Tumor Cell-Derived Extracellular Vesicle-Coated Nanocarriers: An Efficient Theranostic Platform for the Cancer-Specific Delivery of Anti-miR-21 and Imaging Agents.

MicroRNAs are critical regulators of cancer initiation, progression, and dissemination. Extensive evidence suggests that the inhibition of over-expressed oncogenic miRNA function can be a robust strategy for anticancer therapy. However, in vivo targeted delivery of miRNA therapeutics to various types of cancers remains a major challenge. Inspired by their natural synthesis and cargo delivery capabilities, researchers have exploited tumor cell-derived extracellular vesicles (TEVs) for the cancer-targeted delivery of therapeutics and theranostics. Here, we investigate a TEV-based nanoplatform for multimodal miRNA delivery and phototherapy treatments as well as the magnetic resonance imaging of cancer. We demonstrated loading of anti-miR-21 that blocks the function of endogenous oncogenic miR-21 over-expressed in cancer cells into and subsequent delivery by TEVs derived from 4T1 cells. We also produced Cy5-anti-miR-21-loaded TEVs from two other cancer cell lines (HepG2 and SKBR3) and confirmed their robust homologous and heterologous transfection efficiency and intracellular Cy5-anti-miR-21 delivery. Additionally, TEV-mediated anti-miR-21 delivery attenuated doxorubicin (DOX) resistance in breast cancer cells with a 3-fold higher cell kill efficiency than in cells treated with DOX alone. We then investigated TEVs as a biomimetic source for the functionalization of gold-iron oxide nanoparticles (GIONs) and demonstrated nanotheranostic properties of TEV-GIONs in vitro. TEV-GIONs demonstrated excellent T2 contrast in in vitro magnetic resonance (MR) imaging and resulted in efficient photothermal effect in 4T1 cells. We also evaluated the biodistribution and theranostic property of anti-miR-21 loaded TEV-GIONs in vivo by labeling with indocyanine green near-infrared dye. We further validated the tumor specific accumulation of TEV-GIONs using MR imaging. Our findings demonstrate that the distribution pattern of the TEV-anti-miR-21-GIONs correlated well with the tumor-targeting capability as well as the activity and efficacy obtained in response to doxorubicin combination treatments. TEVs and TEV-GIONs are promising nanotheranostics for future applications in cancer molecular imaging and therapy.

Synergistic inhibition of glioma cell proliferation by Withaferin A and tumor treating fields.

Glioblastoma (GBM) is the most aggressive and lethal form of brain cancer. Standard therapies are non-specific and often of limited effectiveness; thus, efforts are underway to uncover novel, unorthodox therapies against GBM. In previous studies, we investigated Withaferin A, a steroidal lactone from Ayurvedic medicine that inhibits proliferation in cancers including GBM. Another novel approach, tumor treating fields (TTFields), is thought to disrupt mitotic spindle formation and stymie proliferation of actively dividing cells. We hypothesized that combining TTFields with Withaferin A would synergistically inhibit proliferation in glioblastoma. Human glioblastoma cells (GBM2, GBM39, U87-MG) and human breast adenocarcinoma cells (MDA-MB-231) were isolated from primary tumors. The glioma cell lines were genetically engineered to express firefly luciferase. Proliferative potential was assessed either by bioluminescence imaging or cell counting via hemocytometer. TTFields (4 V/cm) significantly inhibited growth of the four cancer cell lines tested (n = 3 experiments per time point, four measurements per sample, p < 0.02 at least; 2-way ANOVA, control vs. treatment). The combination of Withaferin A (10-100 nM) with TTFields significantly inhibited the growth of the glioma cells to a degree beyond that of Withaferin A or TTFields alone. The interaction of the Withaferin A and TTFields on glioma cells was found to be synergistic in nature (p < 0.01, n = 3 experiments). These findings were validated by both bioluminescence and hemocytometric measurements. The combination of Withaferin A with TTFields represents a novel approach to treat GBM in a manner that is likely better than either treatment alone and that is synergistic.

Imaging B Cells in a Mouse Model of Multiple Sclerosis Using 64Cu-Rituximab PET.

B lymphocytes are a key pathologic feature of multiple sclerosis (MS) and are becoming an important therapeutic target for this condition. Currently, there is no approved technique to noninvasively visualize B cells in the central nervous system (CNS) to monitor MS disease progression and response to therapies. Here, we evaluated 64Cu-rituximab, a radiolabeled antibody specifically targeting the human B cell marker CD20, for its ability to image B cells in a mouse model of MS using PET. Methods: To model CNS infiltration by B cells, experimental autoimmune encephalomyelitis (EAE) was induced in transgenic mice that express human CD20 on B cells. EAE mice were given subcutaneous injections of myelin oligodendrocyte glycoprotein fragment1-125 emulsified in complete Freund adjuvant. Control mice received complete Freund adjuvant alone. PET imaging of EAE and control mice was performed 1, 4, and 19 h after 64Cu-rituximab administration. Mice were perfused and sacrificed after the final PET scan, and radioactivity in dissected tissues was measured with a γ-counter. CNS tissues from these mice were immunostained to quantify B cells or were further analyzed via digital autoradiography. Results: Lumbar spinal cord PET signal was significantly higher in EAE mice than in controls at all evaluated time points (e.g., 1 h after injection: 5.44 ± 0.37 vs. 3.33 ± 0.20 percentage injected dose [%ID]/g, P < 0.05). 64Cu-rituximab PET signal in brain regions ranged between 1.74 ± 0.11 and 2.93 ± 0.15 %ID/g for EAE mice, compared with 1.25 ± 0.08 and 2.24 ± 0.11 %ID/g for controls (P < 0.05 for all regions except striatum and thalamus at 1 h after injection). Similarly, ex vivo biodistribution results revealed notably higher 64Cu-rituximab uptake in the brain and spinal cord of huCD20tg EAE, and B220 immunostaining verified that increased 64Cu-rituximab uptake in CNS tissues corresponded with elevated B cells. Conclusion: B cells can be detected in the CNS of EAE mice using 64Cu-rituximab PET. Results from these studies warrant further investigation of 64Cu-rituximab in EAE models and consideration of use in MS patients to evaluate its potential for detecting and monitoring B cells in the progression and treatment of this disease. These results represent an initial step toward generating a platform to evaluate B cell-targeted therapeutics en route to the clinic.

Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part II. In Vivo Imaging of Bone Marrow Stromal Cells in Swine with PET/CT and MR Imaging.

Purpose To quantitatively determine the limit of detection of marrow stromal cells (MSC) after cardiac cell therapy (CCT) in swine by using clinical positron emission tomography (PET) reporter gene imaging and magnetic resonance (MR) imaging with cell prelabeling. Materials and Methods Animal studies were approved by the institutional administrative panel on laboratory animal care. Seven swine received 23 intracardiac cell injections that contained control MSC and cell mixtures of MSC expressing a multimodality triple fusion (TF) reporter gene (MSC-TF) and bearing superparamagnetic iron oxide nanoparticles (NP) (MSC-TF-NP) or NP alone. Clinical MR imaging and PET reporter gene molecular imaging were performed after intravenous injection of the radiotracer fluorine 18-radiolabeled 9-[4-fluoro-3-(hydroxyl methyl) butyl] guanine ((18)F-FHBG). Linear regression analysis of both MR imaging and PET data and nonlinear regression analysis of PET data were performed, accounting for multiple injections per animal. Results MR imaging showed a positive correlation between MSC-TF-NP cell number and dephasing (dark) signal (R(2) = 0.72, P = .0001) and a lower detection limit of at least approximately 1.5 × 10(7) cells. PET reporter gene imaging demonstrated a significant positive correlation between MSC-TF and target-to-background ratio with the linear model (R(2) = 0.88, P = .0001, root mean square error = 0.523) and the nonlinear model (R(2) = 0.99, P = .0001, root mean square error = 0.273) and a lower detection limit of 2.5 × 10(8) cells. Conclusion The authors quantitatively determined the limit of detection of MSC after CCT in swine by using clinical PET reporter gene imaging and clinical MR imaging with cell prelabeling. (©) RSNA, 2016 Online supplemental material is available for this article.

AshwaMAX and Withaferin A inhibits gliomas in cellular and murine orthotopic models.

Glioblastoma multiforme (GBM) is an aggressive, malignant cancer Johnson and O'Neill (J Neurooncol 107: 359-364, 2012). An extract from the winter cherry plant (Withania somnifera ), AshwaMAX, is concentrated (4.3 %) for Withaferin A; a steroidal lactone that inhibits cancer cells Vanden Berghe et al. (Cancer Epidemiol Biomark Prev 23: 1985-1996, 2014). We hypothesized that AshwaMAX could treat GBM and that bioluminescence imaging (BLI) could track oral therapy in orthotopic murine models of glioblastoma. Human parietal-cortical glioblastoma cells (GBM2, GBM39) were isolated from primary tumors while U87-MG was obtained commercially. GBM2 was transduced with lentiviral vectors that express Green Fluorescent Protein (GFP)/firefly luciferase fusion proteins. Mutational, expression and proliferative status of GBMs were studied. Intracranial xenografts of glioblastomas were grown in the right frontal regions of female, nude mice (n = 3-5 per experiment). Tumor growth was followed through BLI. Neurosphere cultures (U87-MG, GBM2 and GBM39) were inhibited by AshwaMAX at IC50 of 1.4, 0.19 and 0.22 µM equivalent respectively and by Withaferin A with IC50 of 0.31, 0.28 and 0.25 µM respectively. Oral gavage, every other day, of AshwaMAX (40 mg/kg per day) significantly reduced bioluminescence signal (n = 3 mice, p < 0.02, four parameter non-linear regression analysis) in preclinical models. After 30 days of treatment, bioluminescent signal increased suggesting onset of resistance. BLI signal for control, vehicle-treated mice increased and then plateaued. Bioluminescent imaging revealed diffuse growth of GBM2 xenografts. With AshwaMAX, GBM neurospheres collapsed at nanomolar concentrations. Oral treatment studies on murine models confirmed that AshwaMAX is effective against orthotopic GBM. AshwaMAX is thus a promising candidate for future clinical translation in patients with GBM.

18F-FAZA PET imaging response tracks the reoxygenation of tumors in mice upon treatment with the mitochondrial complex I inhibitor BAY 87-2243.

PURPOSE: We describe a noninvasive PET imaging method that monitors early therapeutic efficacy of BAY 87-2243, a novel small-molecule inhibitor of mitochondrial complex I as a function of hypoxia-inducible factor-1α (HIF1α) activity. EXPERIMENTAL DESIGN: Four PET tracers [(18)F-FDG, (18)F-Fpp(RGD)2, (18)F-FLT, and (18)F-FAZA] were assessed for uptake into tumor xenografts of drug-responsive (H460, PC3) or drug-resistant (786-0) carcinoma cells. Mice were treated with BAY 87-2243 or vehicle. At each point, RNA from treated and vehicle H460 tumor xenografts (n = 3 each) was isolated and analyzed for target genes. RESULTS: Significant changes in uptake of (18)F-FAZA, (18)F-FLT, and (18)F-Fpp(RGD)2 (P < 0.01) occurred with BAY 87-2243 treatment with (18)F-FAZA being the most prominent. (18)F-FDG uptake was unaffected. (18)F-FAZA tumor uptake declined by 55% to 70% (1.21% ± 0.10%ID/g to 0.35 ± 0.1%ID/g; n = 6, vehicle vs. treatment) in both H460 (P < 0.001) and PC3 (P < 0.05) xenografts 1 to 3 days after drug administration. (18)F-FAZA uptake in 786-0 xenografts was unaffected. Decline occurred before significant differences in tumor volume, thus suggesting (18)F-FAZA decrease reflected early changes in tumor metabolism. BAY 87-2243 reduced expression of hypoxia-regulated genes CA IX, ANGPTL4, and EGLN-3 by 99%, 93%, and 83%, respectively (P < 0.001 for all), which corresponds with reduced (18)F-FAZA uptake upon drug treatment. Heterogeneous expression of genes associated with glucose metabolism, vessel density, and proliferation was observed. CONCLUSIONS: Our studies suggest suitability of (18)F-FAZA-PET as an early pharmacodynamic monitor on the efficacy of anticancer agents that target the mitochondrial complex I and intratumor oxygen levels (e.g., BAY 87-2243).

Bioluminescence resonance energy transfer (BRET) imaging of protein-protein interactions within deep tissues of living subjects.

Identifying protein-protein interactions (PPIs) is essential for understanding various disease mechanisms and developing new therapeutic approaches. Current methods for assaying cellular intermolecular interactions are mainly used for cells in culture and have limited use for the noninvasive assessment of small animal disease models. Here, we describe red light-emitting reporter systems based on bioluminescence resonance energy transfer (BRET) that allow for assaying PPIs both in cell culture and deep tissues of small animals. These BRET systems consist of the recently developed Renilla reniformis luciferase (RLuc) variants RLuc8 and RLuc8.6, used as BRET donors, combined with two red fluorescent proteins, TagRFP and TurboFP635, as BRET acceptors. In addition to the native coelenterazine luciferase substrate, we used the synthetic derivative coelenterazine-v, which further red-shifts the emission maxima of Renilla luciferases by 35 nm. We show the use of these BRET systems for ratiometric imaging of both cells in culture and deep-tissue small animal tumor models and validate their applicability for studying PPIs in mice in the context of rapamycin-induced FK506 binding protein 12 (FKBP12)-FKBP12 rapamycin binding domain (FRB) association. These red light-emitting BRET systems have great potential for investigating PPIs in the context of drug screening and target validation applications.

Use of DNA microarray and small animal positron emission tomography in preclinical drug evaluation of RAF265, a novel B-Raf/VEGFR-2 inhibitor.

Positron emission tomography (PET) imaging has become a useful tool for assessing early biologic response to cancer therapy and may be particularly useful in the development of new cancer therapeutics. RAF265, a novel B-Raf/vascular endothelial growth factor receptor-2 inhibitor, was evaluated in the preclinical setting for its ability to inhibit the uptake of PET tracers in the A375M(B-Raf(V600E)) human melanoma cell line. RAF265 inhibited 2-deoxy-2-[(18)F]fluoro-d-glucose (FDG) accumulation in cell culture at 28 hours in a dose-dependent manner. RAF265 also inhibited FDG accumulation in tumor xenografts after 1 day of drug treatment. This decrease persisted for the remaining 2 weeks of treatment. DNA microarray analysis of treated tumor xenografts revealed significantly decreased expression of genes regulating glucose and thymidine metabolism and revealed changes in apoptotic genes, suggesting that the imaging tracers FDG, 3-deoxy-3-[(18)F]fluorothymidine, and annexin V could serve as potential imaging biomarkers for RAF265 therapy monitoring. We concluded that RAF265 is highly efficacious in this xenograft model of human melanoma and decreases glucose metabolism as measured by DNA microarray analysis, cell culture assays, and small animal FDG PET scans as early as 1 day after treatment. Our results support the use of FDG PET in clinical trials with RAF265 to assess early tumor response. DNA microarray analysis and small animal PET studies may be used as complementary technologies in drug development. DNA microarray analysis allows for analysis of drug effects on multiple pathways linked to cancer and can suggest corresponding imaging tracers for further analysis as biomarkers of tumor response.

A pilot toxicology study of single-walled carbon nanotubes in a small sample of mice.

Single-walled carbon nanotubes are currently under evaluation in biomedical applications, including in vivo delivery of drugs, proteins, peptides and nucleic acids (for gene transfer or gene silencing), in vivo tumour imaging and tumour targeting of single-walled carbon nanotubes as an anti-neoplastic treatment. However, concerns about the potential toxicity of single-walled carbon nanotubes have been raised. Here we examine the acute and chronic toxicity of functionalized single-walled carbon nanotubes when injected into the bloodstream of mice. Survival, clinical and laboratory parameters reveal no evidence of toxicity over 4 months. Upon killing, careful necropsy and tissue histology show age-related changes only. Histology and Raman microscopic mapping demonstrate that functionalized single-walled carbon nanotubes persisted within liver and spleen macrophages for 4 months without apparent toxicity. Although this is a preliminary study with a small group of animals, our results encourage further confirmation studies with larger groups of animals.

Evaluation of firefly luciferase bioluminescence mediated photodynamic toxicity in cancer cells.

PURPOSE: This work investigated whether fLuc-catalyzed oxidation of D-luciferin generates sufficient light to induce photodynamic toxicity in cancer cells. PROCEDURES: Light emission was assessed via cooled CCD (charge-coupled device) camera. Parental and fLuc expressing cancer cells were exposed to subtoxic concentrations of photosensitizers (Rose Bengal or hypericin) and D-luciferin, sunlight, or lamplight. Toxicity was assessed by MTT assay. RESULTS: fLuc expressing cells emitted up to 500-fold higher levels of photons than parental cell lines. Although exposure to photosensitizer and sunlight reduced survival of various cell lines, survival of fLuc expressing cells incubated with photosensitizer and D-luciferin, or photosensitizer and lamplight, did not differ significantly from parental or untreated cells. CONCLUSIONS: Contesting recent reports, fLuc bioluminescence does not generate sufficient photons to induce Rose Bengal or hypericin photodynamic toxicity in a range of malignant and nonmalignant cell lines, and is not suitable as a generalizable approach to antineoplastic therapy.

Preclinical safety evaluation of 18F-FHBG: a PET reporter probe for imaging herpes simplex virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk's expression.

UNLABELLED: 9-(4-(18)F-Fluoro-3-[hydroxymethyl]butyl)guanine ((18)F-FHBG) is a sensitive and specific PET reporter probe for imaging the PET reporter genes, herpes simplex 1 thymidine kinase (HSV1-tk) and its mutant HSV1-sr39tk. (18)F-FHBG has suitable pharmacokinetics and dosimetry for clinical applications and imaging of HSV1-TK has been demonstrated in the livers of hepatocellular cancer patients. METHODS: Male and female Sprague-Dawley rats and New Zealand White rabbits were divided into equal groups receiving either 14 microg/kg cold FHBG or carrier solution, for a 14-d acute toxicity assessment. We monitored body weight, food and water consumption, body temperature, cardiovascular electrical and functional indices, respiratory performance and oxygen saturation, comprehensive blood chemistry, complete blood count (CBC), and urinalysis. We conducted daily cage-side examinations for the detection of any clinical abnormalities. Tissues of the animals that were euthanized and necropsied on day 14 were prepared for histopathologic examination. RESULTS: No significant differences in cardiovascular and respiratory parameters, food consumption, body weight, urine components, or clinical signs attributable to test article toxicity were observed between the treatment and control groups. Any differences noted in the blood chemistry and CBC parameters were deemed to be incidental findings unrelated to the administration of the FHBG. CONCLUSION: Acute toxicity evaluation of FHBG at 100 times the expected human dose does not indicate harm to organ function or tissues. The Food and Drug Administration has approved FHBG as an Investigational New Drug.