients. 37 (29.6%) patients had elevated S100B (>100 pg/ml) and 24 (20.2%) had elevated MIA (>10 pg/ml) values. Overall specificities for FDG PET/CT, S100B and MIA were 96.8% (95% CI, 89.1% to 99.1%), 85.7% (75.0% to 92.3%), and 95.2% (86.9% to 98.4%), corresponding sensitivities were 96.8% (89.0% to 99.1%), 45.2% (33.4% to 55.5%), and 36.1% (25.2% to 48.6%), respectively. The negative predictive values (NPV) for PET/CT, S100B, and MIA were 96.8% (89.1% to 99.1%), 61.4% (50.9% to 70.9%), and 60.6% (50.8% to 69.7%). The positive predictive values (PPV) were 96.7% (89.0% to 99.1%), 75.7% (59.9% to 86.6%), and 88.0% (70.0% to 95.8%). Patients with elevated S100B- or MIA values or PET/CT positive findings showed a significantly (p<0.001 each, univariate Cox regression models) higher risk of melanoma associated death which was increased 4.2-, 6.5- or 17.2-fold, respectively. CONCLUSION: PET/CT has a higher prognostic power in the assessment of cancer-associated mortality in melanoma patients compared with S100 and MIA

Quantitative Analysis and Comparison Study of [18F]AlF-NOTA-PRGD2, [18F]FPPRGD2 and [68Ga]Ga-NOTA-PRGD2 Using a Reference Tissue Model

With favorable pharmacokinetics and binding affinity for αvβ3 integrin, 18F-labeled dimeric cyclic RGD peptide ([18F]FPPRGD2) has been intensively used as a PET imaging probe for lesion detection and therapy response monitoring. A recently introduced kit formulation method, which uses an 18F-fluoride-aluminum complex labeled RGD tracer ([18F]AlF-NOTA-PRGD2), provides a strategy for simplifying the labeling procedure to facilitate clinical translation. Meanwhile, an easy-to-prepare 68Ga-labeled NOTA-PRGD2 has also been reported to have promising properties for imaging integrin αvβ3. The purpose of this study is to quantitatively compare the pharmacokinetic parameters of [18F]FPPRGD2, [18F]AlF-NOTA-PRGD2, and [68Ga]Ga-NOTA-PRGD2. U87MG tumor-bearing mice underwent 60-min dynamic PET scans following the injection of three tracers. Kinetic parameters were calculated using Logan graphical analysis with reference tissue. Parametric maps were generated using voxel-level modeling. All three compounds showed high binding potential (BpND = k3/k4) in tumor voxels. [18F]AlF-NOTA-PRGD2 showed comparable BpND value (3.75±0.65) with those of [18F]FPPRGD2 (3.39±0.84) and [68Ga]Ga-NOTA-PRGD2 (3.09±0.21) (p>0.05). Little difference was found in volume of distribution (VT) among these three RGD tracers in tumor, liver and muscle. Parametric maps showed similar kinetic parameters for all three tracers. We also demonstrated that the impact of non-specific binding could be eliminated in the kinetic analysis. Consequently, kinetic parameter estimation showed more comparable results among groups than static image analysis. In conclusion, [18F]AlF-NOTA-PRGD2 and [68Ga]Ga-NOTA-PRGD2 have comparable pharmacokinetics and quantitative parameters compared to those of [18F]FPPRGD2. Despite the apparent difference in tumor uptake (%ID/g determined from static images) and clearance pattern, the actual specific binding component extrapolated from kinetic modeling appears to be comparable for all three dimeric RGD tracers

Computed Tomography Imaging of Primary Lung Cancer in Mice Using a Liposomal-Iodinated Contrast Agent

To investigate the utility of a liposomal-iodinated nanoparticle contrast agent and computed tomography (CT) imaging for characterization of primary nodules in genetically engineered mouse models of non-small cell lung cancer.Primary lung cancers with mutations in K-ras alone (Kras(LA1)) or in combination with p53 (LSL-Kras(G12D);p53(FL/FL)) were generated. A liposomal-iodine contrast agent containing 120 mg Iodine/mL was administered systemically at a dose of 16 µl/gm body weight. Longitudinal micro-CT imaging with cardio-respiratory gating was performed pre-contrast and at 0 hr, day 3, and day 7 post-contrast administration. CT-derived nodule sizes were used to assess tumor growth. Signal attenuation was measured in individual nodules to study dynamic enhancement of lung nodules.A good correlation was seen between volume and diameter-based assessment of nodules (R(2)>0.8) for both lung cancer models. The LSL-Kras(G12D);p53(FL/FL) model showed rapid growth as demonstrated by systemically higher volume changes compared to the lung nodules in Kras(LA1) mice (p<0.05). Early phase imaging using the nanoparticle contrast agent enabled visualization of nodule blood supply. Delayed-phase imaging demonstrated significant differential signal enhancement in the lung nodules of LSL-Kras(G12D);p53(FL/FL) mice compared to nodules in Kras(LA1) mice (p<0.05) indicating higher uptake and accumulation of the nanoparticle contrast agent in rapidly growing nodules.The nanoparticle iodinated contrast agent enabled visualization of blood supply to the nodules during the early-phase imaging. Delayed-phase imaging enabled characterization of slow growing and rapidly growing nodules based on signal enhancement. The use of this agent could facilitate early detection and diagnosis of pulmonary lesions as well as have implications on treatment response and monitoring

Use of Cis-[18F]Fluoro-Proline for Assessment of Exercise-Related Collagen Synthesis in Musculoskeletal Connective Tissue

Protein turnover in collagen rich tissue is influenced by exercise, but can only with difficulty be studied in vivo due to use of invasive procedure. The present study was done to investigate the possibility of applying the PET-tracer, cis-[18F]fluoro-proline (cis-Fpro), for non-invasive assessment of collagen synthesis in rat musculoskeletal tissues at rest and following short-term (3 days) treadmill running. Musculoskeletal collagen synthesis was studied in rats at rest and 24 h post-exercise. At each session, rats were PET scanned at two time points following injection of cis-FPro: (60 and 240 min p.i). SUV were calculated for Achilles tendon, calf muscle and tibial bone. The PET-derived results were compared to mRNA expression of collagen type I and III. Tibial bone had the highest SUV that increased significantly (p<0.001) from the early (60 min) to the late (240 min) PET scan, while SUV in tendon and muscle decreased (p<0.001). Exercise had no influence on SUV, which was contradicted by an increased gene expression of collagen type I and III in muscle and tendon. The clearly, visible uptake of cis-Fpro in the collagen-rich musculoskeletal tissues is promising for multi-tissue studies in vivo. The tissue-specific differences with the highest basal uptake in bone are in accordance with earlier studies relying on tissue incorporation of isotopic-labelled proline. A possible explanation of the failure to demonstrate enhanced collagen synthesis following exercise, despite augmented collagen type I and III transcription, is that SUV calculations are not sensitive enough to detect minor changes in collagen synthesis. Further studies including kinetic compartment modeling must be performed to establish whether cis-Fpro can be used for non-invasive in-vivo assessment of exercise-induced changes in musculoskeletal collagen synthesis

Heterozygosity for Pten Promotes Tumorigenesis in a Mouse Model of Medulloblastoma

BACKGROUND: Recent publications have described an important role for cross talk between PI-3 kinase and sonic hedgehog signaling pathways in the pathogenesis of medulloblastoma. METHODOLOGY/PRINCIPAL FINDINGS: We crossed mice with constitutive activation of Smoothened, SmoA1, with Pten deficient mice. Both constitutive and conditional Pten deficiency doubled the incidence of mice with symptoms of medulloblastoma and resulted in decreased survival. Analysis revealed a clear separation of gene signatures, with up-regulation of genes in the PI-3 kinase signaling pathway, including downstream activation of angiogenesis in SmoA1+/-; Pten +/- medulloblastomas. Western blotting and immunohistochemistry confirmed reduced or absent Pten, Akt activation, and increased angiogenesis in Pten deficient tumors. Down-regulated genes included genes in the sonic hedgehog pathway and tumor suppressor genes. SmoA1+/-; Pten +/+ medulloblastomas appeared classic in histology with increased proliferation and diffuse staining for apoptosis. In contrast, Pten deficient tumors exhibited extensive nodularity with neuronal differentiation separated by focal areas of intense staining for proliferation and virtually absent apoptosis. Examination of human medulloblastomas revealed low to absent PTEN expression in over half of the tumors. Kaplan-Meier analysis confirmed worse overall survival in patients whose tumor exhibited low to absent PTEN expression. CONCLUSIONS/SIGNIFICANCE: This suggests that PTEN expression is a marker of favorable prognosis and mouse models with activation of PI-3 kinase pathways may be important tools for preclinical evaluation of promising agents for the treatment of medulloblastoma