Alpha-1 Antitrypsin Inhibits Tumorigenesis and Progression of Colitis-Associated Colon Cancer through Suppression of Inflammatory Neutrophil-Activated Serine Proteases and IGFBP-3 Proteolysis.

Colitis-associated colon cancer (CAC) accompanies the massive infiltration of neutrophils during tumorigenesis and progression of CAC. Depletion of neutrophils in circulation results in significant inhibition of tumor incidence in CAC. However, the underlying mechanisms are largely unclear. In this study, we provide evidence for the crucial involvement of inflammatory neutrophil-activated serine proteases (NSPs) on the dysregulation of the anti-inflammatory and antitumor IGFBP-3/IGFBP-3R signaling axis in CAC using a chronic AOM/DSS mouse model. We also provide preclinical evidence for α1-antitrypsin (AAT) as a preventive and as a therapeutic for CAC. AAT administration not only prevented colitis-associated tumorigenesis but also inhibited established CAC. AOM/DSS treatment results in the significant activation of NSPs, leading to CAC through increased pro-inflammatory cytokines and decreased anti-inflammatory and antitumor IGFBP-3. Collectively, these data suggest that the NSPs proteolyze IGFBP-3, whereas AAT inhibits chronic colonic inflammation-induced NSP activity and subsequently suppresses IGFBP-3 proteolysis. Therefore, the anti-inflammatory and antitumor functions of the IGFBP-3/IGFBP-3R axis are restored. AAT mimicking small peptides also showed their inhibitory effects on NSP-induced IGFBP-3 proteolysis. These results suggest that targeting the NSP-IGFBP-3/IGFBP-3R axis using NSP inhibitors such as AAT and the AAT mimics and IGFBP-3R agonists could lead to novel approaches for the prevention and treatment of CAC.

Dendritic cell trafficking in tumor-bearing mice.

Prostate cancer is one of the leading causes of cancer deaths, with no curative treatments once it spreads. Alternative therapies, including immunotherapy, have shown limited efficacy. Dendritic cells (DC) have been widely used in the treatment of various malignancies. DC capture antigens and move to the lymphoid organs where they prime naive T cells. Interaction between DC and T cells are most active in lymph nodes and suppression of DC trafficking to lymph nodes impairs the immune response. In this work, we aimed to study trafficking of DC in vivo via various routes of delivery, to optimize the effectiveness of DC-based therapy. A DC labeling system was developed using 1,1'-dioctadecyltetramethyl indotricarbocyanine Iodine for in vivo fluorescent imaging. DC harvested from C57B/6 mice were matured, labeled, and injected intravenously, subcutaneously, or intratumorally, with or without antigen loading with whole tumor lysate, into C57B/6 mice inoculated with RM-1 murine prostate tumor cells. Signal intensity was measured in vivo and ex vivo. Signal intensity at the tumor site increased over time, suggesting trafficking of DC to the tumor with all modes of injection. Subcutaneous injection showed preferential trafficking to lymph nodes and tumor. Intravenous injection showed trafficking to lungs, intestines, and spleen. Subcutaneous injection of DC pulsed with whole tumor lysate resulted in the highest increase in signal intensity at the tumor site and lymph nodes, suggesting subcutaneous injection of primed DC leads to highest preferential trafficking of DC to the immunocompetent organs.

Biodistribution and PET imaging of 89-zirconium labeled cerium oxide nanoparticles synthesized with several surface coatings.

Cerium oxide nanoparticles (CONPs) have unique surface chemistry allowing catalyst-like antioxidant properties, and are being investigated for several disease indications in medicine. Studies have utilized surface modified CONPs toward this application, but have been lacking in comprehensive biodistribution and pharmacokinetic data and a direct comparison to uncoated CONPs. We developed an enhanced single-pot synthesis of several coated CONPs and an efficient intrinsic core labeling of CONPs with the clinical PET isotope, zirconium-89, allowing detailed PET imaging and ex vivo biodistribution. All coated [89Zr]-CONPs showed benefit in terms of biodistribution compared to uncoated [89Zr]-CONPs, while retaining the intrinsic antioxidant properties. Among these, poly(acrylic acid) coated CONPs demonstrated excellent candidacy for clinical implementation due to their enhanced renal clearance and low reticuloendothelial system uptake. This work also demonstrates the value of intrinsic core labeling and PET imaging for evaluation of nanoparticle constructs to better inform future studies towards clinical use.

Sepiapterin Enhances Tumor Radio- and Chemosensitivities by Promoting Vascular Normalization.

Previously, we demonstrated that nitric oxide (NO) synthase (NOS) is uncoupled in a wide range of solid tumors and that restoring NOS coupling with the tetrahydrobiopterin precursor sepiapterin (SP) inhibits tumor progression. Endothelial dysfunction characterizes the poorly functional vasculature of solid tumors, and since NO is critical for regulation of endothelial function we asked whether SP, by recoupling NOS, improves tumor vasculature structure and function-enhancing chemotherapeutic delivery and response to radiotherapy. MMTV-neu mice with spontaneous breast tumors were treated with SP by oral gavage and evaluated by multispectral optoacoustic tomographic analysis of tumor HbO2 and by tissue staining for markers of hypoxia, blood perfusion, and markers of endothelial and smooth muscle proteins. Recoupling tumor NOS activity results in vascular normalization observed as reduced tumor hypoxia, improved tumor percentage of HbO2 and perfusion, as well as increased pericyte coverage of tumor blood vessels. The normalized vasculature and improved tumor oxygenation led to a greater than 2-fold increase in radiation-induced apoptosis compared with radiation or SP alone. High-performance liquid chromatography analysis of tumor doxorubicin levels showed a greater than 50% increase in doxorubicin uptake and a synergistic effect on tumor cell apoptosis. This study highlights for the first time the importance of NOS uncoupling and endothelial dysfunction in the development of tumor vasculature and presents a new approach for improving the tumoricidal efficacies of chemotherapy and radiotherapy.

18F-Labeled Carboplatin Derivative for PET Imaging of Platinum Drug Distribution.

Increasing evidence indicates that reduced intracellular drug accumulation is the parameter most consistently associated with platinum drug resistance, emphasizing the need to directly measure the intratumor drug concentration. In the era of precision medicine and with the advent of powerful imaging and proteomics technologies, there is an opportunity to better understand drug resistance by exploiting these techniques to provide new knowledge on drug-target interactions. Here, we contribute to this endeavor by reporting on the development of an 18F-labeled carboplatin derivative (18F-FCP) that has the potential to image drug uptake and retention, including intratumoral distribution, by PET. Methods: Fluorinated carboplatin (19F-FCP) was synthesized using 19F-labeled 2-(5-fluoro-pentyl)-2-methyl malonic acid (19F-FPMA) as the labeling agent to coordinate with the cisplatin-aqua complex. It was then used to treat cell lines and compared with cisplatin and carboplatin at different concentrations. Manual radiosynthesis and characterization of 18F-FCP were performed using 18F-FPMA for coordination with the cisplatin-aqua complex. Automated radiosynthesis of 18F-FCP was optimized on the basis of manual synthesis procedures. The stability of 18F-FCP was verified using high-performance liquid chromatography. 18F-FCP was evaluated using ex vivo biodistribution and in vivo PET imaging in non-tumor-bearing animals as well as in KB-3-1 and COLO-205 tumor xenograft-bearing nude mice. Results: In vitro cytotoxicity studies demonstrated that 19F-FCP has an antitumor activity profile similar to that of the parent drug carboplatin. In vivo plasma and urine stability analysis showed intact 18F-FCP at 24 h after injection. PET imaging and biodistribution studies showed fast clearance from blood and major accumulation in the kidneys, indicating substantial renal clearance of 18F-FCP. Using 18F-FCP PET, we could image and identify the intratumor drug profile. Conclusion: Our results demonstrated that 19F-FCP, like carboplatin, retains antitumor activity in various cell lines. 18F-FCP could be a useful imaging tool for measuring the intratumor drug distribution. This strategy of using a new therapeutic carboplatin derivative to quantify and track platinum drugs in tumors using PET has the potential to translate into a clinically useful imaging tool for individual patients.

[18F]-Fluorinated Carboplatin and [111In]-Liposome for Image-Guided Drug Delivery.

Radiolabeled liposomes have been employed as diagnostic tools to monitor in vivo distribution of liposomes in real-time, which helps in optimizing the therapeutic efficacy of the liposomal drug delivery. This work utilizes the platform of [111In]-Liposome as a drug delivery vehicle, encapsulating a novel 18F-labeled carboplatin drug derivative ([18F]-FCP) as a dual-molecular imaging tool as both a radiolabeled drug and radiolabeled carrier. The approach has the potential for clinical translation in individual patients using a dual modal approach of clinically-relevant radionuclides of 18F positron emission tomography (PET) and 111In single photon emission computed tomography (SPECT). [111In]-Liposome was synthesized and evaluated in vivo by biodistribution and SPECT imaging. The [18F]-FCP encapsulated [111In]-Liposome nano-construct was investigated, in vivo, using an optimized dual-tracer PET and SPECT imaging in a nude mouse. The biodistribution data and SPECT imaging showed spleen and liver uptake of [111In]-Liposome and the subsequent clearance of activity with time. Dual-modality imaging of [18F]-FCP encapsulated [111In]-Liposome showed significant uptake in liver and spleen in both PET and SPECT images. Qualitative analysis of SPECT images and quantitative analysis of PET images showed the same pattern of activity during the imaging period and demonstrated the feasibility of dual-tracer imaging of a single dual-labeled nano-construct.

Development of a cell delivery system using alginate microbeads for tissue regeneration.

Alginate microbeads incorporating adipose-derived stem cells (ASCs) have potential for delivering viable cells capable of facilitating tissue regeneration. These microbeads are formed in calcium crosslinking solutions containing organic osmolytes to ensure physiological osmolality, but the comparative effects of these osmolytes on the microencapsulated cells are not known. In addition, delivery parameters needed to use microencapsulated cells for tissue regeneration remain unknown. We investigated the following parameters: (1) osmolyte effects on microbead diameter, cell viability and growth factor production; (2) the effect of the number of cells per microbead and the number of microbeads per unit volume on cell viability, growth factor production, and microbead degradation; (3) the ability of both degradable and non-degradable alginate microbeads to localize cells at the delivery site in vivo; and (4) whether alginate microbeads containing alginate-lyase elicit an inflammatory response after repeated exposure. Smallest microbead diameters were achieved using glucose as the osmolyte but cell viability and growth factor production did not depend on osmolyte type. As cell number per microbead or microbead number per well increased, growth factor production per cell decreased although percent cell viability was unchanged. The rate of cell release varied with the number of beads per well and with the number of cells per microbead. At the highest microbead density and at the lowest density of cells per microbead, cell release was delayed. Therefore fewer microbeads may be sufficient for clinical applications. Both degradable (0.22 U g-1) and non-degradable (0 U g-1) alginate microbeads localized cells at the delivery site. Degradable alginate microbeads delivered subcutaneously elicited a mild chronic inflammatory response on second exposure, but how this might impact repeated use of the technology remains to be determined.

Astrocyte Elevated Gene-1 (AEG-1) Regulates Lipid Homeostasis.

Astrocyte elevated gene-1 (AEG-1), also known as MTDH (metadherin) or LYRIC, is an established oncogene. However, the physiological function of AEG-1 is not known. To address this question, we generated an AEG-1 knock-out mouse (AEG-1KO) and characterized it. Although AEG-1KO mice were viable and fertile, they were significantly leaner with prominently less body fat and lived significantly longer compared with wild type (WT). When fed a high fat and cholesterol diet (HFD), WT mice rapidly gained weight, whereas AEG-1KO mice did not gain weight at all. This phenotype of AEG-1KO mice is due to decreased fat absorption from the intestines, not because of decreased fat synthesis or increased fat consumption. AEG-1 interacts with retinoid X receptor (RXR) and inhibits RXR function. In enterocytes of AEG-1KO mice, we observed increased activity of RXR heterodimer partners, liver X receptor and peroxisome proliferator-activated receptor-α, key inhibitors of intestinal fat absorption. Inhibition of fat absorption in AEG-1KO mice was further augmented when fed an HFD providing ligands to liver X receptor and peroxisome proliferator-activated receptor-α. Our studies reveal a novel role of AEG-1 in regulating nuclear receptors controlling lipid metabolism. AEG-1 may significantly modulate the effects of HFD and thereby function as a unique determinant of obesity.

Spag17 deficiency results in skeletal malformations and bone abnormalities.

Height is the result of many growth and development processes. Most of the genes associated with height are known to play a role in skeletal development. Single-nucleotide polymorphisms in the SPAG17 gene have been associated with human height. However, it is not clear how this gene influences linear growth. Here we show that a targeted mutation in Spag17 leads to skeletal malformations. Hind limb length in mutants was significantly shorter than in wild-type mice. Studies revealed differences in maturation of femur and tibia suggesting alterations in limb patterning. Morphometric studies showed increased bone formation evidenced by increased trabecular bone area and the ratio of bone area to total area, leading to reductions in the ratio of marrow area/total area in the femur. Micro-CTs and von Kossa staining demonstrated increased mineral in the femur. Moreover, osteocalcin and osterix were more highly expressed in mutant mice than in wild-type mice femurs. These data suggest that femur bone shortening may be due to premature ossification. On the other hand, tibias appear to be shorter due to a delay in cartilage and bone development. Morphometric studies showed reduction in growth plate and bone formation. These defects did not affect bone mineralization, although the volume of primary bone and levels of osteocalcin and osterix were higher. Other skeletal malformations were observed including fused sternebrae, reduced mineralization in the skull, medial and metacarpal phalanges. Primary cilia from chondrocytes, osteoblasts, and embryonic fibroblasts (MEFs) isolated from knockout mice were shorter and fewer cells had primary cilia in comparison to cells from wild-type mice. In addition, Spag17 knockdown in wild-type MEFs by Spag17 siRNA duplex reproduced the shorter primary cilia phenotype. Our findings disclosed unexpected functions for Spag17 in the regulation of skeletal growth and mineralization, perhaps because of its role in primary cilia of chondrocytes and osteoblasts.

The Role of Nitric Oxide Synthase Uncoupling in Tumor Progression.

UNLABELLED: Here, evidence suggests that nitric oxide synthases (NOS) of tumor cells, in contrast with normal tissues, synthesize predominantly superoxide and peroxynitrite. Based on high-performance liquid chromatography analysis, the underlying mechanism for this uncoupling is a reduced tetrahydrobiopterin:dihydrobiopterin ratio (BH4:BH2) found in breast, colorectal, epidermoid, and head and neck tumors compared with normal tissues. Increasing BH4:BH2 and reconstitution of coupled NOS activity in breast cancer cells with the BH4 salvage pathway precursor, sepiapterin, causes significant shifts in downstream signaling, including increased cGMP-dependent protein kinase (PKG) activity, decreased ß-catenin expression, and TCF4 promoter activity, and reduced NF-κB promoter activity. Sepiapterin inhibited breast tumor cell growth in vitro and in vivo as measured by a clonogenic assay, Ki67 staining, and 2[18F]fluoro-2-deoxy-D-glucose-deoxyglucose positron emission tomography (FDG-PET). In summary, using diverse tumor types, it is demonstrated that the BH4:BH2 ratio is lower in tumor tissues and, as a consequence, NOS activity generates more peroxynitrite and superoxide anion than nitric oxide, resulting in important tumor growth-promoting and antiapoptotic signaling properties. IMPLICATIONS: The synthetic BH4, Kuvan, is used to elevate BH4:BH2 in some phenylketonuria patients and to treat diseases associated with endothelial dysfunction, suggesting a novel, testable approach for correcting an abnormality of tumor metabolism to control tumor growth.

Near-infrared imaging of adoptive immune cell therapy in breast cancer model using cell membrane labeling.

The overall objective of this study is to non-invasively image and assess tumor targeting and retention of directly labeled T-lymphocytes following their adoptive transfer in mice. T-lymphocytes obtained from draining lymph nodes of 4T1 (murine breast cancer cell) sensitized BALB/C mice were activated in-vitro with Bryostatin/Ionomycin for 18 hours, and were grown in the presence of Interleukin-2 for 6 days. T-lymphocytes were then directly labeled with 1,1-dioctadecyltetramethyl indotricarbocyanine Iodide (DiR), a lipophilic near infrared fluorescent dye that labels the cell membrane. Assays for viability, proliferation, and function of labeled T-lymphocytes showed that they were unaffected by DiR labeling. The DiR labeled cells were injected via tail vein in mice bearing 4T1 tumors in the flank. In some cases labeled 4T1 specific T-lymphocytes were injected a week before 4T1 tumor cell implantation. Multi-spectral in vivo fluorescence imaging was done to subtract the autofluorescence and isolate the near infrared signal carried by the T-lymphocytes. In recipient mice with established 4T1 tumors, labeled 4T1 specific T-lymphocytes showed marked tumor retention, which peaked 6 days post infusion and persisted at the tumor site for up to 3 weeks. When 4T1 tumor cells were implanted 1-week post-infusion of labeled T-lymphocytes, T-lymphocytes responded to the immunologic challenge and accumulated at the site of 4T1 cell implantation within two hours and the signal persisted for 2 more weeks. Tumor accumulation of labeled 4T1 specific T-lymphocytes was absent in mice bearing Meth A sarcoma tumors. When lysate of 4T1 specific labeled T-lymphocytes was injected into 4T1 tumor bearing mice the near infrared signal was not detected at the tumor site. In conclusion, our validated results confirm that the near infrared signal detected at the tumor site represents the DiR labeled 4T1 specific viable T-lymphocytes and their response to immunologic challenge can be imaged in vivo.

Intrinsically radiolabelled [(59)Fe]-SPIONs for dual MRI/radionuclide detection.

Towards the development of iron oxide nanoparticles with intrinsically incorporated radionuclides for dual Positron Emission Tomography/Magnetic Resonance Imaging (PET/MRI) and more recently of Single Photon Emission Computed Tomography/Magnetic Resonance Imaging (SPECT/MRI), we have developed intrinsically radiolabeled [(59)Fe]-superparamagnetic iron oxide nanoparticles ([(59)Fe]-SPIONs) as a proof of concept for an intrinsic dual probe strategy. (59)Fe was incorporated into Fe3O4 nanoparticle crystal lattice with 92±3% efficiency in thermal decomposition synthesis. Multidentate poly(acrylic acid)-dopamine-poly(ethylene-glycol-2000) (PAA-DOP-PEG) ligands were designed and synthesized based on facile EDC chemistry and utilized to functionalize the [(59)Fe]-SPIONs. The transverse relaxivity of [(59)Fe]-SPIONs (97±3 s(-1)mM(-1)) was characterized and found to be similar to non-radioactive SPIONs (72±10 s(-1)mM(-1)), indicating that (59)Fe incorporation does not alter the SPIONs' MRI contrast properties. [(59)Fe]-SPIONs were used to evaluate the nanoparticle biodistribution by ex vivo gamma counting and MRI. Nude mice (n=15) were injected with [(59)Fe]-SPIONs and imaged at various time points with 7T small animal MRI scanner. Ex vivo biodistribution was evaluated by tissue-based gamma counting. MRI signal contrast qualitatively correlates with the %ID/g of [(59)Fe]-SPIONs, with high contrast in liver (45±6%), medium contrast in kidneys (21±5%), and low contrast in brain (4±6%) at 24 hours. This work demonstrates the synthesis and in vivo application of intrinsically radiolabeled [(59)Fe]-SPIONs for bimodal detection and provides a proof of concept for incorporation of both gamma- and positron-emitting inorganic radionuclides into the core of metal based MRI contrast agent nanoparticles.

Creation of an in vitro biomechanical model of the trachea using rapid prototyping.

Previous in vitro models of the airways are either rigid or, if flexible, have not matched in vivo compliance characteristics. Rapid prototyping provides a quickly evolving approach that can be used to directly produce in vitro airway models using either rigid or flexible polymers. The objective of this study was to use rapid prototyping to directly produce a flexible hollow model that matches the biomechanical compliance of the trachea. The airway model consisted of a previously developed characteristic mouth-throat region, the trachea, and a portion of the main bronchi. Compliance of the tracheal region was known from a previous in vivo imaging study that reported cross-sectional areas over a range of internal pressures. The compliance of the tracheal region was matched to the in vivo data for a specific flexible resin by iteratively selecting the thicknesses and other dimensions of tracheal wall components. Seven iterative models were produced and illustrated highly non-linear expansion consisting of initial rapid size increase, a transition region, and continued slower size increase as pressure was increased. Thickness of the esophageal interface membrane and initial trachea indention were identified as key parameters with the final model correctly predicting all phases of expansion within a value of 5% of the in vivo data. Applications of the current biomechanical model are related to endotracheal intubation and include determination of effective mucus suctioning and evaluation of cuff sealing with respect to gases and secretions.

An alternative approach to histopathological validation of PET imaging for radiation therapy image-guidance: a proof of concept.

PURPOSE: In radiotherapy, PET images can be used to guide the delivery of selectively escalated doses to biologically relevant tumour subvolumes. Validation of PET for such applications requires demonstration of spatial coincidence between PET tracer uptake pattern and the histopathologically confirmed target. This study introduces a novel approach to histopathological validation of PET image segmentation for radiotherapy guidance. METHODS AND MATERIALS: Sequential tissue sections from surgically excised whole-tumour specimens were used to acquire full 3D-sets of both histopathological images (microscopy) and PET tracer distribution images (autoradiography). After these datasets were accurately registered, a full 3D autoradiographic distribution of PET tracer was reconstructed and used to obtain synthetic PET images (sPET) by simulating the image deterioration induced by processes involved in PET image formation. To illustrate the method, sPET images were used in this study to investigate spatial coincidence between high FDG uptake areas and the distribution of viable tissue in two small animal tumour models. RESULTS: The reconstructed 3D autoradiographic distribution of the PET tracer was spatially coherent, as indicated by the high average value of the normalised pixel-by-pixel correlation of intensities between successive slices (0.84 ± 0.05 and 0.94 ± 0.02). The loss of detail in the sPET images versus the 3D autoradiography was significant as indicated by Dice coefficient values corresponding to the two tumours (0 and 0.1 at 70% threshold). The maximum overlap between the FDG segmented volumes and the extent of the viable tissue as indicated by Dice coefficient values, was 0.8 for one tumour (for the image thresholded at 22% of max intensity) and 0.88 for the other (threshold of 14% of max intensity). CONCLUSION: It was demonstrated that the use of synthetic PET images for histopathological validation allows for bypassing a technically challenging and error-prone step of registering non-invasive PET images with histopathology.

Highly stable intrinsically radiolabeled indium-111 quantum dots with multidentate zwitterionic surface coating: dual modality tool for biological imaging.

Here we describe a novel strategy to incorporate indium-111 into near infrared (NIR) emitting Cu-In-Se quantum dots (CIS-QDs) to synthesize intrinsically radiolabeled QDs (rQDs), as a quantitative tool for in vivo SPECT/fluorescence imaging. Multidentate zwitterionic polymer ligands were used to functionalize and improve the stability of CIS-rQDs and reduce nonspecific binding with plasma proteins/cell membrane. CIS-rQDs were taken up by colorectal adenocarcinoma (COLO-205) and human epidermoid carcinoma (KB-3-1) cells at low uptake rate (∼0.4%, 2 × 105 QDs per cell at 24 h) and reduced nonspecific interaction of zwitterionic CIS-rQDs with cells was observed by fluorescence microscopy. The cytotoxicity of CIS-rQDs was reduced due to the low toxic inorganic composition of QDs and multidentate zwitterionic surface coating. In 5 out of 6 nude mice bearing either COLO-205 or KB-3-1 tumor, both SPECT and fluorescence imaging demonstrated passive localization of CIS-rQDs in the tumor as early as 6 h post-injection. In these mice the passive accumulation of CIS-rQDs in the tumor, due to leaky vasculature, ranged from ∼0.3% ID per g to ∼4.6% ID per g at 48 h post-injection (from region of interest analysis of SPECT imaging). This intrinsic radio-labeling strategy provides a nanoparticle platform which incorporates imaging and potentially therapeutic radionuclides with retention of fluorescence intensity. It also provides complimentary quantitative data capabilities for both in vivo SPECT imaging and radiotracer ex vivo analysis.

Microfluidic radiosynthesis and biodistribution of [18 F] 2-(5-fluoro-pentyl)-2-methyl malonic acid.

Microfluidics technology has emerged as a powerful tool for the radiosynthesis of positron emission tomography (PET) and single-photon emission computed tomography radiolabeled compounds. In this work, we have exploited a continuous flow microfluidic system (Advion, Inc., USA) for the [(18) F]-fluorine radiolabeling of the malonic acid derivative, [(18) F] 2-(5-fluoro-pentyl)-2-methyl malonic acid ([(18) F]-FPMA), also known as [(18) F]-ML-10, a radiotracer proposed as a potential apoptosis PET imaging agent. The radiosynthesis was developed using a new tosylated precursor. Radiofluorination was initially optimized by manual synthesis and served as a basis to optimize reaction parameters for the microfluidic radiosynthesis. Under optimized conditions, radio-thin-layer chromatography analysis showed 79% [(18) F]-fluorine incorporation prior to hydrolysis and purification. Following hydrolysis, the [(18) F]-FPMA was purified by C18 Sep-Pak, and the final product was analyzed by radio-HPLC (high-performance liquid chromatography). This resulted in a decay-corrected 60% radiochemical yield and ≥98% radiochemical purity. Biodistribution data demonstrated rapid blood clearance with less than 2% of intact [(18) F]-FPMA radioactivity remaining in the circulation 60 min post-injection. Most organs showed low accumulation of the radiotracer, and radioactivity was predominately cleared through kidneys (95% in 1 h). Radio-HPLC analysis of plasma and urine samples showed a stable radiotracer at least up to 60 min post-injection.

Sperm-associated antigen-17 gene is essential for motile cilia function and neonatal survival.

Primary ciliary dyskinesia (PCD), resulting from defects in cilia assembly or motility, is caused by mutations in a number of genes encoding axonemal proteins. PCD phenotypes are variable, and include recurrent respiratory tract infections, bronchiectasis, hydrocephaly, situs inversus, and male infertility. We generated knockout mice for the sperm-associated antigen-17 (Spag17) gene, which encodes a central pair (CP) protein present in the axonemes of cells with "9 + 2" motile cilia or flagella. The targeting of Spag17 resulted in a severe phenotype characterized by immotile nasal and tracheal cilia, reduced clearance of nasal mucus, profound respiratory distress associated with lung fluid accumulation and disruption of the alveolar epithelium, cerebral ventricular expansion consistent with emerging hydrocephalus, failure to suckle, and neonatal demise within 12 hours of birth. Ultrastructural analysis revealed the loss of one CP microtubule in approximately one quarter of tracheal cilia axonemes, an absence of a C1 microtubule projection, and other less frequent CP structural abnormalities. SPAG6 and SPAG16 (CP proteins that interact with SPAG17) were increased in tracheal tissue from SPAG17-deficient mice. We conclude that Spag17 plays a critical role in the function and structure of motile cilia, and that neonatal lethality is likely explained by impaired airway mucociliary clearance.

Intrinsically radiolabeled multifunctional cerium oxide nanoparticles for in vivo studies.

Cerium oxide nanoparticles (CONPs) have demonstrated protection properties against oxidation in various cells and tissues. The mechanism of this, however, is poorly understood. Monitoring the interaction of CONPs with biological compartments 'in situ' is crucial to understand their biochemical and physiological properties in vivo. In this paper, a multifunctional nanoparticle platform was obtained through an intrinsic radiolabeling strategy and extrinsic surface functionalization to combine dual imaging components (Single Photon Emission Computed Tomography/Optical Imaging, SPECT/OI) in one nanoparticle. The cell viability, cell uptake and overall in vivo biodistribution of CONPs were also manipulated through surface functionalization. The intrinsic radiolabeling strategy is demonstrated by incorporating radionuclides (141Ce, 111In or 65Zn) into CONPs and a radiolabeled CONP (rCONP) was coated with biocompatible polymers including Dextran T10 (DT10), poly(acrylic acid) (PAA), or functionalized DT10 (DT10-NH2, DT10-PEG and DT10-sulfobetaine). Fluorescent CONPs were obtained through conjugation of fluorescein isothiocyanate (FITC) with DT10-NH2 rCONP and used for cell imaging. The DT10 and DT10-NH2 rCONP did not show decreased viability up to 120 µg mL-1 whilst the PAA rCONP showed decreased viability beyond 40 µg mL-1. Variations in blood circulation and renal/hepatic clearance of rCONPs were demonstrated and were dependent on surface coating and the hydrodynamic size of nanoparticles. The ex vivo biodistribution results were reflected in SPECT imaging of 141Ce-rCONPs, showing accumulation in the liver and spleen of a living mouse over a one week period. The intrinsic radiolabeling and extrinsic surface modifications together determine the biophysical properties of CONPs and their potential applications for in vivo studies and biomedical imaging.

Synthesis and characterization of intrinsically radiolabeled quantum dots for bimodal detection.

A novel approach was developed to synthesize radioactive quantum dots (r-QDs) thereby enabling both optical and radionuclide signals to be detected from the same intrinsic bimodal probe. This proof-of-concept is exemplified by the incorporation of the radionuclide (109)Cadmium into the core/shell of the nanoparticle. Green and near infrared (NIR) emission intrinsic r-QDs were synthesized and characterized. Zwitterionic and Poly-polyethlene glycol (PEGylated) ligands were synthesized and used to coat r-QDs. Zwitterionic NIR r-QDs (quantum yield = 11%) and PEGylated NIR r-QDs (quantum yield = 14%) with an average size of 13.8 nm and 16.8 nm were obtained respectively. The biodistribution of NIR zwitterionic and PEGylated r-QDs in nude mice was investigated and zwitterionic r-QDs showed longer blood circulation (t(1/2) = 21.4±1.1 hrs) than their PEGylated counterparts (t(1/2) = 6.4±0.5 min). Both zwitterionic and PEGylated r-QDs exhibited progressive accumulation in the liver and spleen, but the magnitude of the accumulation (%ID/g) was about 3-6 fold higher with the PEGylated r-QDs at all the time points. The results demonstrated the feasibility of r-QDs synthesis in quantitative yield and retention of fluorescence following incorporation of radioactivity into the core/shell of the nanoparticle. The gamma signal from the same fluorescent elemental material enabled quantitative and robust pharmacokinetic measurements and how these changed depended on the type of coating ligands used. This strategy for intrinsically radio-labeling the QDs is currently being implemented in our laboratory for the incorporation of other radiometals.

Tumour microenvironment heterogeneity affects the perceived spatial concordance between the intratumoural patterns of cell proliferation and 18F-fluorothymidine uptake.

BACKGROUND AND PURPOSE: PET imaging with (18)F-fluorothymidine ((18)F-FLT) can potentially be used to identify tumour subvolumes for selective dose escalation in radiation therapy. The purpose of this study is to analyse the co-localization of intratumoural patterns of cell proliferation with (18)F-FLT tracer uptake. MATERIALS AND METHODS: Mice bearing FaDu or SQ20B xenograft tumours were injected with (18)F-FLT, and bromodeoxyuridine (proliferation marker). Ex vivo images of the spatial pattern of intratumoural (18)F-FLT uptake and that of bromodeoxyuridine DNA incorporation were obtained from thin tumour tissue sections. These images were segmented by thresholding and Relative Operating Characteristic (ROC) curves and Dice similarity indices were evaluated. RESULTS: The thresholds at which maximum overlap occurred between FLT-segmented areas and areas of active cell proliferation were significantly different for the two xenograft tumour models, whereas the median Dice values were not. However, ROC analysis indicated that segmented FLT images were more specific at detecting the proliferation pattern in FaDu tumours than in SQ20B tumours. CONCLUSION: Highly dispersed patterns of cell proliferation observed in certain tumours can affect the perceived spatial concordance between the spatial pattern of (18)F-FLT uptake and that of cell proliferation even when high-resolution ex vivo autoradiography imaging is used for (18)F-FLT imaging.