Mary J. Cole

Oncogenic BRAF Is Required for Tumor Growth and Maintenance in Melanoma Models

The usual paradigm for developing kinase inhibitors in oncology is to use a high-affinity proof-of-concept inhibitor with acceptable metabolic properties for key target validation experiments. This approach requires substantial medicinal chemistry and can be confounded by drug toxicity and off-target activities of the test molecule. As a better alternative, we have developed inducible short-hairpin RNA xenograft models to examine the in vivo efficacy of inhibiting oncogenic BRAF. Our results show that tumor regression resulting from BRAF suppression is inducible, reversible, and tightly regulated in these models. Analysis of regressing tumors showed the primary mechanism of action for BRAF to be increased tumor cell proliferation and survival. In a metastatic melanoma model, conditional BRAF suppression slowed systemic tumor growth as determined by in vivo bioluminescence imaging. Taken together, gain-of-function BRAF signaling is strongly associated with in vivo tumorigenicity, confirming BRAF as an important target for small-molecule and RNA interference-based therapeutics.

Imaging Tumors with an Albumin-Binding Fab, a Novel Tumor-Targeting Agent

Association with albumin as a means to improve biodistribution and tumor deposition of a Fab was investigated using AB.Fab4D5, a bifunctional molecule derived from trastuzumab (HERCEPTIN) capable of binding albumin and tumor antigen HER2 (erbB2) simultaneously. AB.Fab4D5 was compared with trastuzumab and a trastuzumab-derived Fab (Fab4D5) for the ability to target tumors overexpressing HER2 in mouse mammary tumor virus/HER2 allograft models. Biodistribution was monitored using intravital microscopy, histology, and integrated single-photon emission computed tomography/computed tomography analysis. Fab4D5 tumor deposition was characterized by rapid but transient appearance in tumor at 2 h with little retention, followed by rapid accumulation in kidney by 6 h. Trastuzumab was slow to accumulate in tumors and slow to clear from normal tissues, although significant tumor deposition was achieved by 24 h. In contrast, AB.Fab4D5 was observed at 2 h in tumor and its presence was sustained beyond 24 h similar to trastuzumab. Intravital microscopy revealed that at peak tumor accumulation, tumor cell staining by AB.Fab4D5 was more uniform than for Fab4D5 or trastuzumab. Similar tumor deposition was achieved for both AB.Fab4D5 and trastuzumab at 48 h (35.9 +/- 1.8% and 38.2 +/- 3.1% injected dose/g); however, AB.Fab4D5 targeted tumors more rapidly and quickly cleared from blood, leading to a lower overall normal tissue exposure. Importantly, unlike Fab4D5, AB.Fab4D5 did not accumulate in kidney, suggesting that association with albumin leads to an altered route of clearance and metabolism. Rapid targeting, excellent tumor deposition and retention, coupled with high tumor to blood ratios may make AB.Fab an exceptional molecule for imaging and cancer therapy.

pHUSH: a single vector system for conditional gene expression

BackgroundConditional expression vectors have become a valuable research tool to avoid artefacts that may result from traditional gene expression studies. However, most systems require multiple plasmids that must be independently engineered into the target system, resulting in experimental delay and an increased potential for selection of a cell subpopulation that differs significantly from the parental line. We have therefore developed pHUSH, an inducible expression system that allows regulated expression of shRNA, miRNA or cDNA cassettes on a single viral vector.ResultsBoth Pol II and Pol III promoters have been successfully combined with a second expression cassette containing a codon-optimized tetracycline repressor and selectable marker. We provide examples of how pHUSH has been successfully employed to study the function of target genes in a number of cell types within in vitro and in vivo assays, including conditional gene knockdown in a murine model of brain cancer.ConclusionWe have successfully developed and employed a single vector system that enables Doxycycline regulated RNAi or transgene expression in a variety of in vitro and in vivo model systems. These studies demonstrate the broad application potential of pHUSH for conditional genetic engineering in mammalian cells.

Quantification of viable tumor microvascular characteristics by multispectral analysis

Tumor heterogeneity complicates the quantification of tumor microvascular characteristics assessed by dynamic contrast‐enhanced MRI (DCE‐MRI). To address this issue a novel approach was developed that combines DCE‐MRI with diffusion‐based multispectral (MS) analysis to quantify the microvascular characteristics of specific tumor tissue populations. Diffusion‐based MS segmentation (feature space: apparent diffusion coefficient, T2 and proton density) was performed to identify tumor tissue populations and the DCE‐MRI characteristics were determined for each tissue class. The ability of this MS DCE‐MRI technique to detect microvascular changes due to treatment with an antibody (G6‐31) to vascular endothelial growth factor‐A (VEGF) was evaluated in a tumor xenograft mouse model. Anti‐VEGF treatment resulted in a significant reduction in Ktrans for the MS viable tumor tissue class (−0.0034 ± 0.0022 min−1, P < 0.01) at 24 hr posttreatment that differ significantly from the change observed in the control group (0.0002 ± 0.0025 min−1). Viable tumor Ktrans for the anti‐VEGF group was also reduced 62% relative to the pretreatment values (P < 0.01). Necrotic tissue classes were found to add only noise to DCE‐MRI estimates. This approach provides a means to measure physiological parameters within the viable tumor and address the issue of tumor heterogeneity that complicates DCE‐MRI analysis. Magn Reson Med, 2008.

Shedding light on bioscience. Symposium on Optical Imaging: Applications to Biology and Medicine.

This dynamic symposium, held on 11-16 February 2003 in Taos, New Mexico, was the first Keystone meeting to focus on optical techniques and their use in biology and medicine. It was organized by D. Becker, D. Farkas and S. Fraser and attracted almost 100 participants from both academia and industry. Fluorescence imaging and its applications, ranging from nano-bioscience to small-animal imaging and imaging of disease progression in humans, were the main topics, with opportunities for further discussion in the cantinas of the town and on the ski slopes of Taos mountain.