Andrea L. Levine

Nanoparticles as image enhancing agents for ultrasonography

Nanoparticles have drawn great attention as targeted imaging and/or therapeutic agents. The small size of the nanoparticles allows them to target cells that are beyond capillary vasculature, such as cancer cells. We investigated the effect of solid nanoparticles for enhancing ultrasonic grey scale images in tissue phantoms and mouse livers in vivo. Silica nanospheres (100 nm) were dispersed in agarose at 1-2.5% mass concentration and imaged by a high-resolution ultrasound imaging system (transducer centre frequency: 30 MHz). Polystyrene particles of different sizes (500-3000 nm) and concentrations (0.13-0.75% mass) were similarly dispersed in agarose and imaged. Mice were injected intravenously with nanoparticle suspensions in saline. B-mode images of the livers were acquired at different time points after particle injection. An automated computer program was used to quantify the grey scale changes. Ultrasonic reflections were observed from nanoparticle suspensions in agarose gels. The image brightness, i.e., mean grey scale level, increased with particle size and concentration. The mean grey scale of mouse livers also increased following particle administration. These results indicated that it is feasible to use solid nanoparticles as contrast enhancing agents for ultrasonic imaging.

Parathyroid hormone-related protein and ezrin are up-regulated in human lung cancer bone metastases

Lung cancer often metastasizes to bone in patients with advanced disease. Identification of the factors involved in the interactions between lung cancer cells and bone will improve the prevention and treatment of bone metastases. We identified changes in metastasis-related gene expression of human HARA lung squamous carcinoma cells co-cultured with neonatal mouse calvariae using a pathway-specific microarray analysis. Nine genes were up-regulated and two genes down-regulated in HARA cells co-cultured with mouse calvariae. Five of the nine up-regulated genes, including caveolin 1, CD44, EphB2, ezrin, and Parathyroid hormone-related protein (PTHrP), and one down-regulated gene, SLPI, were further confirmed by Reverse transcription-polymerase chain reaction (RT-PCR). A mouse model was subsequently used to study the role of PTHrP and ezrin in bone metastasis in vivo. PTHrP (all three isoforms) and ezrin were up-regulated in HARA cells at sites of bone metastasis as detected by RT-PCR and immunohistochemistry. The PTHrP 141 mRNA isoform was increased by the greatest extent (13.9-fold) in bone metastases compared to PTHrP 139 and PTHrP 173 mRNA. We then generated a HARA cell line in which PTHrP expression was inducibly silenced by RNA interference. Silencing of PTHrP expression caused significant reduction of submembranous F-actin and decreased HARA cell invasion. Ezrin up-regulation was confirmed by Western blots on HARA cells co-cultured with adult mouse long bones. Further, Transforming growth factor beta (TGF-β) was identified as one of the factors in the bone microenvironment that was responsible for the up-regulation of ezrin. The identification of PTHrP and ezrin as important regulators of lung cancer bone metastasis offers new mechanistic insights into the metastasis of lung cancer and provides potential targets for the prevention and treatment of lung cancer metastasis.

Adenovirus-mediated expression of TIMP-1 and TIMP-2 in bone inhibits osteolytic degradation by human prostate cancer

Matrix metalloproteinases (MMPs) are proteolytic enzymes that play critical roles in the pathogenesis of human cancers. Clinical trials using synthetic small molecule MMP inhibitors have been carried out but with little success. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors that block the extracellular matrix‐degrading activity of MMPs. Here, we investigated the possibilities of genetically modifying human bones with TIMPs to create a high‐TIMP bone microenvironment, which is hostile to metastatic prostate cancer cells using adenovirus‐mediated gene transfer technology and SCID‐hu end‐organ colonization mouse model. Two strategies were used to achieve bone‐specific TIMP expression: (i) ex vivo bone adenoviral infection followed by in vivo bone implantation; and (ii) ex vivo BMS cell infection followed by injection into in vivo implanted human fetal bones. PC‐3 prostate cancer cells were injected into human fetal bones 4 weeks after implantation in SCID mice. In vitro, adenovirus‐mediated expression of TIMP‐1 or TIMP‐2 in bone fragments inhibited MMP‐2 activity, bone turnover and prostate cancer cell‐induced proteolytic degradation as determined by gelatin zymography, calcium measurement and DQ protein quenched fluorescence assay, respectively. In vivo, immunohistochemistry confirmed TIMP‐2 expression in AdTIMP‐2‐infected bone implants 4 weeks after implantation in SCID mice. Mice receiving AdTIMP‐treated bone fragments showed significantly reduced PC‐3‐induced osteolysis, osteoclast recruitment and bone turnover in the implanted bones. We propose that adenoviral gene transfer of TIMP‐1 and TIMP‐2 can prevent the proteolytic activity of prostate cancer cells in bone and that enhancing anti‐proteolytic defense mechanisms in target organs represents a promising form of prostate cancer gene therapy.

Feline head and neck squamous cell carcinoma: a natural model for the human disease and development of a mouse model

Head and neck squamous cell carcinoma (H/N SCC) is a devastating disease in humans and cats, and shares similar features between the two species. The large population of pet cats in the United States, along with the high incidence of oral SCC in the cat, makes the cat an attractive candidate as a natural model for the human disease. There are similarities in pathology, progression, outcome, resistance to treatment, possible aetiologies and p53 expression, and we discuss the benefits of the cat as a natural model. We describe the development of a nude mouse xenograft model of feline oral SCC using the SCCF1 cell line transfected with a luciferase expression construct. In vivo tumour growth and metastasis were measured using serial bioluminescent imaging, and tumours grew best in the subcutis. The cat and nude mouse models will be useful to investigate the pathogenesis and the molecular basis of H/N SCC, and for preclinical drug screening.

Assessing prostate volume by magnetic resonance imaging: A comparison of different measurement approaches for organ volume analysis

Objectives:We sought to evaluate the capabilities of different magnetic resonance imaging (MRI)-based methodologies for measuring prostate volume. Materials and Methods:Twenty-four male beagles with benign prostatic hyperplasia were enrolled in a drug trial and imaged at 5 time points. A total of 120 prostate volumes were determined by MRI-based semiautomated segmentation. For planimetric assessment, 8 diameter locations were determined in the axial and coronal plane of the MRI slice with maximum extension of the prostate. Thirteen calculation models based on these diameters were determined by comparison to the reference volume and evaluated during treatment. Results:The segmented MRI prostate volume significantly correlated with post necropsy volume. The best diameter-based model also worked very well for monitoring prostate volume of dogs under treatment. Conclusions:MRI-based segmentation is highly accurate in assessing prostate volume. Diameter-based measurements are closely correlated to the segmented prostate volume and are feasible to monitor therapy.

Benign prostate hyperplasia: evaluation of treatment response with DCE MRI.

Benign prostate hyperplasia (BPH) is a major disease and its non-surgical therapy a major area of interest. The purpose of this study was to establish perfusion parameters in beagles with BPH using dynamic contrast-enhanced (DCE) MRI and to investigate changes due to the effects of finasteride treatment. Twelve male beagles (mean age 4.4±0.9,years) were divided into a control and treatment group that received a daily dose of 1 mg/kg finasteride. DCE MRI was carried out in a clinical scanner using a 3D spoiled gradient echo sequence prior to and during treatment. 0.2 mmol/kg contrast agent (gadoteridol) was administered with an injection rate of 0.2 ml/s followed by a 15 ml flush of saline. Contrast enhancement was evaluated by pharmacokinetic mapping of a two-compartment model with colour overlay images in addition to regional ROI analysis. Quantitative parameters were defined by the amplitude of contrast enhancement A, the exchange rate kep and the time to maximum signal enhancement. Dynamic contrast-enhanced MRI investigations of the prostate revealed two distinct zones, an inner, periurethral zone and an outer, parenchymal zone. The periurethral zone is highly vascularized, whereas the parenchymal zone is moderately vascularized when compared to other parenchymal organs. During treatment, in the parenchymal zone the intensity of enhancement (amplitude A) and the time to maximum signal enhancement increased, while the exchange rate kep decreased. Dynamic contrast-enhanced MRI of BPH reveals distinct differences between individual zones within the prostate. Moreover, changes during successful treatment suggest increased blood volume per volume of tissue and decreased vessel leakiness.

Rapid calcitonin response to experimental hypercalcemia in healthy horses

Calcium has important physiological functions, and disorders of calcium homeostasis are frequent in horses. We have made important progress understanding equine calcium homeostasis; however, limited information on equine calcitonin (CT) is available, in part because of the lack of validated CT assays. To determine the CT response to high ionized calcium (Ca(2+)) concentrations in healthy horses, we induced hypercalcemia in 10 healthy horses using a calcium gluconate 23% solution (5mg/kg; 120 mL/500 kg horse) infused over 4 min. Four horses were infused with 120 mL of 0.9% NaCl and used as controls. We validated a human-specific CT radioimmunoassay for use in horses. Serum Ca(2+) concentrations increased from 6.2+/-0.3mg/dL to 9.9+/-0.5mg/dL (4 min; P<0.01). Serum CT increased from 16.7+/-8.0 pg/mL to 87.1+/-55.8 pg/mL at 2 min, and 102.5+/-51.1 pg/mL at 4 min (P<0.01). Serum CT returned to baseline by 20 min, whereas serum Ca(2+) returned to baseline by 40 min. Of interest, CT concentrations returned to baseline despite hypercalcemia, suggesting thyroid gland C-cell CT depletion. Resting CT values higher than 40 pg/mL were considered abnormally elevated. No significant changes in serum Ca(2+) or CT concentrations were found in control horses. The coefficients of variation for the CT radioimmunoassay were lower than 11.9%. We conclude that the equine thyroid gland C-cell responds quickly to changes in extracellular Ca(2+) concentrations by secreting large quantities of CT into the systemic circulation, indicating that CT is important in equine calcium homeostasis. The human CT radioimmunoassay can be used to measure changes in equine CT.