Supplementary Figures 1-3 from Oncogenic BRAF Is Required for Tumor Growth and Maintenance in Melanoma Models

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, T(2) 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 K(trans) 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 K(trans) 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.

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.

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 3096 Hepsin, a type II transmembrane serine protease, is highly upregulated in prostate cancer. Hepsin promotes tumor metastasis in a mouse model of prostate cancer, suggesting a role in the later tumor stages. However, hepsin protein levels are already high in pre-cancerous lesions and remain elevated in the carcinoma and metastasis stages. In this study, we investigated the role of hepsin in primary tumor growth in an orthotopic prostate cancer model by use of hepsin-transfected LnCaP cell lines, which also stably expressed luciferase for bioluminescence imaging. The LnCaP-34 cells stably expressed hepsin protein on the cell surface at about 5-fold higher levels than the LnCaP-17 cells, which only expressed endogenous hepsin. The higher hepsin levels on LnCaP-34 correlated with increased hepsin enzymatic activity towards pro-urokinase type plasminogen activator. 1x106 LnCaP-17 or LnCaP-34 cells were injected into the left anterior lobe of the prostate of SCID-beige mice (20 animals per group) and tumors were analyzed after 16 weeks. We found a higher tumor occurrence (80% vs 58%) and lower body weights (20.8±0.8g vs. 23.5±0.9g; p<0.05) in the LnCaP-34 group compared to the LnCaP-17 group. In addition, animals of the LnCaP-34 group had 2.4-fold higher normalized prostate tissue weights and larger tumors than the LnCaP-17 group (tumor wet weights of 2.12±0.22g and 1.20±0.27g, respectively; p<0.02). Most strikingly, histological examination revealed that the LnCaP-17 tumors were confined to the left anterior lobe of the prostate (7/7 animals), i.e. the lobe where tumor cells were injected at the start of the study, whereas the LnCaP-34 tumors had spread from the left to the right anterior lobes (7/7 animals). These results suggested that hepsin overexpression endowed LnCaP-34 cells with increased invasive capacity concomitant with enhanced tumor growth. Moreover, the in-vitro proliferation rates of LnCaP-17 and LnCaP-34 cells were identical, indicating that the prostate microenvironment was important in mediating the hepsin-driven tumor growth. Unlike the differences in primary tumor growth, the occurrence of lung metastatic lesions as determined by ex-vivo bioluminescence imaging was similar in the LnCaP-17 and LnCaP-34 groups (50% vs 44%). This is in agreement with the presence of numerous tumor cell infiltrates in the tumor vasculature in both groups. However, lymph node metastasis was increased in the LnCaP-34 animals (22% vs 0% in LnCaP-17 animals), based on periaortic lymph node bioluminescence imaging at the time of tissue harvest. In conclusion, the finding that hepsin overexpression enhances invasive tumor growth suggests that elevated hepsin expression in pre-cancerous lesions and in prostate carcinoma may contribute to tumor invasiveness and growth.

BACKGROUND:Conditional 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.RESULTS:Both 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.CONCLUSION:We 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.

Recombinant human rhApo2L/TRAIL selectively stimulates apoptosis in various cancer cells through its receptors DR4 and DR5, and is currently in clinical trials. Preclinical studies have established antitumor activity of rhApo2L/TRAIL in models of epithelial cancers; however, efficacy in non-Hodgkin lymphoma (NHL) models is not well studied. Of 7 NHL cell lines tested in vitro, rhApo2L/TRAIL stimulated apoptosis in BJAB, Ramos RA1, and DoHH-2 cells. Rituximab, a CD20 antibody used to treat certain types of NHL, augmented rhApo2L/TRAIL-induced caspase activation in Ramos RA1 and DoHH2 but not BJAB or SC-1 cells, through modulation of intrinsic rather than extrinsic apoptosis signaling. In vivo, rhApo2L/TRAIL and rituximab cooperated to attenuate or reverse growth of tumor xenografts of all 4 of these cell lines. Depletion of natural killer (NK) cells or serum complement substantially reduced combined efficacy against Ramos RA1 tumors, suggesting involvement of antibody-dependent cell- and complement-mediated cytotoxicity. Both agents exhibited greater activity against disseminated than subcutaneous BJAB xenografts, and worked together to inhibit or abolish disseminated tumors and increase survival. Moreover, rhApo2L/TRAIL helped circumvent acquired rituximab resistance of a Ramos variant. These findings provide a strong rationale for clinical investigation of rhApo2L/TRAIL in combination with rituximab as a novel strategy for NHL therapy.

Abstract 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. (Cancer Res 2006; 66(2): 999-1006)

Obesity and overfat is a growing health condition that contributes to many chronic diseases including heart disease, diabetes, high blood pressure, some cancers. Assessing body fatness is a necessary means of measuring progress in reducing obesity. Utilizing accurate and convenient methods of body composition assessment is desirable. PURPOSE The purpose of this study was to compare the body fat readings of middle aged women utilizing DEXA and bioelectrical impedance. METHODS Female volunteers aged 30–64 years were recruited from Northwest Arkansas (N=340). Subjects' weight was measured on a double-beam balance scale and height by a stadiometer with subjects wearing no shoes and light clothing. Two body fat measures were completed; one with a hand-held bioelectrical impedance method (Omron, HBF-308); the other with a bone densitometer (Lunar, Prodigy). Dual-energy electrical impedance (DEXA) was used to assess body composition including body fat. Subjects lay supine on the scanner table and were wearing no metal and light clothing when the scan was performed. All measurements were conducted by a single certified densitometry technologist. Descriptive statistics and regression procedures were conducted using SPSS version 10.0. RESULTS The mean age of the participants was 49 years. The mean height was 64 inches and mean weight was 154 pounds. The mean percent of body fat measured by bioelectrical impedance was 34%, while by DEXA it was 40%. Multiple regression procedures indicated that bioelectrical impedance was significantly correlated with DEXA (R2 = .764, p=0.001). When subjects were stratified according to body fat categories, underweight (p=0.04) normal weight (p=0.001), overweight (p=0.001) and obese (p=0.001) showed significant results, however and extremely obese was not significant (p=0.549). CONCLUSIONS The results are consistent with other research that indicates that bioelectrical impedance is a valid measure for assessing body fat in normal or overweight female population. Utilizing bioelectrical impedance is desirable due to the convenience, low cost of device, small space needed for storage and use as well as simple training procedures. Benefits also exists for utilizing a bone densitometer. First, it is the gold standard for body composition assessment and can therefore be utilizing for all populations, including the excessively obese. Additionally, body fat, lean mass and bone tissue measures can be obtained, providing participants will more health information. Unlike bioelectrical impedance, DEXA is not influenced by hydration.

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.