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The following select list of references describes the core VisualSonics' technology and its multiple applications for small animal research. The body of references spans a number of years and the current technology offering may be vastly advanced from certain techniques utilized or cited in earlier papers or posters.

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	Targeted Contrast-Enhanced Ultrasound Imaging of Tumor Angiogenesis with Contrast Microbubbles Conjugated to Integrin-Binding Knottin Peptides. Jürgen K. Willmann ¹, Richard H. Kimura ², Nirupama Deshpande ¹, Amelie M. Lutz ¹, Jennifer R. Cochran ³, and Sanjiv S. Gambhir ² ¹ Molecular Imaging Program at Stanford, Department of Radiology, School of Medicine, Stanford University, Stanford, California ² Molecular Imaging Program at Stanford, Department of Radiology, School of Medicine, Stanford University, Stanford, California; Department of Bioengineering, School of Medicine, Stanford University, Stanford, California ³ Department of Bioengineering, School of Medicine, Stanford University, Stanford, California The Journal of Nuclear Medicine. 2010 Feb 11. [Epub ahead of print] Targeted contrast-enhanced ultrasound imaging is increasingly being recognized as a powerful imaging tool for the detection and quantification of tumor angiogenesis at the molecular level. The purpose of this study was to develop and test a new class of targeting ligands for targeted contrast-enhanced ultrasound imaging of tumor angiogenesis with small, conformationally constrained peptides that can be coupled to the surface of ultrasound contrast agents. METHODS: Directed evolution was used to engineer a small, disulfide-constrained cystine knot (knottin) peptide that bound to alphavbeta3 integrins with a low nanomolar affinity (KnottinIntegrin). A targeted contrast-enhanced ultrasound imaging contrast agent was created by attaching KnottinIntegrin to the shell of perfluorocarbon-filled microbubbles (MB-KnottinIntegrin). A knottin peptide with a scrambled sequence was used to create control microbubbles (MB-KnottinScrambled). The binding of MB-KnottinIntegrin and MB-KnottinScrambled to alphavbeta3 integrin-positive cells and control cells was assessed in cell culture binding experiments and compared with that of microbubbles coupled to an anti-alphavbeta3 integrin monoclonal antibody (MBalphavbeta3) and microbubbles coupled to the peptidomimetic agent c(RGDfK) (MBcRGD). The in vivo imaging signals of contrast-enhanced ultrasound with the different types of microbubbles were quantified in 42 mice bearing human ovarian adenocarcinoma xenograft tumors by use of a high-resolution 40-MHz ultrasound system. RESULTS: MB-KnottinIntegrin attached significantly more to alphavbeta3 integrin-positive cells (1.76 +/- 0.49 [mean +/- SD] microbubbles per cell) than to control cells (0.07 +/- 0.006). Control MB-KnottinScrambled adhered less to alphavbeta3 integrin-positive cells (0.15 +/- 0.12) than MB-KnottinIntegrin. After blocking of integrins, the attachment of MB-KnottinIntegrin to alphavbeta3 integrin-positive cells decreased significantly. The in vivo ultrasound imaging signal was significantly higher after the administration of MB-KnottinIntegrin than after the administration of MBalphavbeta3 or control MB-KnottinScrambled. After in vivo blocking of integrin receptors, the imaging signal after the administration of MB-KnottinIntegrin decreased significantly (by 64%). The imaging signals after the administration of MB-KnottinIntegrin were not significantly different in the groups of tumor-bearing mice imaged with MB-KnottinIntegrin and with MBcRGD. Ex vivo immunofluorescence confirmed integrin expression on endothelial cells of human ovarian adenocarcinoma xenograft tumors. CONCLUSION: Integrin-binding knottin peptides can be conjugated to the surface of microbubbles and used for in vivo targeted contrast-enhanced ultrasound imaging of tumor angiogenesis. Our results demonstrate that microbubbles conjugated to small peptide-targeting ligands provide imaging signals higher than those provided by a large antibody molecule. KEY WORDS: targeted ultrasound imaging, contrast-enhanced ultrasound imaging, ovarian cancer, knottin peptides

	Paracrine Regulation of Pancreatic Cancer Cell Invasion by Peripheral Nerves. Ziv Gil, Oren Cavel, Kaitlyn Kelly, Peter Brader, Avigail Rein, Sizhi P. Gao, Diane L. Carlson, Jatin P. Shah, Yuman Fong, Richard J. Wong The Laboratory for Applied Cancer Research, Tel Aviv Sourasky Medical Center, Tel Aviv University, Tel Aviv, Israel (ZG, OC, AR); Department of Surgery (ZG, KK, AR, JPS, PB, YF, RJW), Department of Medicine (SPG), and Department of Pathology (DLC), Memorial Sloan-Kettering Cancer Center, New York, NY; Department of Radiology, Division of Pediatric Radiology, Medical University Graz, Austria (PB) Journal of the National Cancer Institute. 2010 Jan 12. [Epub ahead of print] BACKGROUND: The ability of cancer to infiltrate along nerves is a common clinical observation in pancreas, head and neck, prostate, breast, and gastrointestinal carcinomas. For these tumors, nerves may provide a conduit for local cancer progression into the central nervous system. Although neural invasion is associated with poor outcome, the mechanism that triggers it is unknown. METHODS: We used an in vitro Matrigel dorsal root ganglion and pancreatic cancer cell coculture model to assess the dynamic interactions between nerves and cancer cell migration and the role of glial cell-derived neurotrophic factor (GDNF). An in vivo murine sciatic nerve model was used to study how nerve invasion affects sciatic nerve function. RESULTS: Nerves induced a polarized neurotrophic migration of cancer cells (PNMCs) along their axons, which was more efficient than in the absence of nerves (migration distance: mean = 187.1 µm, 95% confidence interval [CI] = 148 to 226 µm vs 14.4 µm, 95% CI = 9.58 to 19.22 µm, difference = 143 µm; P < .001; n = 20). PNMC was induced by secretion of GDNF, via phosphorylation of the RET-Ras–mitogen-activated protein kinase pathway. Nerves from mice deficient in GDNF had reduced ability to attract cancer cells (nerve invasion index: wild type vs gdnf+/–, mean = 0.76, 95% CI = 0.75 to 0.77 vs 0.43, 95% CI = 0.42 to 0.44; P < .001; n = 60–66). Tumor specimens excised from patients with neuroinvasive pancreatic carcinoma had higher expression of the GDNF receptors RET and GRF1 as compared with normal tissue. Finally, systemic therapy with pyrazolopyrimidine-1, a tyrosine kinase inhibitor targeting the RET pathway, suppressed nerve invasion toward the spinal cord and prevented paralysis in mice. CONCLUSION: These data provide evidence for paracrine regulation of pancreatic cancer invasion by nerves, which may have important implications for potential therapy directed against nerve invasion by cancer.

	Quantifying Antivascular Effects of Monoclonal Antibodies to Vascular Endothelial Growth Factor: Insights from Imaging. James P.B. O'Connor^1,2, Richard A.D. Carano³, Andrew R. Clamp², Jed Ross³, Calvin C.K. Ho³, Alan Jackson¹, Geoff J.M. Parker¹, Chris J. Rose¹, Franklin V. Peale⁴, Michel Friesenhahn⁵, Claire L. Mitchell^1,2, Yvonne Watson¹, Caleb Roberts¹, Lynn Hope², Sue Cheung¹, Hani Bou Reslan³, Mary Ann T. Go⁶, Glenn J. Pacheco⁶, Xiumin Wu⁷, Tim C. Cao³, Sarajane Ross⁶, Giovanni A. Buonaccorsi¹, Karen Davies¹, Jurjees Hasan², Paula Thornton², Olivia del Puerto⁸, Napoleone Ferrara⁷, Nicholas van Bruggen³ and Gordon C. Jayson² ¹ Imaging Science and Biomedical Engineering, School of Cancer and Imaging Sciences, University of Manchester and ² Cancer Research UK and University of Manchester Department of Medical Oncology, Christie Hospital, Manchester, United Kingdom ³ Biomedical Imaging Group, Department of Tumor Biology and Angiogenesis, Departments of ⁴ Pathology, ⁵ Biostatistics, ⁶ Translational Oncology and ⁷ Ferrara Laboratory, Genentech, Inc., South San Francisco, California; and ⁸ Roche Products Limited, Hertfordshire, United Kingdom Clinical Cancer Research. 2009 Nov 2; 15(21):6674–82 PURPOSE: Little is known concerning the onset, duration, and magnitude of direct therapeutic effects of anti–vascular endothelial growth factor (VEGF) therapies. Such knowledge would help guide the rational development of targeted therapeutics from bench to bedside and optimize use of imaging technologies that quantify tumor function in early-phase clinical trials. EXPERIMENTAL DESIGN: Preclinical studies were done using ex vivo microcomputed tomography and in vivo ultrasound imaging to characterize tumor vasculature in a human HM-7 colorectal xenograft model treated with the anti-VEGF antibody G6-31. Clinical evaluation was by quantitative magnetic resonance imaging in 10 patients with metastatic colorectal cancer treated with bevacizumab. RESULTS: Microcomputed tomography experiments showed reduction in perfused vessels within 24 to 48 h of G6-31 drug administration (P ≤ 0.005). Ultrasound imaging confirmed reduced tumor blood volume within the same time frame (P = 0.048). Consistent with the preclinical results, reductions in enhancing fraction and fractional plasma volume were detected in patient colorectal cancer metastases within 48 h after a single dose of bevacizumab that persisted throughout one cycle of therapy. These effects were followed by resolution of edema (P = 0.0023) and tumor shrinkage in 9 of 26 tumors at day 12. CONCLUSION: These data suggest that VEGF-specific inhibition induces rapid structural and functional effects with downstream significant antitumor activity within one cycle of therapy. This finding has important implications for the design of early-phase clinical trials that incorporate physiologic imaging. The study shows how animal data help interpret clinical imaging data, an important step toward the validation of image biomarkers of tumor structure and function. KEY WORDS: angiogenesis, biomarker, imaging, VEGF

	In vivo, Noninvasive, Label-Free Detection and Eradication of Circulating Metastatic Melanoma Cells Using Two-Color Photoacoustic Flow Cytometry with a Diode Laser. Ekaterina I. Galanzha ^{1, 2}, Evgeny V. Shashkov ¹, Paul M. Spring ², James Y. Suen ², and Vladimir P. Zharov ^{1, 2} ¹ Phillips Classic Laser and Nanomedicine Laboratories ² Department of Otolaryngology-Head and Neck Surgery, University of Arkansas for Medical Sciences, Little Rock, Arkansas Cancer Research. 2009 Oct 15;69(20):7926-34. Epub 2009 Oct 13 The circulating tumor cell (CTC) count has been shown as a prognostic marker for metastasis development. However, its clinical utility for metastasis prevention remains unclear, because metastases may already be present at the time of initial diagnosis with existing assays. Their sensitivity ex vivo is limited by a small blood sample volume, whereas in vivo examination of larger blood volumes may be clinically restricted by the toxicity of labels used for targeting of CTCs. We introduce a method for in vivo photoacoustic blood cancer testing with a high-pulse-repetition-rate diode laser that, when applied to melanoma, is free of this limitation. It uses the overexpression of melanin clusters as intrinsic, spectrally-specific cancer markers and signal amplifiers, thus providing higher photoacoustic contrast of melanoma cells compared with a blood background. In tumor-bearing mouse models and melanoma-spiked human blood samples, we showed a sensitivity level of 1 CTC/mL with the potential to improve this sensitivity 103-fold in humans in vivo, which is impossible with existing assays. Additional advances of this platform include decreased background signals from blood through changes in its oxygenation, osmolarity, and hematocrit within physiologic norms, assessment of CTCs in deep vessels, in vivo CTC enrichment, and photoacoustic-guided photothermal ablation of CTCs in the bloodstream. These advances make feasible the early diagnosis of melanoma during the initial parallel progression of primary tumor and CTCs, and laser blood purging using noninvasive or hemodialysis-like schematics for the prevention of metastasis. KEY WORDS: circulating tumor cells, melanoma, photoacoustic detection

	Morphological ultrasound micro-imaging of thyroid in living mice. Marcello Mancini, Emilia Vergara, Giuliana Salvatore, Adelaide Greco, Giancarlo Troncone, Andrea Affuso, Raffaele Liuzzi, Paolo Salerno, Maria Scotto di Santolo, Massimo Santoro, Arturo Brunetti, and Marco Salvatore Institute of Biostructure and Bioimaging, Italian National Research Council (CNR), Naples, Italy; SDN Foundation IRCCS, Naples, Italy; Department of Biomorphological and Functional Sciences, Universita' "Federico II" Naples, Italy; Dipartimento di Studi delle Istituzioni e dei Sistemi Territoriali, Università "Parthenope", Naples, Italy; CEINGE-Biotecnologie Avanzate s.c.a.r.l., Naples, Italy; Biogem (Biologia e genetica molecolare) Ariano Irpino, Avellino, Italy; Dipartimento di Biologia e Patologia Cellulare e Molecolare "L. Califano", Università "Federico II", Naples, Italy Endocrinology. 2009 Oct;150(10):4810-5. Epub 2009 Jul 9 To explore high-frequency ultrasound (HFUS) for non-invasive micro-imaging of thyroid in living mice. Thyroid examination was performed by HFUS in 10 normal C57BL/6 mice, 8 mice treated by propil-thyouracil (PTU), and 22 Tg-TRK-T1 transgenic mice. The dimension of the gland and the presence of nodules were evaluated. Nodules were classified as malignant (hypoechogenicity, poorly defined margins, internal microcalcification, irregular shapes and extra glandular extension) or not and the findings were compared to histological data. Thyroid images were successfully obtained in all the animals analyzed. Normal thyroid reached a volume of 4.92 microl (range 2.11-4.92 microl). Mice with PTU-induced goiter showed diffuse thyroid enlargement (median volume 6.67 microl, range 4.09-8.82 microl). In 19 of 22 (86%) Tg-TRK-T1 mice, HFUS identified a nodular process (the smallest detected nodule had a diameter of 0.46 mm). Eleven nodules were classified as malignant and 8 as benign. Compared to histological analysis, HFUS showed a sensitivity of 100% in the detection of thyroid nodules and a specificity of 60% (2 of the nodules identified by HFUS were not confirmed at the histology). The specificity and sensitivity of HFUS in predicting the malignancy of the thyroid nodules were 83% and 91%, respectively. Thus, HFUS is an accurate imaging modality that can potentially replace more invasive techniques, and, therefore, it represents a significant advancement in phenotypic assessment of mouse models of thyroid cancer.

	Inhibition of Hsp90 via 17-DMAG induces apoptosis in a p53-dependent manner to prevent medulloblastoma. Olivier Ayrault¹, Michael D. Godeny², Christopher Dillon², Frederique Zindy¹, Patrick Fitzgerald², Martine F. Roussel¹ and Helen M. Beere² Departments of ¹Genetics and Tumor Cell Biology and ²Immunology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105 Proceedings of the National Academy of Sciences of the United States of America (PNAS). 2009 Oct 6;106(40):17037-42. Epub 2009 Sep 23 Elevated expression of HSP90 is observed in many tumor types and is associated with a limited clinical response. Targeting HSP90 using inhibitors such as 17-DMAG (17-desmethoxy-17-N,N-dimethylaminoethylaminogeldanamycin) has shown limited therapeutic success. HSP90 regulates the function of several proteins implicated in tumorigenesis although the precise mechanism through which 17-DMAG regulates tumor cell survival remains unclear. We observed a requirement for p53 in mediating 17-DMAG-induced cell death. The sensitivity of primary mouse embryonic fibroblasts and tumor cells to 17-DMAG-induced apoptosis depended on the p53 status. Wild-type MEFs underwent 17-DMAG-induced caspase-dependent cell death, whilst those lacking p53 failed to do so. Interestingly p53-dependent cell death occurred independently of Atm or Arf. Primary tumor cells derived from two models of murine medulloblastoma (Ptch1+/-;Ink4c-/- and p53FL/FL;Nestin-Cre+; Ink4c-/-) that retain and lack p53 function, respectively, displayed a dependence on functional p53 to engage 17-DMAG-induced apoptosis. Strikingly, 17-DMAG treatment in an allograft model of Ptch1+/-;Ink4c-/- but not p53FL/FL;Nestin-Cre+; Ink4c-/- tumor cells prevented tumor growth in vivo. Our data suggest that p53 status is a likely predictor of the sensitivity of tumors to 17-DMAG.

	Inhibition of Tumor Growth Progression by Antiandrogens and mTOR Inhibitor in a Pten-Deficient Mouse Model of Prostate Cancer. Weisheng Zhang ¹, Joe Zhu ¹, Clay L. Efferson ², Chris Ware ³, Jennifer Tammam ², Minilik Angagaw ⁴, Jason Laskey ², Kimberly A. Bettano ¹, Shailaja Kasibhatla ², John F. Reilly ³, Cyrille Sur ⁵, and Pradip K. Majumder ² Departments of ¹Imaging, ²Oncology, ³Pharmacology, and ⁴Laboratory Animal Research, Merck Research Laboratories, Boston, Massachusetts; and ⁵Imaging Department, Merck Research Laboratories, West Point, Pennsylvania Cancer Research. 2009 Sep 15;69(18):7466-72. Epub 2009 Sep 8 Androgen receptors have been shown to play a critical role in prostate cancer. We used ultrasound imaging techniques to track tumor response to antiandrogen and rapamycin treatment in a prostate-specific Pten-deleted mouse model of cancer. Depletion of androgens by either surgical or chemical castration significantly inhibited tumor growth progression without altering the activation of Akt and mammalian target of rapamycin (mTOR). We also showed for the first time that targeting mTOR along with antiandrogen treatment exhibited additive antitumor effects in vivo when compared with single agents. Our preclinical data suggest that combination of antiandrogens with mTOR inhibitors might be more effective in treating androgen-dependent prostate cancer patients. KEYWORDS: PTEN, prostate tumor, ultrasound imaging, androgen receptor, Casodex, mTOR

	Tumor Vascular Changes Mediated by Inhibition of Oncogenic Signaling. Naseer Qayum ¹, Ruth J. Muschel ¹, Jae Hong Im ¹, Lukxmi Balathasan ¹, Cameron J. Koch ², Sonal Patel ³, W. Gillies McKenna ¹, and Eric J. Bernhard ¹ ¹ Gray Institute for Radiation Oncology and Biology, Oxford University, Oxford, United Kingdom ² Department of Radiation Oncology, University of Pennsylvania, Philadelphia, Pennsylvania ³ PIramed Pharma, Slough, United Kingdom Cancer Research. 2009 Aug 1;69(15):6347-54. Epub 2009 Jul 21 Many inhibitors of the epidermal growth factor receptor (EGFR)-RAS-phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway are in clinical use or under development for cancer therapy. Here, we show that treatment of mice bearing human tumor xenografts with inhibitors that block EGFR, RAS, PI3K, or AKT resulted in prolonged and durable enhancement of tumor vascular flow, perfusion, and decreased tumor hypoxia. The vessels in the treated tumors had decreased tortuosity and increased internodal length accounting for the functional alterations. Inhibition of tumor growth cannot account for these results, as the drugs were given at doses that did not alter tumor growth. The tumor cell itself was an essential target, as HT1080 tumors that lack EGFR did not respond to an EGFR inhibitor but did respond with vascular alterations to RAS or PI3K inhibition. We extended these observations to spontaneously arising tumors in MMTV-neu mice. These tumors also responded to PI3K inhibition with decreased tumor hypoxia, increased vascular flow, and morphologic alterations of their vessels, including increased vascular maturity and acquisition of pericyte markers. These changes are similar to the vascular normalization that has been described after the antiangiogenic treatment of xenografts. One difficulty in the use of vascular normalization as a therapeutic strategy has been its limited duration. In contrast, blocking tumor cell RAS-PI3K-AKT signaling led to persistent vascular changes that might be incorporated into clinical strategies based on improvement of vascular flow or decreased hypoxia. These results indicate that vascular alterations must be considered as a consequence of signaling inhibition in cancer therapy. KEYWORDS: signaling inhibition, tumor vasculature, hypoxia, EGFR, RAS, PI3K, AKT

	Prostate-specific Klf6 Inactivation Impairs Anterior Prostate Branching Morphogenesis through Increased Activation of the Shh Pathway. Ching Ching Leow¹, Bu-er Wang¹, Jed Ross², Sara M. Chan³, Jiping Zha³, Richard A. D. Carano², Gretchen Frantz³, Michael M. Shen⁴, Frederic J. de Sauvage¹, and Wei-Qiang Gao¹ From the From the Departments of ¹Molecular Biology, ²Tumor Biology and Angiogenesis, and , ³Pathology, Genentech Inc., South San Francisco, California 94080 and , the ⁴Herbert Irving Comprehensive Cancer Center, Columbia University College of Physicians and Surgeons, New York, New York 10032 The Journal of biological chemistry. 2009 Jul 31;284(31):21057-65. Epub 2009 Jun 3 Kruppel-like factor 6 (Klf6) belongs to a family of zinc finger transcription factors known to play a role in development and tumor suppression. Although Klf6 is highly mutated in prostate cancer, its function in prostate development is unknown. We have generated a prostate-specific Klf6-deficient mouse model and report here a novel role for Klf6 in the regulation of prostate branching morphogenesis. Importantly, our study reveals a novel relationship between Klf6 and the Shh pathway. Klf6-deficiency leads to elevated levels of hedgehog pathway components (Shh, Ptc and Gli) and loss of their localized expression, which in turn causes impaired lateral branching. ABBREVIATIONS: Hh, hedgehog • BMP, bone morphogenic protein • FGF, fibroblastic growth factor • Shh, sonic hedgehog • Micro-CT, micro-computed tomography • SMA, smooth muscle actin • H&E, hematoxylin and eosin • TCRD, T-cell receptor delta chain.

	Coregistration of ultrasonography and magnetic resonance imaging with a preliminary investigation of the spatial colocalization of vascular endothelial growth factor receptor 2 expression and tumor perfusion in a murine tumor model. Mary E. Loveless, Jennifer G. Whisenant, Kevin Wilson, Andrej Lyshchik, Tuhin K. Sinha, John C. Gore, Thomas E. Yankeelov Institute of Imaging Science, Vanderbilt University, Nashville, Tennessee 37232-2675, USA Molecular Imaging. 2009 Jul-Aug;8(4):187-98 We present an ultrasonography (US)-magnetic resonance imaging (MRI) coregistration technique and examine its application in a preliminary multimodal, multiparametric study in a preclinical model of breast cancer. Nine mice were injected with 67NR breast cancer cells and imaged 6 and 9 days later with 4.7 T MRI and high-frequency US. Tumor volumes from each data set were segmented independently by two investigators and coregistered using an iterative closest point algorithm. In addition to anatomic images, vascular endothelial growth factor receptor 2 (VEGFR2) distribution images from the central tumor slice using VEGFR2-targeted ultrasound contrast agent (UCA) and measurements of perfusion and extravascular-extracellular volume fraction using dynamic contrast-enhanced MRI were acquired from five mice for multiparametric coregistration. Parametric maps from each modality were coregistered and examined for spatial correlation. Average registration root mean square (RMS) error was 0.36 +/- 0.11 mm, less than approximately two voxels. Segmented volumes were compared between investigators to minimize interobserver variability; the average RMS error was 0.23 +/- 0.09 mm. In the preliminary study, VEGFR2-targeted UCA data did not demonstrate direct spatial correlation with magnetic resonance measures of vascular properties. In summary, a method for accurately coregistering small animal US and MRI has been presented that allows for comparison of quantitative metrics provided by the two modalities.

	Inhibition of Hedgehog Signaling Enhances Delivery of Chemotherapy in a Mouse Model of Pancreatic Cancer. Kenneth P. Olive ¹, Michael A. Jacobetz ¹, Christian J. Davidson ², Aarthi Gopinathan ³, Dominick McIntyre ¹, Davina Honess ¹, Basetti Madhu ¹, Mae A. Goldgraben ¹, Meredith E. Caldwell ¹, David Allard ¹, Kristopher K. Frese ¹, Gina DeNicola ³, Christine Feig ¹, Chelsea Combs ², Stephen P. Winter ¹, Heather Ireland ¹, Stefanie Reichelt ¹, William J. Howat ¹, Alex Chang ⁴, Mousumi Dhara ⁴, Lifu Wang ⁵, Felix Rückert ⁶, Robert Grützmann ⁶, Christian Pilarsky ⁶, Kamel Izeradjene ⁷, Sunil R. Hingorani ⁷, Pearl Huang ⁸, Susan E. Davies ⁹, William Plunkett ¹⁰, Merrill Egorin ¹¹, Ralph H. Hruban ⁴, Nigel Whitebread ¹², Karen McGovern ¹², Julian Adams ¹², Christine Iacobuzio-Donahue ⁴, John Griffiths ¹, David A. Tuveson ¹ ¹ Cancer Research UK, Cambridge Research Institute, The Li Ka Shing Centre, Robinson Way, Cambridge, CB2 ORE, UK. ² Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104, USA. ³ Cancer Research UK, Cambridge Research Institute, The Li Ka Shing Centre, Robinson Way, Cambridge, CB2 ORE, UK.; Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104, USA. ⁴ Departments of Oncology and Pathology, The Sol Goldman Pancreatic Cancer Research Center, Sidney Cancer Center and Johns Hopkins University, Baltimore, MD 21287, USA. ⁵ Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104, USA.; Department of Gastroenterology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China. ^{6/sup> Department of Surgery, University Hospital Dresden, Fetscherstr. 74, 01307 Dresden, Germany. ⁷ Clinical Research and Public Health Sciences Division, Fred Hutchinson Cancer Research Center, and University of Washington, Seattle, WA 98109, USA. ⁸ Oncology Franchise, Merck and Co, North Wales, PA 19454, USA. ⁹ Department of Histopathology, Addenbrooke’s Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge, CB2 2QQ, UK. ¹⁰ Univ. of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. ¹¹ Division of Hematology and Oncology, University of Pittsburgh Medical Center, Pittsburgh, PA 15213, USA. ¹² Infinity Pharmaceuticals Inc, Cambridge, MA 01239, USA. Sciencexpress. 2009 Jun 12;324(5933):1457-61. Epub 2009 May 21 Pancreatic ductal adenocarcinoma (PDA) is among the most lethal human cancers, in part because it is insensitive to many chemotherapeutic drugs. Studying a mouse model of PDA that is refractory to the clinically used drug gemcitabine, we found that the tumors in this model were poorly perfused and poorly vascularized, properties that are shared with human PDA. We tested whether the delivery and efficacy of gemcitabine in the mice could be improved by coadministration of IPI-926, a drug that depletes tumor-associated stromal tissue by inhibiting the Hedgehog cellular signaling pathway. The combination therapy produced a transient increase in intratumoral vascular density and intratumoral concentration of gemcitabine, leading to transient stabilization of disease. Thus, inefficient drug delivery may be an important contributor to chemoresistance in pancreatic cancer.}

	Sunitinib and PF-562,271 (FAK/Pyk2 inhibitor) effectively block growth and recovery of human hepatocellular carcinoma in a rat xenograft model. Cedo M. Bagi,¹ James Christensen,² Darrel P. Cohen,² Walter G. Roberts,³ Dean Wilkie,⁴ Terri Swanson,¹ Theresa Tuthill⁵ and Catharine J. Andresen¹ ¹ Global Science & Technology; and ⁴ Investigative Pathology; PGRD; Pfizer, Inc.; Groton, CT USA; ² Oncology; PGRD; Pfizer Inc; La Jolla, CA USA; ³ Safety and Risk Management; and ⁵ Molecular Medicine; PGRD; Pfizer Inc; New London, CT USA Cancer Biology & Therapy. 2009 May;8(9):856-65 EXPERIMENTAL DESIGN: To investigate the antitumor effect of sunitinib and FAK/Pyk2 tyrosine kinase inhibitor (PF-562,271)combination therapy in vivo, utilizing human hepatocellular carcinoma (HCC) cells Huh7.5. Nude rats were inoculated subcutaneously with Huh7.5 hepatoma cells. Dosing for Phase 1 was initiated on day 5 post tumor inoculations with Vehicle(Group 1), sunitinib (25 mg/kg/day; Group 2) and sunitinib plus PF-562,271 combination (15 mg/kg/day; Group 3). Phase 2 of the study started on day 26, and each of the three original groups was divided in two subgroups; half of the rats remained on original therapy (Groups 1A and 2A) with the exception of Group 3A that was euthanized after Phase 1. The other half of the rats were switched to sunitinib and PF-562,271 combination (Group 1B) or vehicle (Groups 2B and 3B). Tumor volume and weight, serum alpha feto-protein (AFP), contrast-enhanced ultrasound imaging (CEUS) and tumor histology were used to evaluate effects of treatment on tumor growth. RESULTS: The results from this study indicate that the combination of sunitinib and PF-562,271 TKI has the potential to target different aspects of angiogenesis and tumor aggressiveness and may have significantly greater effect than relevant single agent, blocking not only tumor growth, but also impacting the ability of the tumor to recover upon withdrawal of the therapy. KEYWORDS: sunitinib, FAK/Pyk2, angiogenesis, metastasis, hepatocellular carcinoma, biomarkers

	Complementarity of ultrasound and fluorescence imaging in an orthotopic mouse model of pancreatic cancer. Cynthia S Snyder¹ , Sharmeela Kaushal¹ , Yuko Kono² , Hop S Tran Cao³ , Robert M Hoffman³,⁴ and Michael Bouvet¹,³ ¹ Moores UCSD Cancer Center, University of California, La Jolla, CA, USA ² UCSD Department of Medicine, La Jolla, CA, USA ³ UCSD Department of Surgery, La Jolla, CA, USA ⁴ AntiCancer Inc., San Diego, CA, USA BMC Cancer. 2009 Apr 8;9(1):106. [Epub ahead of print] BACKGROUND: Pancreatic cancer is a devastating disease characterized by dismal 5-year survival rates and limited treatment options. In an effort to provide useful models for preclinical evaluation of new experimental therapeutics, we and others have developed orthotopic mouse models of pancreatic cancer. The utility of these models for pre-clinical testing is dependent upon quantitative, noninvasive methods for monitoring in vivo tumor progression in real time. Toward this goal, we performed whole-body fluorescence imaging and ultrasound imaging to evaluate and to compare these noninvasive imaging modalities for assessing tumor burden and tumor progression in an orthotopic mouse model of pancreatic cancer. METHODS: The human pancreatic cancer cell line XPA-1, engineered for stable, high-level expression of red fluorescent protein (RFP), was implanted into the pancreas of nude mice using orthotopic implantation. The tumors were allowed to grow over a period of one to several weeks during which time the mice were imaged using both fluorescence imaging and ultrasound imaging to measure tumor burden and to monitor tumor growth. RESULTS: Whole-body fluorescence imaging and ultrasound imaging both allowed for the visualization and measurement of orthotopic pancreatic tumor implants in vivo. The imaging sessions were well-tolerated by the mice and yielded data which correlated well in the quantitative assessment of tumor burden. Whole-body fluorescence and two-dimensional ultrasound imaging showed a strong correlation for measurement of tumor size over a range of tumor sizes (R2 = 0.6627, P = 0.003 for an exposure time of 67 msec and R2 = 0.6553, P = 0.003 for an exposure time of 120 msec). CONCLUSIONS: Our findings suggest a complementary role for fluorescence imaging and ultrasound imaging in assessing tumor burden and tumor progression in orthotopic mouse models of human cancer.

	The selective hypoxia inducible factor-1 inhibitor PX-478 provides in vivo radiosensitization through tumor stromal effects. David L. Schwartz^1,2, Garth Powis³, Arun Thitai-Kumar², Yi He¹, James Bankson⁴, Ryan Williams³, Robert Lemos³, Junghwan Oh¹, Andrei Volgin², Suren Soghomonyan², Ryuichi Nishii², Mian Alauddin², Uday Mukhopadhay², Zhenghong Peng³, William Bornmann³ and Juri Gelovani² Departments of ¹Radiation Oncology, ²Experimental Diagnostic Imaging, ³Experimental Therapeutics, and ⁴Imaging Physics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas Molecular Cancer Therapeutics. 2009 Apr 1; 8(4):947–58 Hypoxia inducible factor-1 (HIF-1) promotes tumor cell adaptation to microenvironmental stress. HIF-1 is up-regulated in irradiated tumors and serves as a promising target for radiosensitization. We initially confirmed that the orally bioavailable HIF-1 inhibitor PX-478 reduces HIF-1 protein levels and signaling in vitro in a dose-dependent manner and provides direct radiosensitization of hypoxic cancer cells in clonogenic survival assays using C6 glioma, HN5 and UMSCCa10 squamous cells, and Panc-1 pancreatic adenocarcinoma cell lines. However, PX-478 yields striking in vivo tumor sensitization to single-dose irradiation, which cannot be explained by incremental improvement in direct tumor cell killing. We show that PX-478 prevents postradiation HIF-1 signaling and abrogates downstream stromal adaptation in C6 and HN5 reporter xenografts as measured by serial ultrasound, vascular magnetic resonance imaging, and hypoxia response element–specific micro–positron emission tomography imaging. The primacy of indirect PX-478 in vivo effects was corroborated by our findings that (a) either concurrent or early postradiation sequencing of PX-478 provides roughly equivalent sensitization and (b) constitutive vascular endothelial growth factor expression maintains refractory tumor vessel function and progression following combined radiation and PX-478. These results confirm that disruption of postradiation adaptive HIF-1 signaling by PX-478 imparts increased therapeutic efficacy through blockade of HIF-1–dependent reconstitution of tumor stromal function. Successful translation of targeted HIF-1 radiosensitization to the clinical setting will require specific consideration of tumor microenvironmental effects and mechanisms. Keywords: PX-478, Hypoxia, HIF-1, Radiation, Radiosensitization

	Effective prostate cancer chemopreventive intervention with green tea polyphenols in the TRAMP model depends on the stage of the disease. Vaqar Mustafa Adhami, Imtiaz Ahmad Siddiqui, Sami Sarfaraz, Sabih Islam Khwaja, Bilal Bin Hafeez, Nihal Ahmad and Hasan Mukhtar Department of Dermatology, University of Wisconsin-Madison, WI 53706 Clinical Cancer Research. 2009 Mar 15;15(6):1947-53. Epub 2009 Mar 10 Purpose: We have previously shown that oral feeding of green tea polyphenols (GTP) to TRAMP (transgenic adenocarcinoma of the mouse prostate) mice in a purely chemopreventive setting significantly inhibits prostate cancer (PCa) development. To translate this to a human situation, present study was designed to identify the stage of PCa that is most vulnerable to chemopreventive intervention by GTP. Experimental Design: GTP infusion (0.1% in drinking water) to TRAMP was initiated at ages representing different stage of the disease: (i) 6 weeks (group 1, normal prostate), ii) 12 weeks (group 2, prostatic intraepithelial neoplasia), (iii) 18 weeks (group 3, well differentiated adenocarcinoma and, (iv) 28 weeks (group 4, moderately differentiated adenocarcinoma. At 32 weeks of age, subsets of animals were evaluated by MRI, ultrasound, prostate weight, and for serum IGF-I/IGFBP-3 and IGF signaling. Results: Tumor free survival was extended to 38 weeks (p<0.001) in group 1, 31 weeks (p<0.01) in group 2 and to 24 weeks (p<0.05) in group 3 compared to 19 weeks in water-fed controls. Median life expectancy was 68 weeks in group 1, 63 weeks in group 2, 56 weeks in group 3 and 51 weeks in group 4 compared to 42 weeks in the control mice. IGF-I and its downstream targets including PI3K, pAkt and pErk were significantly inhibited only when intervention was initiated early when PIN lesions were common. Conclusions: Our studies indicate that chemopreventive potential of GTP decreases with advancing stage of the disease and underscore the need to design appropriate chemoprevention clinical trails.

	Quantitative Ultrasound Characterization of Responses to Radiotherapy in Cancer Mouse Models. Roxana M. Vlad ¹, ³, Sebastian Brand ⁵, Anoja Giles ³, Michael C. Kolios ¹, ⁴, Gregory J. Czarnota ¹, ², ³ Authors' Affiliations: Departments of ¹Medical Biophysics and ²Radiation Oncology, University of Toronto, ³Sunnybrook Health Sciences Centre, and ⁴Department of Physics, Ryerson University, Toronto, Ontario, Canada; and ⁵Orthopedics Department, Q-BAM Laboratory, University of Halle, Halle, Germany Clinical Cancer Research. 2009 Mar 15;15(6):2067-75. Epub 2009 Mar 10 PURPOSE: Currently, no imaging modality is used routinely to assess tumor responses to radiotherapy within hours to days after the delivery of treatment. In this study, we show the application of quantitative ultrasound methods to characterize tumor responses to cancer radiotherapy in vivo, as early as 24 hours after treatment administration. EXPERIMENTAL DESIGN: Three mouse models of head and neck cancer were exposed to radiation doses of 0, 2, 4, and 8 Gray. Data were collected with an ultrasound scanner using frequencies of 10 to 30 MHz. Ultrasound estimates calculated from normalized power spectra and parametric images (spatial maps of local estimates of ultrasound parameters) were used as indicators of response. RESULTS: Two of the mouse models (FaDu and C666-1) exhibited large hyperechoic regions at 24 hours after radiotherapy. The ultrasound integrated backscatter increased by 6.5 to 8.2 dB (P < 0.001) and the spectral slopes increased from 0.77 to 0.90 dB/MHz for the C666-1 tumors and from 0.54 to 0.78 dB/MHz for the FaDu tumors (P < 0.05), in these regions compared with preirradiated tumors. The hyperechoic regions in the ultrasound images corresponded in histology to areas of cell death. Parametric images could discern the tumor regions that responded to treatment. The other cancer mouse model (Hep-2) was resistant to radiotherapy. CONCLUSIONS: The results indicate that cell structural changes after radiotherapy have a significant influence on ultrasound spectral parameters. This provides a foundation for future investigations regarding the use of ultrasound in cancer patients to individualize treatments noninvasively based on their responses to specific interventions. KEY WORDS: ultrasound imaging, spectral parameters, radiation therapy, tumor response, apoptosis, parametric imaging

	Cytosolic Phospholipase A2: Targeting Cancer through the Tumor Vasculature. Amanda Linkous, Ling Geng, Andrej Lyshchik, Dennis E. Hallahan, Eugenia M. Yazlovitskaya Departments of 1Radiation Oncology, 2Cancer Biology, 3Radiology and Radiological Sciences, and 4Cell and Developmental Biology, and 5Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee Clinical Cancer Research. 2009 Mar 1;15(5):1635-44. Epub 2009 Feb 24 Purpose: In vascular endothelial cells, low doses of ionizing radiation trigger the immediate activation of cytosolic phospholipase A2 (cPLA2). This event initiates prosurvival signaling that could be responsible for radioresistance of tumor vasculature. Thus, the development of radiosensitizers targeting these survival pathways may enhance tumor response to radiation therapy. Arachidonyltrifluoromethyl Ketone (AACOCF3), a specific cPLA2 inhibitor, was studied as a potential radiosensitizer. Experimental Design: Vascular endothelial cells (3B11 and MPMEC) and lung tumor cells (LLC and H460) were treated with 1 µmol/L AACOCF3 for 30 minutes prior to irradiation. Treatment response was evaluated by clonogenic survival, activation of extracellular signal-regulated kinase 1/2 (ERK1/2), tubule formation, and migration assays. For in vivo experiments, mice with LLC or H460 tumors in the hind limbs were treated for 5 consecutive days with 10 mg/kg AACOCF3 administered daily 30 minutes prior to irradiation. Treatment response was assessed by tumor growth delay, Power Doppler Sonography, and immunohistochemistry. Results: In cell culture experiments, inhibition of cPLA2 with AACOCF3 prevented radiation-induced activation of ERK1/2 and decreased clonogenic survival of irradiated vascular endothelial cells but not the lung tumor cells. Treatment with AACOCF3 also attenuated tubule formation and migration in irradiated vascular endothelial cells. In both tumor mouse models, treatment with AACOCF3 prior to irradiation significantly suppressed tumor growth and decreased overall tumor blood flow and vascularity. Increased apoptosis in both tumor cells and tumor vascular endothelium was determined as a possible mechanism of the observed effect. Conclusion: These findings identify cPLA2 as a novel molecular target for tumor sensitization to radiation therapy through the tumor vasculature. Key Words: cytosolic phospholipase A2, radiosensitization, endothelium, lung cancer, microvasculature

	Suppression of Tumor Growth In vivo by the Mitocan {alpha}-tocopheryl Succinate Requires Respiratory Complex II. Lan-Feng Dong, Ruth Freeman, Ji Liu, Renata Zobalova, AlvaroMarin-Hernandez, Marina Stantic, Jakub Rohlena, Karel Valis, Sara Rodriguez-Enriquez, Bevan Butcher, Jacob Goodwin, Ulf T. Brunk, Paul K. Witting, RafaelMoreno-Sanchez, Immo E. Scheffler, Stephen J. Ralph, and Jiri Neuzil Authors' Affiliations: Apoptosis Research Group and Genomic Research Centre, School of Medical Science, Griffith University, Southport, Queensland, Australia; Molecular Therapy Group, Institute of Biotechnology, Academy of Sciences of the Czech Republic, Prague, Czech Republic; Department of Biochemistry, National Institute of Cardiology, Mexico City, Mexico; Division of Pharmacology, University of Linkoping, Linkoping, Sweden; ANZAC Research Institute, Concord Hospital, Concord, Sydney, New South Wales, Australia; and Division of Biology, University of California, San Diego, California. Clinical Cancer Research 2009 Mar 1;15(5):1593-600. Epub 2009 Feb 17 PURPOSE: Vitamin E analogues are potent novel anticancer drugs. The purpose of this study was to elucidate the cellular target by which these agents, represented by alpha-tocopoheryl succinate (alpha-TOS), suppress tumors in vivo, with the focus on the mitochondrial complex II (CII).EXPERIMENTAL DESIGN: Chinese hamster lung fibroblasts with functional, dysfunctional, and reconstituted CII were transformed using H-Ras. The cells were then used to form xenografts in immunocompromized mice, and response of the cells and the tumors to alpha-TOS was studied.RESULTS: The CII-functional and CII-reconstituted cells, unlike their CII-dysfunctional counterparts, responded to alpha-TOS by reactive oxygen species generation and apoptosis execution. Tumors derived from these cell lines reciprocated their responses to alpha-TOS. Thus, growth of CII-functional and CII-reconstituted tumors was strongly suppressed by the agent, and this was accompanied by high level of apoptosis induction in the tumor cells. On the other hand, alpha-TOS did not inhibit the CII-dysfuntional tumors.CONCLUSIONS: We document in this report a novel paradigm, according to which the mitochondrial CII, which rarely mutates in human neoplasias, is a plausible target for anticancer drugs from the group of vitamin E analogues, providing support for their testing in clinical trials.

	A Method for Assessing the Microvasculature in a Murine Tumor Model Using Contrast-Enhanced Ultrasonography. Mary E. Loveless, MS, Xia Li, PhD, Jessica Huamani, MS, Andrej Lyshchik, MD, PhD, Benoit Dawant, PhD, Dennis Hallahan, MD, John C. Gore, PhD, Thomas E. Yankeelov, PhD Institute of Imaging Science (M.E.L., X.L., B.D., J.C.G., T.E.Y.) and Departments of Biomedical Engineering (M.E.L., D.H., J.C.G., T.E.Y.), Electrical Engineering and Computer Science (X.L., B.D.), Radiation Oncology (J.H., D.H.), Radiology and Radiological Sciences (A.L., J.C.G., T.E.Y.), Physics and Astronomy (J.C.G., T.E.Y.), and Cancer Biology (T.E.Y.), Vanderbilt University, Nashville, Tennessee USA Journal of ultrasound in medicine. 2008 Dec; 27(12):1699-709 OBJECTIVE: The purpose of this study was to develop a method for assessing tumor vascularity in a preclinical model of breast cancer using contrast-enhanced ultrasonography. METHODS: Eight mice were injected with 67NR breast cancer cells on their hind limbs and imaged with ultrasonography 8 days later. Mice were injected with an ultrasound contrast agent (UCA), and a sequence of images of the resultant backscattered echoes was recorded before and after high-power "destruction" pulses for each of multiple parallel planes. From these, data maps of the maximum contrast enhancement (within each time course) were constructed for each pixel, which enabled reconstruction of high-resolution coregistered sections into a 3-dimensional (3D) volume reflecting tumor vascularity. Additional studies were performed to determine the duration and repeatability of image enhancement, and images were correlated with conventional 3D power Doppler measurements. RESULTS: The lifetime of the UCA in vivo was found to be 4.3 +/- 1.09 minutes (mean +/- SD). The 3D contrast-enhanced ultrasonographic technique produced images that correlated well with power Doppler images in specific regions but also depicted additional regions of flow surrounding the power Doppler signal. The mean correlation coefficient between voxel measurements of the central slice for each animal was 0.64 +/- 0.07 (P < .01). In addition, sequential studies in each animal were reproducible. CONCLUSIONS: A method producing high-resolution volumetric assessments of tumor vascularity in a preclinical model of breast cancer is shown that correlates with other ultrasonographic measures of blood flow, which may provide greater sensitivity to the microvasculature.

	Interstitial Doppler Optical Coherence Tomography as a Local Tumor Necrosis Predictor in Photodynamic Therapy of Prostatic Carcinoma: An In vivo Study. Beau A. Standish, Kenneth K.C. Lee, Xiao Jin, Adrian Mariampillai, Nigel R. Munce, Michael F.G. Wood, Brian C. Wilson, I. Alex Vitkin, and Victor X.D. Yang Department of Medical Biophysics and Radiation Oncology, University of Toronto, Ontario Cancer Institute/University Health Network, Department Electrical and Computer Engineering, Ryerson University, and Imaging Research, Sunnybrook Health Sciences Center, Toronto, Canada Cancer Research. 2008 Dec 1;68(23):9987-95 We have tested the feasibility of real-time localized blood flow measurements, obtained with interstitial (IS) Doppler optical coherence tomography (DOCT), to predict photodynamic therapy (PDT)-induced tumor necrosis deep within solid Dunning rat prostate tumors. IS-DOCT was used to quantify the PDT-induced microvascular shutdown rate in s.c. Dunning prostate tumors (n=28). Photofrin (12.5 mg/kg) was administered 20 to 24 hours before tumor irradiation, with 635 nm surface irradiance of 8 to 133 mWcm(-2) for 25 minutes. High frequency ultrasound and calipers were used to measure the thickness of the skin covering the tumor and the location of the echogenic IS probe within it. A two-layer Monte Carlo model was used to calculate subsurface fluence rates within the IS-DOCT region of interest (ROI). Treatment efficacy was estimated by percent tumor necrosis within the ROI, as quantified by H&E staining, and correlated to the measured microvascular shutdown rate during PDT treatment. IS-DOCT measured significant PDT-induced vascular shutdown within the ROI in all tumors. A strong relationship (R2=0.723) exists between the percent tumor necrosis at 24 hours posttreatment and the vascular shutdown rate: slower shutdown corresponded to higher treatment efficacy, i.e., more necrosis. Controls (needle+light, no drug, n=3) showed minimal microvascular changes or necrosis (4%+/-1%). This study has correlated a biological end point with a direct and localized measurement of PDT-induced microvascular changes, suggesting a potential clinical role of on-line, real-time microvascular monitoring for optimizing treatment efficacy in individual patients.

	ODC1 Is a Critical Determinant of MYCN Oncogenesis and a Therapeutic Target in Neuroblastoma. Michael D. Hogarty^1,2, Murray D. Norris⁵, Kimberly Davis¹, Xueyuan Liu¹, Nicholas F. Evageliou¹, Candace S. Hayes⁷, Bruce Pawel³, Rong Guo⁴, Huaqing Zhao⁴, Eric Sekyere⁵, Joanna Keating⁵, Wayne Thomas⁵, Ngan Ching Cheng⁵, Jayne Murray⁵, Janice Smith⁵, Rosemary Sutton⁵, Nicola Venn⁵, Wendy B. London⁸, Allen Buxton⁸, Susan K. Gilmour⁷, Glenn M. Marshall^5,6 and Michelle Haber⁵ ¹ Division of Oncology, The Children's Hospital of Philadelphia; Departments of ² Pediatrics, ³ Pathology, and ⁴ Biostatistics and Epidemiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; ⁵ Children's Cancer Institute Australia for Medical Research; ⁶ Centre for Children's Cancer and Blood Disorders, Sydney Children's Hospital, Randwick, New South Wales, Australia; ⁷ Lankenau Institute for Medical Research, Wynnewood, Pennsylvania; and ⁸ Department of Statistics, University of Florida and Children's Oncology Group Statistics and Data Center Department, Gainesville, Florida Cancer Research. 2008 Dec 1;68(23):9735-45 Neuroblastoma is a frequently lethal childhood tumor in which MYC gene deregulation, commonly as MYCN amplification, portends poor outcome. Identifying the requisite biopathways downstream of MYC may provide therapeutic opportunities. We used transcriptome analyses to show that MYCN-amplified neuroblastomas have coordinately deregulated myriad polyamine enzymes (including ODC1, SRM, SMS, AMD1, OAZ2, and SMOX) to enhance polyamine biosynthesis. High-risk tumors without MYCN amplification also overexpress ODC1, the rate-limiting enzyme in polyamine biosynthesis, when compared with lower-risk tumors, suggesting that this pathway may be pivotal. Indeed, elevated ODC1 (independent of MYCN amplification) was associated with reduced survival in a large independent neuroblastoma cohort. As polyamines are essential for cell survival and linked to cancer progression, we studied polyamine antagonism to test for metabolic dependence on this pathway in neuroblastoma. The Odc inhibitor alpha-difluoromethylornithine (DFMO) inhibited neuroblast proliferation in vitro and suppressed oncogenesis in vivo. DFMO treatment of neuroblastoma-prone genetically engineered mice (TH-MYCN) extended tumor latency and survival in homozygous mice and prevented oncogenesis in hemizygous mice. In the latter, transient Odc ablation permanently prevented tumor onset consistent with a time-limited window for embryonal tumor initiation. Importantly, we show that DFMO augments antitumor efficacy of conventional cytotoxics in vivo. This work implicates polyamine biosynthesis as an arbiter of MYCN oncogenesis and shows initial efficacy for polyamine depletion strategies in neuroblastoma, a strategy that may have utility for this and other MYC-driven embryonal tumors. KEY WORDS: Embryonal tumors • metabolomics • polyamines • oncogene • experimental therapeutics

	Molecular Imaging of Therapeutic Response to Epidermal Growth Factor Receptor Blockade in Colorectal Cancer. H. Charles Manning, Nipun B. Merchant, A. Coe Foutch, John M. Virostko, Shelby K. Wyatt, Chirayu Shah, Eliot T. McKinley, Jingping Xie, Nathan J. Mutic, M. Kay Washington, Bonnie LaFleur, Mohammed Noor Tantawy, Todd E. Peterson, M. Sib Ansari, Ronald M. Baldwin, Mace L. Rothenberg, Darryl J. Bornhop, John C. Gore, and Robert J. Coffey 1Vanderbilt Institute of Imaging Science, Departments of 2Radiology and Radiological Sciences, 3Neurological Surgery, 4Surgery, 5Pathology, and 6Biostatistics, and 7Program in Chemical and Physical Biology, Vanderbilt University Medical Center; Departments of 8Biomedical Engineering, 9Chemistry, and 10Cell and Developmental Biology, Vanderbilt University; 11Department of Medicine,Vanderbilt University Medical School; 12Department of Veterans AffairsMedical Center, Nashville,Tennessee Clin. Cancer Res. 2008 Nov 15;14(22):7413-22 PURPOSE: To evaluate noninvasive molecular imaging methods as correlative biomarkers of therapeutic efficacy of cetuximab in human colorectal cancer cell line xenografts grown in athymic nude mice. The correlation between molecular imaging and immunohistochemical analysis to quantify epidermal growth factor (EGF) binding, apoptosis, and proliferation was evaluated in treated and untreated tumor-bearing cohorts. EXPERIMENTAL DESIGN: Optical imaging probes targeting EGF receptor (EGFR) expression (NIR800-EGF) and apoptosis (NIR700-Annexin V) were synthesized and evaluated in vitro and in vivo. Proliferation was assessed by 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) positron emission tomography. Assessment of inhibition of EGFR signaling by cetuximab was accomplished by concomitant imaging of NIR800-EGF, NIR700-Annexin V, and [18F]FLT in cetuximab-sensitive (DiFi) and insensitive (HCT-116) human colorectal cancer cell line xenografts. Imaging results were validated by measurement of tumor size and immunohistochemical analysis of total and phosphorylated EGFR, caspase-3, and Ki-67 immediately following in vivo imaging. RESULTS: NIR800-EGF accumulation in tumors reflected relative EGFR expression and EGFR occupancy by cetuximab. NIR700-Annexin V accumulation correlated with cetuximab-induced apoptosis as assessed by immunohistochemical staining of caspase-3. No significant difference in tumor proliferation was noted between treated and untreated animals by [18F]FLT positron emission tomography or Ki-67 immunohistochemistry. CONCLUSIONS: Molecular imaging can accurately assess EGF binding, proliferation, and apoptosis in human colorectal cancer xenografts. These imaging approaches may prove useful for serial, noninvasive monitoring of the biological effects of EGFR inhibition in preclinical studies. It is anticipated that these assays can be adapted for clinical use.

	Early therapy evaluation of combined anti-death receptor 5 antibody and gemcitabine in orthotopic pancreatic tumor xenografts by diffusion-weighted magnetic resonance imaging. Kim H, Morgan DE, Buchsbaum DJ, Zeng H, Grizzle WE, Warram JM, Stockard CR, McNally LR, Long JW, Sellers JC, Forero A, Zinn KR. Department of Radiology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0012, USA Cancer Research. 2008 Oct 15;68(20):8369-76. Early therapeutic efficacy of anti-death receptor 5 antibody (TRA-8) combined with gemcitabine was measured using diffusion-weighted magnetic resonance imaging (DWI) in an orthotopic pancreatic tumor model. Groups 1 to 4 of severe combined immunodeficient mice (n = 5-7 per group) bearing orthotopically implanted, luciferase-positive human pancreatic tumors (MIA PaCa-2) were subsequently (4-5 weeks thereafter) injected with saline (control), gemcitabine (120 mg/kg), TRA-8 (200 mug), or TRA-8 combined with gemcitabine, respectively, on day 0. DWI, anatomic magnetic resonance imaging, and bioluminescence imaging were done on days 0, 1, 2, and 3 after treatment. Three tumors from each group were collected randomly on day 3 after imaging, and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining was done to quantify apoptotic cellularity. At just 1 day after starting therapy, the changes of apparent diffusion coefficient (ADC) in tumor regions for group 3 (TRA-8) and group 4 (TRA-8/Gem) were 21 +/- 9% (mean +/- SE) and 27 +/- 3%, respectively, significantly higher (P < 0.05) than those of group 1 (-1 +/- 5%) and group 2 (-2 +/- 4%). There was no statistical difference in tumor volumes for the groups at this time. The mean ADC values of groups 2 to 4 gradually increased over 3 days, which were concurrent with tumor volume regressions and bioluminescence signal decreases. Apoptotic cell densities of tumors in groups 1 to 4 were 0.7 +/- 0.4%, 0.6 +/- 0.2%, 3.1 +/- 0.9%, and 4.7 +/- 1.0%, respectively, linearly proportional to the ADC changes on day 1. Further, the ADC changes were highly correlated with the previously reported mean survival times of animals treated with the same agents and doses. This study supports the clinical use of DWI for pancreatic tumor patients for early assessment of drug efficacy.

	Ultrasound imaging of apoptosis in tumor response: novel preclinical monitoring of photodynamic therapy effects. Banihashemi B, Vlad R, Debeljevic B, Giles A, Kolios MC, Czarnota GJ. Department of Radiation Oncology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada Cancer Research. 2008 Oct 15;68(20):8590-6. High-frequency ultrasound is a novel method to detect apoptotic cell death based on changes in cell morphology that cause alterations in the viscoelastic and, consequently, the acoustic properties of cell ensembles and tissues. In this study, we evaluated the first preclinical tumor-based use of high-frequency ultrasound spectroscopy to noninvasively monitor tumor treatment by following xenograft malignant melanoma tumor responses to photodynamic therapy (PDT) in vivo. We observed a time-dependant increase in ultrasound backscatter variables after treatment. The observed increases in spectroscopic variables correlated with morphologic findings, indicating increases in apoptotic cell death, which peaked at 24 hours after PDT. We analyzed the changes in spectral slope and backscatter in relation to apoptosis and histologic variations in cell nuclear size. Changes in spectral slope strongly correlated with the changes in mean nuclear size over time, associated with apoptosis, after PDT (P < 0.05). At 48 hours, a decrease in ultrasound backscatter was observed, which could be explained by an increase in cell nuclear degradation. In summary, we show that high-frequency ultrasound spectroscopic variables can be used noninvasively to monitor response after treatment in a preclinical tumor cancer model. These findings provide a foundation for future investigations regarding the use of ultrasound to monitor and aid the customization of treatments noninvasively based on responses to specific interventions.

	Biomedical imaging in the safety evaluation of new drugs. Yi-Xiang J Wang and Sen-Xiang Yan Department of Diagnostic Radiology and Organ Imaging, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, China; Department of Radiation Oncology, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China Laboratory Animals. 2008 Oct;42(4):433-41. Toxicology accounts for approximately one-third of attrition in new drug development and is a major concern in the pharmaceutical industry. This paper reviews the role of biomedical imaging in the safety evaluation of new candidate drugs. Ex vivo high-resolution threedimensional imaging of specimens can provide a quick overview of the specimens. Volumetric measurements of tissue structures and lesions can be made with higher precision and reproducibility than histology approaches. As opposed to histology, in vivo animal imaging permits longitudinal studies of the same animals over an extended period of time, with individual animals serving as their own control. Therefore, the number of animals required for a study can be significantly reduced and the intra-subject variability is minimized. Repeated in vivo imaging allows monitoring of the occurrence and progression, or regression, of various structural and functional abnormalities. Compared with other biological assays, imaging can provide anatomically specific information about tissue abnormality. Imaging offers the opportunity to carry forward the same methodology in animal experiments into human studies and has an important role in clinical trials when other safety biomarkers for early toxicities are not available.

	Targeted Microbubbles for Imaging Tumor Angiogenesis: Assessment of Whole-Body Biodistribution with Dynamic Micro-PET in Mice. Jürgen K. Willmann, MD; Zhen Cheng, PhD; Corrine Davis, DVM, PhD; Amelie M. Lutz, MD; Meike L. Schipper, MD; Carsten H. Nielsen, BS; Sanjiv S. Gambhir, MD, PhD Molecular Imaging Program at Stanford, Department of Radiology and Bio-X Program (J.K.W., Z.C., A.M.L., M.L.S., C.H.N., S.S.G.), Department of Comparative Medicine (C.D.), and Department of Bioengineering (S.S.G.), Stanford University School of Medicine, James H. Clark Center, 318 Campus Dr, East Wing, 1st Floor, Stanford, CA 94305-5427 Radiology. 2008 Oct; 249(1): 212-9 PURPOSE: To evaluate in vivo whole-body biodistribution of microbubbles (MBs) targeted to tumor angiogenesis-related vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) by using dynamic micro-positron emission tomography (PET) in living mice. MATERIALS AND METHODS: Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. Lipid-shell perfluorocarbon-filled MBs, targeted to VEGFR2 via anti-VEGFR2 antibodies, were radiolabeled by conjugating the radiofluorination agent N-succinimidyl-4-[(18)F]fluorobenzoate (SFB) to the anti-VEGFR2 antibodies. These MBs were then injected intravenously into nude mice (n = 4) bearing angiosarcomas, and the whole-body biodistribution of these probes was assessed for 60 minutes by using dynamic micro-PET. Results were compared with ex vivo gamma counting (n = 6) and immunofluorescence staining (n = 6). Control studies in angiosarcoma-bearing mice were performed with injection of the radiolabeled antibodies alone (n = 3) or free SFB (n = 3). A mixed-effects regression of MB accumulation on fixed effects of time and tissue type (tumor or muscle) and random effect of animal was performed. RESULTS: VEGFR2-targeted MBs rapidly cleared from the blood circulation (50% blood clearance after approximately 3.5 minutes) and accumulated in the liver (mean, 33.4% injected dose [ID]/g +/- 13.7 [standard deviation] at 60 minutes) and spleen (mean, 9.3% ID/g +/- 6.5 at 60 minutes) on the basis of micro-PET imaging. These findings were confirmed with ex vivo gamma counting. Uptake of targeted MBs was significantly higher (P < .0001) in tumor than in adjacent skeletal muscle tissue. Immunofluorescence staining demonstrated accumulation of the targeted MBs within hepatic Kupffer cells and splenic macrophages. Biodistribution of the radiolabeled antibodies and free SFB differed from the distribution of the targeted MBs. CONCLUSION: Dynamic micro-PET allows assessment of in vivo biodistribution of VEGFR2-targeted MBs. (c) RSNA, 2008.

	An orally bioavailable small-molecule inhibitor of Hedgehog signaling inhibits tumor initiation and metastasis in pancreatic cancer. Georg Feldmann,, Volker Fendrich, Karen McGovern, Djahida Bedja, Savita Bisht, Hector Alvarez,, Jan-Bart M. Koorstra, Nils Habbe,, Collins Karikari, Michael Mullendore,, Kathleen L. Gabrielson, Rajni Sharma, William Matsui, and Anirban Maitra Departments of Pathology, Comparative Medicine, Oncology, and Surgery, and The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland and Infinity Pharmaceuticals, Cambridge, Massachusetts Molecular Cancer Therapeutics. 2008 Sep; 7(9):2725-35 Recent evidence suggests that blockade of aberrant Hedgehog signaling can be exploited as a therapeutic strategy for pancreatic cancer. Our previous studies using the prototype Hedgehog small-molecule antagonist cyclopamine had shown the striking inhibition of systemic metastases on Hedgehog blockade in spontaneously metastatic orthotopic xenograft models. Cyclopamine is a natural compound with suboptimal pharmacokinetics, which impedes clinical translation. In the present study, a novel, orally bioavailable small-molecule Hedgehog inhibitor, IPI-269609, was tested using in vitro and in vivo model systems. In vitro treatment of pancreatic cancer cell lines with IPI-269609 resembled effects observed using cyclopamine (i.e., Gli-responsive reporter knockdown, down-regulation of the Hedgehog target genes Gli1 and Ptch, as well as abrogation of cell migration and colony formation in soft agar). Single-agent IPI-269609 profoundly inhibited systemic metastases in orthotopic xenografts established from human pancreatic cancer cell lines, although Hedgehog blockade had minimal effect on primary tumor volume. The only discernible phenotype observed within the treated primary tumor was a significant reduction in the population of aldehyde dehydrogenase-bright cells, which we have previously identified as a clonogenic tumor-initiating population in pancreatic cancer. Selective ex vivo depletion of aldehyde dehydrogenase-bright cells with IPI-269609 was accompanied by significant reduction in tumor engraftment rates in athymic mice. Pharmacologic blockade of aberrant Hedgehog signaling might prove to be an effective therapeutic strategy for inhibition of systemic metastases in pancreatic cancer, likely through targeting subsets of cancer cells with tumor-initiating ("cancer stem cell") properties.

	Dual-targeted Contrast Agent for US Assessment of Tumor Angiogenesis in Vivo. Jürgen K. Willmann, MD; Amelie M. Lutz, MD; Ramasamy Paulmurugan, PhD; Manishkumar R. Patel, PhD; Pauline Chu, BA; Jarrett Rosenberg, PhD; Sanjiv S. Gambhir, MD, PhD Molecular Imaging Program at Stanford, Department of Radiology and Bio-X Program (J.K.W., A.M.L., R.P., M.R.P., J.R., S.S.G.) and Department of Bioengineering (S.S.G.), Stanford University School of Medicine, the James H Clark Center, 318 Campus Dr, East Wing, 1st Floor, Stanford, CA 94305-5427; and Department of Comparative Medicine, Stanford University, Stanford, Calif (P.C.) Radiology. 2008 Sep;248(3):936-44 PURPOSE: To develop and validate a dual-targeted ultrasonographic (US) imaging agent with microbubbles (MBs) that attaches to both vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and alpha(v)beta(3) integrin and to compare the US imaging signal obtained from dual-targeted MBs (MB(D)) with that from single-targeted MBs (MB(S)) in a murine model of tumor angiogenesis. MATERIALS AND METHODS: Animal protocols were approved by the institutional Administrative Panel on Laboratory Animal Care. Single- and dual-targeted US imaging agents were prepared by attaching anti-VEGFR2, anti-alpha(v)beta(3) integrin, or both antibodies to the shell of perfluorocarbon-filled MBs. Binding specificities of targeted MBs compared with isotype-matched immunoglobulin G-labeled control MBs (MB(C)) and nontargeted nonlabeled MBs (MB(N)) were tested with VEGFR2-positive and alpha(v)beta(3) integrin-positive cells (mouse SVR cells) and control cells (mouse 4T1 cells). In vivo imaging signals of contrast material-enhanced US by using anti-VEGFR2-targeted MBs (MB(V)), anti-alpha(v)beta(3) integrin-targeted MBs (MB(I)), MB(D), and MB(C) were quantified in 49 mice bearing SK-OV-3 tumors (human ovarian cancer). Tumor tissue was stained for VEGFR2, alpha(v)beta(3) integrin, and CD31. RESULTS: Attachment of MB(D) to SVR cells (mean, 0.74 MBs per cell +/- 0.05 [standard deviation]) was significantly higher than attachment to 4T1 cells (mean, 0.04 +/- 0.03), and attachment to SVR cells was higher for MB(D) than for MB(V) (mean, 0.58 +/- 0.09), MB(I) (mean, 0.42 +/- 0.21), MB(C) (mean, 0.11 +/- 0.13), and MB(N) (mean, 0.01 +/- 0.01) (P < .05). Imaging signal in the murine tumor angiogenesis model was significantly higher (P < .001) for MB(D) (mean, 16.7 +/- 7.2) than for MB(V) (mean, 11.3 +/- 5.7), MB(I) (mean, 7.8 +/- 5.3), MB(C) (mean, 2.8 +/- 0.9), and MB(N) (mean, 1.1 +/- 0.4). Immunofluorescence confirmed expression of VEGFR2 and alpha(v)beta(3) integrin on tumor vasculature. CONCLUSION: Dual-targeted contrast-enhanced US directed at both VEGFR2 and alpha(v)beta(3) integrin improves in vivo visualization of tumor angiogenesis in a human ovarian cancer xenograft tumor model in mice. SUPPLEMENTAL MATERIAL: http://radiology.rsnajnls.org/cgi/content/full/248/3/936/DC1. RSNA, 2008

	Vessel Fractions in Tumor Xenografts Depicted by Flow- or Contrast-Sensitive Three-Dimensional High-Frequency Doppler Ultrasound Respond Differently to Antiangiogenic Treatment. Moritz Palmowski, Jochen Huppert, Peter Hauff, Michael Reinhardt, Karin Schreiner, Michaela A. Socher, Peter Hallscheidt, Guenter W. Kauffmann, Wolfhard Semmler, and Fabian Kiessling Medical Physics in Radiology, German Cancer Research Center; Division of Experimental Molecular Imaging, RWTH-Aachen University, Aachen, Germany; Global Drug Discovery, Bayer Schering Pharma AG, Berlin, Germany; and Department of Diagnostic Radiology, University of Heidelberg, Heidelberg, Germany Cancer Research. 2008 Sep 1;68(17):7042-9 High-frequency volumetric Power Doppler ultrasound (HF-VPDU) captures flow-dependent signals in blood vessels and can be used to assess antiangiogenic therapy effects in rodent tumors. However, the sensitivity is limited to vessels larger than capillaries. Contrast-enhanced HF-VPDU reveals all perfused vessels by assessing stimulated acoustic emissions from disintegrating microbubbles. Thus, we investigated whether flow-sensitive and contrast-enhanced HF-VPDU can depict different vessel fractions and assess their early response to antiangiogenic therapy. Mice with A431 tumors were scanned before and after administration of polybutylcyanoacrylate microbubbles by HF-VPDU. Animals received either antiangiogenic treatment (SU11248) or a control substance and were imaged repeatedly over 9 days. At each time point, tumors were removed for immunohistochemical analysis. During growth of untreated tumors, vascularization decreased correspondingly on flow-sensitive and contrast-enhanced scans. Treated tumors showed a significantly (P < 0.05) stronger decline in vascularization than controls, which was more pronounced in contrast-enhanced scans. Surprisingly, whereas vascularization remained low in contrast-enhanced scans, flow-sensitive ultrasound indicated a reincrease after day 6 with a higher vascularization than the controls at day 9. Histologic evaluation indicated that immature vessels degraded markedly on therapy, whereas large mature vessels on the tumor periphery were more therapy resistant and drew closer due to tumor shrinkage. In conclusion, contrast-enhanced HF-VPDU and flow-sensitive HF-VPDU are both capable of assessing the effects of antiangiogenic therapy. Because contrast-sensitive ultrasound is more sensitive for small immature vessels and flow-sensitive ultrasound mostly captures large vessels at the tumor periphery, the combination of both methods can provide evidence of vascular maturity in tumors.

	Imaging Hypoxia in Orthotopic Rat Liver Tumors with Iodine 124-labeled Iodoazomycin Galactopyranoside PET. Christopher C. Riedl,MD; Peter Brader,MD; Pat B. Zanzonico, PhD; Yun Shin Chun,MD; Yanghee Woo, MD; Paramjeet Singh, MD; Sean Carlin, PhD; Bixiu Wen,MD; C. Clifton Ling, PhD; Hedvig Hricak,MD; Yuman Fong, MD Departments of Radiology (C.C.R., P.B., H.H.), Medical Physics (P.B.Z., S.C., B.W., C.C.L.), and Surgery (Y.S.C., Y.W., P.S., Y.F.), Memorial Sloan-Kettering Cancer Center, 1275 York Ave, Room C-278, New York, NY 10021. Radiology. 2008 Aug;248(2):561-70 PURPOSE: To evaluate iodine 124 (124I)-labeled iodoazomycin galactopyranoside (IAZGP) positron emission tomography (PET) in the detection of hypoxia in an orthotopic rat liver tumor model by comparing regions of high (124)I-IAZGP uptake with independent measures of hypoxia and to determine the optimal time after injection to depict hypoxia. MATERIALS AND METHODS: The institutional animal care and use committee approved this study. Morris hepatoma tumors were established in the livers of 15 rats. Tumor oxygenation was measured in two rats with a fluorescence fiberoptic oxygen probe. (124)I-IAZGP was coadministered with the established hypoxia markers pimonidazole and EF5 in nine rats; 12-hour PET data acquisition was performed 24 hours later. Tumor cryosections were analyzed with immunofluorescence and autoradiography. In the four remaining rats, serial 20- and 60-minute PET data acquisition was peformed up to 48 hours after tracer administration. RESULTS: Oxygen probe measurements showed severe hypoxia (<1 mm Hg) distributed evenly throughout tumor tissue. Analysis of cryosections showed diffuse homogeneous uptake of (124)I-IAZGP throughout all tumors. The (124)I-IAZGP distribution correlated positively with pimonidazole (r = 0.78) and EF5 (r = 0.76) distribution. Tracer uptake in tumors was detectable with PET after 24 hours in seven of nine rats. In rats that underwent serial PET, tumor-to-liver contrast was sufficient to enable detection of hypoxia between 6 and 48 hours after tracer administration. The optimal ratio between signal intensity and tumor-to-liver contrast occurred 6 hours after tracer administration. CONCLUSION: Regions of high (124)I-IAZGP uptake in orthotopic rat liver tumors are consistent with independent measures of hypoxia; visualization of hypoxia with (124)I-IAZGP PET is optimal 6 hours after injection.

	Attenuated multimutated herpes simplex virus-1 effectively treats prostate carcinomas with neural invasion while preserving nerve function. Kelly K, Brader P, Rein A, Shah JP, Wong RJ, Fong Y, Gil Z Department of Surgery, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA The Journal of the Federation of American Societies for Experimental Biology. 2008 Jun;22(6):1839-48. Many cancers can cause disability and pain by invading nerves. In particular, prostate carcinoma has a high propensity for neural invasion (NI) at an early stage. Attempted surgical treatment of tumors with NI often leads to erectile dysfunction and deteriorated quality of life. Therefore, there is a need for novel modalities that will selectively target cancer cells while preserving neural function. Herpes simplex viruses (HSVs) have a natural trophism for peripheral nerves. We hypothesized that oncolytic therapy using HSV engineered to minimize neurotoxicity would be appropriate for this clinical setting. Attenuated HSV (NV1023) injected to sciatic nerves of nude mice had no toxic effect on nerve function (n=30). NV1023 had significant oncolytic effect on prostate carcinoma cells (PC3, DU145, and LNCap) in vitro. An in vivo model of NI was established by implanting prostate carcinoma cells in the sciatic nerves of nude mice. Mice were treated with NV1023 or saline 7 days after establishment of tumors. Significant reduction in tumor size and inhibition of NI was found 6-8 wk after treatment (P<0.005). All animals treated with saline developed complete paralysis <5 wk post-treatment, whereas most NV1023-treated animals had preserved nerve function >12 wk after treatment (P<0.0001). We conclude that oncolytic therapy effectively treats prostate carcinomas with NI in an in vivo murine model while preserving neural function. These findings may hold significant clinical implications for patients with prostate cancer or other neurotrophic tumors.

	MetMAb, the one-armed 5D5 anti-c-Met antibody, inhibits orthotopic pancreatic tumor growth and improves survival. Jin H, Yang R, Zheng Z, Romero M, Ross J, Bou-Reslan H, Carano RA, Kasman I, Mai E, Young J, Zha J, Zhang Z, Ross S, Schwall R, Colbern G, Merchant M Department of Translational Oncology, Genentech, Inc., South San Francisco, California 94080, USA Cancer Research. 2008 Jun 1;68(11):4360-8. The hepatocyte growth factor (HGF) and its receptor, c-Met, have been implicated in driving proliferation, invasion, and poor prognosis in pancreatic cancer. Here, we investigated the expression of HGF and c-Met in primary pancreatic cancers and described in vitro and in vivo models in which MetMAb, a monovalent antibody against c-Met, was evaluated. First, expression of HGF and MET mRNA was analyzed in 59 primary pancreatic cancers and 51 normal samples, showing that both factors are highly expressed in pancreatic cancer. We next examined HGF responsiveness in pancreatic cancer lines to select lines that proliferate in response to HGF. Based on these studies, two lines were selected for further in vivo model development: BxPC-3 (c-Met(+), HGF(-)) and KP4 (c-Met(+), HGF(+)) cells. As BxPC-3 cells are responsive to exogenous HGF, s.c. tumor xenografts were grown in a paracrine manner with purified human HGF provided by osmotic pumps, wherein MetMAb treatment significantly inhibited tumor growth. KP4 cells are autocrine for HGF and c-Met, and MetMAb strongly inhibited s.c. tumor growth. To better model pancreatic cancer and to enable long-term survival studies, an orthotopic model of KP4 was established. MetMAb significantly inhibited orthotopic KP4 tumor growth in 4-week studies monitored by ultrasound and also improved survival in 90-day studies. MetMAb significantly reduced c-Met phosphorylation in orthotopic KP4 tumors with a concomitant decrease in Ki-67 staining. These data suggest that the HGF/c-Met axis plays an important role in the progression of pancreatic cancer and that targeting c-Met therein may have therapeutic value.

	Volumetric high-frequency Doppler ultrasound enables the assessment of early antiangiogenic therapy effects on tumor xenografts in nude mice. Jugold M, Palmowski M, Huppert J, Woenne EC, Mueller MM, Semmler W, Kiessling F. German Cancer Research Center, Heidelberg, Germany. Eur Radiol. 2008 Apr;18(4):753-8. Epub 2007 Dec 15 The sensitivity of Doppler ultrasound below 10 MHz to assess antiangiogenic therapy effects in tumor xenografts has been shown to be limited. Thus, our aim was to evaluate high-frequency volumetric power-Doppler ultrasound (HF-VPDU) for monitoring antiangiogenic treatments. Squamous cell carcinoma xenografts grown in nude mice were scanned with HF-VPDU at a center frequency of 30 MHz (VisualSonics Vevo 770 micro-ultrasound system). Images with 200-mum slice thicknesses were recorded and merged into a three-dimensional dataset, of which the relative color pixel density was determined. Animals received either VEGFR2 antibodies or 0.9% NaCl and were examined at days 0, 3 and 6 of treatment. After the last examination, tumors were resected and their vascularization characterized by immunohistology. At day 6, the volumes of treated and untreated tumors were not significantly different yet. In contrast, mean tumor vascularization in treated animals had decreased to 44%, while in the control group it had increased to 152% (P < 0.01). In correspondence, vessel density, as determined by CD31 staining, was 0.19 +/- 0.10% in treated and 0.92 +/- 0.41% in untreated tumors (P < 0.01). Additionally, the fraction of mature (SMA-positive) vessels increased under therapy. Thus, HF-VPDU can be considered as an easily applicable and fast method to screen high animal numbers for antiangiogenic therapy effects.

	Urocortin2 inhibits tumor growth via effects on vascularization and cell proliferation. Zhengrong Hao, Yan Huang, Jake Cleman, Ion S. Jovin, Wylie W. Vale, Tracy L. Bale, and Frank J. Giordano Department of Medicine, Yale University, New Haven, CT 06520; Peptide Biology Laboratories, Salk Institute, La Jolla, CA 92037; and Department of Animal Biology, University of Pennsylvania, Philadelphia, PA 19104 Proceedings of the National Academy of Sciences. 2008 March 11;105(10): 3939-3944 The corticotropin-releasing factor (CRF) receptor CRFR2 is expressed widely in peripheral tissues and in the vasculature, although its functional roles in those tissues have only recently begun to be elucidated. Previously we found that genetic deletion of CRFR2 resulted in profound postnatal hypervascularization in mice, characterized by both an increase in total vessel number and a dramatic increase in vessel diameter. These data strongly suggested that ligands for CRFR2 act to limit tissue vascularity, perhaps as a counterbalance to factors that promote neovascularization. Urocortin 2 (Ucn2) is a specific ligand for the CRFR2. We hypothesized that activation of CRFR2 by Ucn2 might thus suppress tumor vascularization and consequently limit tumor growth. Here, we show that viral-mediated expression of Ucn2 strikingly inhibits the growth and vascularization of Lewis Lung Carcinoma Cell (LLCC) tumors in vivo. Further, we found that this effect on tumor growth inhibition was independent of whether exposure to Ucn2 occurred before or after establishment of measurable tumors. In vitro, Ucn2 directly inhibited the proliferation of LLCC, suggesting that the tumor-suppressing effects of CRFR2 activation involve a dual mechanism of both a direct inhibition of tumor cell cycling and the suppression of tumor vascularization. These results establish that Ucn2 inhibits tumor growth, suggesting a potential therapeutic role for CRFR2 ligands in clinical malignancies.

	Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma. Yan Ding, Elissa A. Boguslawski, Bree D. Berghuis, John J. Young, Zhongfa Zhang, Kim Hardy, Kyle Furge, Eric Kort, Arthur E. Frankel, Rick V. Hay, James H. Resau and Nicholas S. Duesbery Laboratories of Cancer and Developmental Cell Biology, Analytical, Cellular and Molecular Microscopy, Cancer Genetics, Noninvasive Imaging and Radiation Biology, and Computational Biology, Van Andel Research Institute, Grand Rapids, Michigan and 6 Cancer Research Institute of Scott & White Memorial Hospital, Temple, Texas Molecular Cancer Therapeutics. 2008 March 1;7: 648-658. We hypothesized that signaling through multiple mitogen-activated protein kinase (MAPK) kinase (MKK) pathways is essential for the growth and vascularization of soft-tissue sarcomas, which are malignant tumors derived from mesenchymal tissues. We tested this using HT-1080, NCI, and Shac fibrosarcoma-derived cell lines and anthrax lethal toxin (LeTx), a bacterial toxin that inactivates MKKs. Western blots confirmed that LeTx treatment reduced the levels of phosphorylated extracellular signal-regulated kinase and p38 MAPK in vitro. Although short treatments with LeTx only modestly affected cell proliferation, sustained treatment markedly reduced cell numbers. LeTx also substantially inhibited the extracellular release of angioproliferative factors including vascular endothelial growth factor, interleukin-8, and basic fibroblast growth factor. Similar results were obtained with cell lines derived from malignant fibrous histiocytomas, leiomyosarcomas, and liposarcomas. In vivo, LeTx decreased MAPK activity and blocked fibrosarcoma growth. Growth inhibition correlated with decreased cellular proliferation and extensive necrosis, and it was accompanied by a decrease in tumor mean vessel density as well as a reduction in serum expression of angioproliferative cytokines. Vital imaging using high-resolution ultrasound enhanced with contrast microbubbles revealed that the effects of LeTx on tumor perfusion were remarkably rapid (<24 h) and resulted in a marked reduction of perfusion within the tumor but not in nontumor tissues. These results are consistent with our initial hypothesis and lead us to propose that MKK inhibition by LeTx is a broadly effective strategy for targeting neovascularization in fibrosarcomas and other similar proliferative lesions.

	{alpha}2{beta}1 integrin expression in the tumor microenvironment enhances tumor angiogenesis in a tumor cell-specific manner. Zhang Z, Ramirez NE, Yankeelov TE, Li Z, Ford LE, Qi Y, Pozzi A, Zutter MM Department of Pathology, Vanderbilt University School of Medicine, Nashville, TN 37232-2561, USA Blood. 2008 Feb 15;111(4):1980-8. To define the role of the alpha2beta1 integrin in pathologic angiogenesis, we investigated tumor-associated growth and angiogenesis in wild-type and alpha2-null mice. Our findings reveal that the alpha2beta1 integrin plays an important role in angiogenesis via regulation of VEGFR1 expression. When challenged with B16F10 melanoma cells, mice lacking alpha2beta1 integrin ex-pression exhibit increased tumor angiogenesis associated with up-regulated VEGFR1 expression. In contrast, there was no alpha2beta1 integrin-dependent difference in the angiogenic response to Lewis lung carcinoma (LLC) cells. Interestingly, whereas B16F10 cells secrete high levels of placental growth factor (PLGF), LLC cells produce high levels of VEGF, but low levels of PLGF. The alpha2beta1 integrin-dependent difference in angiogenesis was restored to LLC cells by expression of PLGF, strongly suggesting that the angiogenic phenotype and tumor growth in the alpha2-null host is dependent on specific interactions between the tumor cell and the genetically defined integrin repertoire of the host microenvironment. Thus integrin alpha2-null mice represent an example of genetic alterations of "the soil" determining response to the "seed."

	US Imaging of Tumor Angiogenesis with Microbubbles Targeted to Vascular Endothelial Growth Factor Receptor Type 2 in Mice. Jürgen K. Willmann, MD; Ramasamy Paulmurugan, PhD; Kai Chen, PhD; Olivier Gheysens, MD; Martin Rodriguez-Porcel, MD; Amelie M. Lutz, MD; Ian Y. Chen, MSE; Xiaoyuan Chen, PhD; Sanjiv S. Gambhir, MD, PhD Molecular Imaging Program at Stanford, Department of Radiology and Bio-X Program (J.K.W., R.P., K.C., O.G., M.R., A.M.L., I.Y.C., X.C., S.S.G.), and Department of Bioengineering (I.Y.C., S.S.G.), Stanford University School of Medicine, the James H. Clark Center, 318 Campus Dr, East Wing, 1st Floor, Stanford, CA 94305-5427 Radiology. 2008 Feb; 246(2):508-18. Epub 2008 Jan 7 PURPOSE: To prospectively evaluate contrast material-enhanced ultrasonography (US) with microbubbles targeted to vascular endothelial growth factor receptor type 2 (VEGFR2) for imaging tumor angiogenesis in two murine tumor models. MATERIALS AND METHODS: Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. A US contrast agent, consisting of encapsulated gaseous microbubbles, was developed specifically to bind to VEGFR2 (by using anti-VEGFR2 antibodies and biotin-streptavidin interaction) which is up-regulated on endothelial cells of tumor blood vessels. VEGFR2-targeted microbubbles (MB(V)), control microbubbles (MB(C)), and nonlabeled microbubbles (MB(N)) were tested for binding specificity on cells expressing VEGFR2 (mouse angiosarcoma SVR cells) and control cells (mouse skeletal myoblast C2C12 cells). Expression of mouse VEGFR2 in culture cells was tested with immunocytochemical and Western blot analysis. Contrast-enhanced US imaging with MB(V) and MB(C) was performed in 28 tumor-bearing nude mice (mouse angiosarcoma, n = 18; rat malignant glioma, n = 10). Differences were calculated by using analysis of variance. RESULTS: In cell culture, adherence of MB(V) on SVR cells (2.1 microbubbles per SVR cell) was significantly higher than adherence of control microbubbles (0.01-0.10 microbubble per SVR cell; P < .001) and significantly more MB(V) attached to SVR cells than to C2C12 cells (0.15 microbubble per C2C12 cell; P < .001). In vivo, contrast-enhanced US imaging showed significantly higher average video intensity when using MB(V) compared with MB(C) for angiosarcoma and malignant glioma tumors (P < .001). Results of immunohistochemical analysis confirmed VEGFR2 expression on vascular endothelial cells of both tumor types. CONCLUSION: US imaging with contrast microbubbles targeted to VEGFR2 allows noninvasive visualization of VEGFR2 expression in tumor vessels in mice. (c) RSNA, 2008

	Measurement of Human Choroidal Melanoma Xenograft Volume in Rats Using High-Frequency Ultrasound. Rod D. Braun and Kerry S. Vistisen Department of Anatomy and Cell Biology and the Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit, Michigan Investigative Ophthalmology and Visual Science. 2008 Jan;49(1):16-22 PURPOSE. The purpose of this study was to test the hypothesis that the volume of primary orthotopic human choroidal melanoma xenografts can be quantified noninvasively in the same nude rat over time using a portable, high-resolution, high-frequency ultrasound (HF-US) system. METHODS. C918 human choroidal melanoma spheroids were implanted in the superior suprachoroidal space of 26 WAG/RijHsd-rnu nude rats. Fourteen rats were anesthetized 14 days after tumor implantation, and HF-US B-scan images of the tumor-bearing eye were captured at 250-{micro}m intervals. Tumor areas were measured on each image and numerically integrated to calculate volume. Tumor volumes were also estimated from serial histologic sections in six rats. Twelve other rats were anesthetized and weighed every 4 to 5 days after implantation for 2 weeks, and HF-US B-scan image series were acquired for subsequent measurement of tumor volume. RESULTS. Tumors could be visualized as heterogeneous, relatively hyperechoic regions in the superior portion of the eye. These regions were verified as tumor by comparison with histologic sections, and histologic and HF-US volumes were highly correlated (r = 0.961; P = 0.002). For the determination of HF-US volume, the intraobserver variability was 9.7% {+/-} 5.1% (n = 8), and the coefficient of variation for multiple measurements was 12.1% {+/-} 6.8% (n = 12). Tumor volume could be repeatedly measured in the same rat every 4 to 5 days for 2 weeks without significant weight loss. CONCLUSIONS. HF-US is a safe, practical method to measure tumor volume in the same nude rat over time in this orthotopic xenograft model of human choroidal melanoma.

	Vitamin E analogues inhibit angiogenesis by selective induction of apoptosis in proliferating endothelial cells: the role of oxidative stress. Lan-Feng Dong, Emma Swettenham, Johanna Eliasson, Xiu-Fang Wang, Mikhal Gold, Yasmine Medunic, Marina Stantic, Pauline Low, LubomirPr ochazka, Paul K. Witting, Jaroslav Turanek, Emmanuel T. Akporiaye, Stephen J. Ralph, and Jiri Neuzil Apoptosis Research Group, School of Medical Science, Griffith University, Southport, Queensland, Australia. Cancer Research. 2007 Dec 15;67(24):11906-13. "Mitocans" from the vitamin E group of selective anticancer drugs, alpha-tocopheryl succinate (alpha-TOS) and its ether analogue alpha-TEA, triggered apoptosis in proliferating but not arrested endothelial cells. Angiogenic endothelial cells exposed to the vitamin E analogues, unlike their arrested counterparts, readily accumulated reactive oxygen species (ROS) by interfering with the mitochondrial redox chain and activating the intrinsic apoptotic pathway. The vitamin E analogues inhibited angiogenesis in vitro as assessed using the "wound-healing" and "tube-forming" models. Endothelial cells deficient in mitochondrial DNA (mtDNA) were resistant to the vitamin E analogues, both in ROS accumulation and apoptosis induction, maintaining their angiogenic potential. alpha-TOS inhibited angiogenesis in a mouse cancer model, as documented by ultrasound imaging (Vevo 770 micro-ultrasound, VisualSonics). We conclude that vitamin E analogues selectively kill angiogenic endothelial cells, suppressing tumor growth, which has intriguing clinical implications. NOTE: The Vevo 770 micro- ultrasound imaging device is equipped with the Power Doppler function, which makes it possible to follow circulation in blood vessels. This was applied to assess the extent of angiogenesis in the FVB/N c-neu mice carcinomas. The control and treated mice were assessed by ultrasound imaging using the same respiratory gating, and the volume of blood vessels within tumors was expressed as percentage of vascularization of individual tumors.

	TRA-8 anti-DR5 monoclonal antibody and gemcitabine induce apoptosis and inhibit radiologically validated orthotopic pancreatic tumor growth. Derosier LC, Vickers SM, Zinn KR, Huang Z, Wang W, Grizzle WE, Sellers J, Stockard CR Jr, Zhou T, Oliver PG, Arnoletti P, Lobuglio AF, Buchsbaum DJ. University of Alabama at Birmingham, 1530 3rd Avenue South, Wallace Tumor Institute 674, Birmingham, AL Mol Cancer Ther. 2007 Dec;6(12):3198-207. Purpose: To evaluate agonistic TRA-8 monoclonal antibody to human death receptor 5 (DR5) and gemcitabine in vitro and in an orthotopic pancreatic cancer model. Experimental Design: Pancreatic cancer cell lines were screened for DR5 expression, cytotoxicity, and apoptosis induced by TRA-8, gemcitabine, or gemcitabine and TRA-8. An orthotopic model of pancreatic cancer was established in severe combined immunodeficient mice. Mice were treated with TRA-8, gemcitabine, or a combination for one or two cycles of therapy. Tumor growth (Vevo 660 micro-ultrasound, VisualSonics) and survival were analyzed. Results: All five pancreatic cancer cell lines showed DR5 protein expression and varying sensitivity to TRA-8-mediated cytotoxicity. MIA PaCa-2 cells were very sensitive to TRA-8, moderately resistant to gemcitabine, with additive cytotoxicity to the combination. S2-VP10 cells were resistant to TRA-8 and sensitive to gemcitabine with synergistic sensitivity to the combination. Combination treatment in vitro produced enhanced caspase-3 and caspase-8 activation. A single cycle of therapy produced comparable efficacy for single-agent TRA-8 and the combination of TRA-8 and gemcitabine, with significant reduction in tumor size and prolonged survival compared with gemcitabine alone or control animals. With two cycles of therapy, TRA-8 and combination therapy produced enhanced inhibition of tumor growth compared with single-agent gemcitabine or untreated animals. However, the combination regimen showed enhanced survival as compared with single-agent TRA-8. Conclusions: Pancreatic cancer cell lines express varying levels of DR5 and differ in their sensitivity to TRA-8 and gemcitabine-induced cytotoxicity. TRA-8 with two cycles of gemcitabine therapy produced the best overall survival.

	Rapid reversal of interleukin-6-dependent epithelial invasion in a mouse model of microbially induced colon carcinoma. Theofilos Poutahidis^1,5, Kevin M. Haigis^2,6, Varada P. Rao¹, Prashant R. Nambiar¹, Christie L. Taylor¹, Zhongming Ge¹, Koichiro Watanabe¹, Anne Davidson^3,7, Bruce H. Horwitz⁴, James G. Fox¹ and Susan E. Erdman¹ ¹ Division of Comparative Medicine; ² Center for Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA; ³ Department of Medicine and Microbiology, Columbia University, 1130 St Nicholas Avenue, Room 918, New York, NY 10032; ⁴ Immunology Research Division, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Avenue, Boston, MA 02115, USA; ⁵ Present address: Laboratory of Pathology, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece; ⁶ Present address: Unit for Molecular Pathology, Center for Cancer Research, Massachusetts General Hospital, Bldg 149, Rm 7148, 13th Street, Charlestown, MA 02129, USA; ⁷ Present address: Feinstein Institute for Medical Research, 350 Community Drive, Manhasset NY 11030, USA Carcinogenesis. 2007 Dec;28(12):2614-23. Epub 2007 Aug 27 Chronic inflammation of mucosal surfaces renders them increasingly susceptible to epithelial cancers both in humans and mice. We have previously shown that anti-inflammatory CD4(+)CD45RB(lo)CD25(+) regulatory (Treg or T(R)) lymphocytes down-regulate inflammation and block development of bacteria-triggered colitis and colorectal cancer (CRC) in 129/SvEv Rag2-/- mice. Interestingly, T(R) cells collected from Interleukin (IL)-10-deficient cell donors not only failed to suppress carcinogenesis but instead promoted invasive mucinous colonic carcinoma with a strong gender bias expressing in male mice. We found we show that peritoneal invasion in this model is dependent on pleiotropic cytokine IL-6. Mucinous carcinoma arose rapidly and consistently after treatment with IL10-/- T(R) cells, which were found to express Foxp3+ and localize throughout tumor tissue. Carcinogenesis was rapidly reversible with transfer of wild type IL10-competent T(R) cells. Likewise, treatment with IL10-Ig fusion protein was sufficient to revert the lesions histologically, and restore inflammatory cytokine and oncogene expression to base line levels. These studies indicate an essential role for IL 6 in this CRC phenotype. Furthermore, immune-competent T(R) cells were important not only for preventing pathology but also for constructive remodeling of bowel following tumorigenic microbial insults. These data provide insights into etiopathogenesis of inflammation-associated epithelial invasion and maintenance of epithelial homeostasis.

	Molecular Imaging Of Vascular Endothelial Growth Factor Receptor 2 Expression Using Targeted Contrast-Enhanced High-Frequency Ultrasonography. Lyshchik A, Fleischer AC, Huamani J, Hallahan DE, Brissova M, Gore JC Department of Radiology and Radiological Sciences, Vanderbilt University Medical Center, Nashville, TN J Ultrasound Med. 2007 Nov;26(11):1575-86. OBJECTIVE: Dilated cardiomyopathy (DCM) leads to dilation of the cardiac chambers and congestive heart failure. Recent reports have associated mutations in the SCN5A gene, which codes for the major cardiac sodium channel Nav1.5, with DCM. Although DCM is the most common form of cardiomyopathy, no animal studies have established this functional connection. METHODS AND RESULTS: We have produced transgenic mice that ectopically express the transcriptional repressor Snail in heart. These animals display severe DCM, ECG abnormalities, conduction defects, revealed by voltage-sensitive dye imaging, and significantly reduced voltage-gated sodium current as measured by patch clamping. There is a concomitant decrease in expression of the major cardiac sodium channel gene Scn5a, which we show by gene reporter assays and electrophoretic mobility shift assays is a direct target of Snail. CONCLUSIONS: Our findings indicate that a decrease in Scn5a expression and significant reduction in sodium current can result in DCM, and support the hypothesis that some mutations in the human SCN5A gene can lead to DCM. NOTE: Ultrasound imaging to support this research paper was performed using a Vevo 660 micro-ultrasound system (VisualSonics).

	Imaging of hypoxia-driven gene expression in an orthotopic liver tumor model. Peter Brader¹, Christopher Cesare Riedl¹, Yanghee Woo², Vladimir Ponomarev¹, Pat Zanzonico³, Bixiu Wen³, Shangde Cai⁴, Hedvig Hricak¹, Yuman Fong², Ronald Blasberg1,⁵ and Inna Serganova⁵ Departments of ¹ Radiology, ² Surgery, and ³ Medical Physics; ⁴ Cyclotron and Radiochemistry Core Facility; and ⁵ Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, New York Molecular Cancer Therapeutics. 2007 Nov;6(11):2900-8. Epub 2007 Nov 7 The purpose of this study was to monitor hypoxia in an orthotopic liver tumor model using a hypoxia-sensitive reporter imaging system and to image enhanced gene expression after clamping the hepatic artery. C6 and RH7777 Morris hepatoma cells were transduced with a triple reporter gene (HSV1-tk/green fluorescent protein/firefly luciferase-triple fusion), placed under the control of a HIF-1-inducible hypoxia responsive element (HRE). The cells showed inducible luciferase activity and green fluorescent protein expression in vitro. Isolated reporter-transduced Morris hepatoma cells were used to produce tumors in livers of nude rats, and the effect of hepatic artery clamping was evaluated. Tumor hypoxia was shown by immunofluorescence microscopy with the hypoxia marker EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl acetamide)] and the fluorescent perfusion marker Hoechst 33342, and by pO(2) electrode measurements. For tumor hypoxia imaging with the HRE-responsive reporter, both luciferase bioluminescence and [(18)F]2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil positron emission tomography was done, and the presence of hypoxia in Morris hepatoma tumors were successfully imaged by both techniques. Transient clamping of the hepatic artery caused cessation of tumor perfusion and severe hypoxia in liver tumors, but not in adjacent liver tissue. These results show that the orthotopic reporter-transduced RH7777 Morris hepatomas are natively hypoxic and poorly perfused in this animal model, and that the magnitude of hypoxia can be monitored using a HRE-responsive reporter system for both bioluminescence and positron emission tomography imaging. However, the severity of tumor ischemia after permanent ligation of the hepatic artery limits our ability to image severe hypoxia in this animal model.

	Ovarian Volume Measurements in Mice With High-Resolution Ultrasonography. Andrej Lyshchik, MD, PhD, Stephen B. Hobbs, Arthur C. Fleischer, MD, Dineo Khabele, MD, Deok-Soo Son, DVM, PhD, John C. Gore, PhD and Ronald R. Price, PhD Department of Radiology and Radiological Sciences, Vanderbilt University Medical Center, Nashville, Tennessee USA (A.L., S.B.H., A.C.F., J.C.G., R.R.P.); Vanderbilt University Institute of Imaging Science, Nashville, Tennessee USA (A.L., J.C.G.); and Department of Obstetrics and Gynecology, Meharry Medical College, Nashville, Tennessee USA (D.K., D.-S.S.). J Ultrasound Med. 2007 Oct; 26(10), PP 1419-1425 Objective. The aim of our study was to evaluate the intraobserver and interobserver variability of ovarian volume measurements in mice with high-resolution 2-dimensional ultrasonography (2DUS) and 3-dimensional ultrasonography (3DUS). Methods. Ovaries of 10 nude mice were visualized with a small-animal ultrasound scanner and a 40-MHz probe (Vevo 770 micro-ultrasound, VisualSonics). For each ovary, volume was measured 3 times by 2 independent readers using both 2DUS and 3DUS methods. The 2DUS method used a biplane ellipsoid model. The 3DUS method estimated the volume by integrating 10 to 12 parallel image planes of the ovary after semiautomated outlining of the boundaries. For each type of measurement, intraobserver and interobserver standard error of measurement (SEM) values and minimal detectable volume changes were calculated by analysis of variance. Results. Two-dimensional ultrasonography showed much poorer reproducibility, with higher absolute intraobserver and interobserver SEM values (0.50 and 0.61 mm3, respectively) than 3DUS (0.20 and 0.35 mm3; P < .01). Relative intraobserver and interobserver SEM values were also much higher for 2DUS (12.20% and 14.88%) than for 3DUS (5.12% and 8.97%; P < .01). The minimal volume changes that could be detected with a 95% confidence level in successive measurements by the same (or different) observers were 33.90% (41.22%) for 2DUS and 14.10% (24.87%) for 3DUS. Conclusions. High-resolution 3DUS can provide a reliable tool for noninvasive, longitudinal ovarian volume measurements in mice.

	Microultrasound Molecular Imaging of Vascular Endothelial Growth Factor Receptor 2 in a Mouse Model of Tumor Angiogenesis. Joshua J. Rychak, James Graba, Alison M.Y. Cheung, Bina S. Mystry, Jonathan R. Lindner, Robert S. Kerbel, and F. Stuart Foster Molecular Imaging. 2007 Sep-Oct; 6(5): PP. 289–296 High-frequency micro-ultrasound imaging of tumor progression in mice enables noninvasive anatomic and functional imaging at excellent spatial and temporal resolution, although micro-ultrasonography alone does not offer molecular scale data. In the current study, we investigated the use of microbubble ultrasound contrast agents bearing targeting ligands specific for molecular markers of tumor angiogenesis using high-frequency micro-ultrasound imaging. A xenograft tumor model in the mouse was used to image vascular endothelial growth factor receptor 2 (VEGFR-2) expression with microbubbles conjugated to an anti-VEGFR-2 monoclonal antibody or an isotype control. Micro-ultrasound imaging (Vevo 770, VisualSonics) was accomplished at a center frequency of 40 MHz, which provided lateral and axial resolutions of 40 and 90 mm, respectively. The B-mode (two-dimensional mode) acoustic signal from microbubbles bound to the molecular target was determined by an ultrasound-based destruction-subtraction scheme. Quantification of the adherent microbubble fraction in nine tumor-bearing mice revealed significant retention of VEGFR-2-targeted microbubbles relative to control targeted microbubbles. These data demonstrate that contrast-enhanced micro-ultrasound imaging is a useful method for assessing molecular expression of tumor angiogenesis in mice at high resolution. NOTE: Please note that original testing and validation of contrast and molecular imaging was performed on the Vevo 770 with multiple contrast agents, including the results desrcibed in this paper. Since launching our molecular imaging program, VisualSonics has partnered with Bracco, the world leader in contrast agents, to develop and commercially manufacture MicroMarker™ Contrast Agent Kits.MicroMarker kits are available in fully QC-ed kits with detailed and simple protocols, biological support from VisualSonics’ specialized Applications Support team, and detailed training including procedural videos. MicroMarker kits are available for (1) perfusion and image enhancement imaging applications,(2) for targeted molecular biomarkers (ie. VEGFR2 for angiogenesis or P-selectin for inflammation, for example), and (3) for cardiovascular myocardial viability studies.

	Combination Treatment with TRA-8 Anti Death Receptor 5 Antibody and CPT-11 Induces Tumor Regression in an Orthotopic Model of Pancreatic Cancer. Derosier LC, Buchsbaum DJ, Oliver PG, Huang ZQ, Sellers JC, Grizzle WE, Wang W, Zhou T, Zinn KR, Long JW, Vickers SM Department of Radiation Oncology, University of Alabama at Birmingham, Birmingham, Alabama Clin Cancer Res. 2007 Sep 15;13(18):5535s-43s Purpose: Evaluate the response of human pancreatic cancer cell lines and orthotopic tumors to TRA-8, an agonistic antibody to death receptor 5, in combination with irinotecan (CPT-11). Experimental Design: MIA PaCa-2 and S2VP10 cells were treated with TRA-8 and/or CPT 11. Cell viability was determined by ATP assay. JC-1 mitochondrial depolarization and Annexin V assays confirmed cell death by apoptosis. Immunoblotting was used to evaluate protein changes. MIA PaCa-2 cells were injected into the pancreas of severe combined immunodeficient mice. Mice underwent abdominal ultrasound (Vevo 660 micro-ultrasound, VisualSonics) to quantitate tumor size before and after treatment with twice weekly injections of 200 µg TRA-8 and/or 25 mg/kg CPT-11 for one or two treatment cycles, each lasting 2 weeks. Results: MIA PaCa-2 cells were more sensitive to TRA-8 and showed additive cytotoxicity, whereas S2VP10 cells showed synergistic cytotoxicity when treated with TRA-8 and CPT-11. Cell death occurred via apoptosis with increased cleavage of caspase-3, caspase-8, and caspase-9 and proapoptotic proteins Bid and poly(ADP)ribose polymerase after combination treatment compared with either agent alone. XIAP and Bcl-XL inhibitors of apoptosis were down-regulated. After a single cycle of in vivo combination therapy, tumor sizes had diminished significantly (P < 0.001) at 8 days posttreatment compared with no treatment, CPT-11, and TRA-8; and there was a 50-day increase in survival with combination treatment over untreated controls (P = 0.0002), 30 days over TRA-8, and a 36-day increase over CPT-11 monotherapy (P = 0.0003). With two cycles of TRA-8/CPT-11 treatment, mean survival time increased significantly (P < 0.001) to 169 days versus untreated controls, TRA-8 or CPT-11 (76, 121, or 108 days, respectively). Conclusions: Combination TRA-8 and CPT-11 therapy produced enhanced cytotoxicity and survival in the MIA PaCa-2 orthotopic model of pancreatic cancer.

	Biodegradable nanoparticles for targeted ultrasound imaging of breast cancer cells in vitro. Jun Liu, Jie Li, Thomas J Rosol, Xueliang Pan and Jeffrey L Voorhees Department of Biomedical Engineering, Ohio State University PHYSICS IN MEDICINE AND BIOLOGY,2007 Aug 21;52(16):4739-47. Epub 2007 Jul 24 Disease-specific enhanced imaging through a targeted agent promises to improve the specificity of medical ultrasound. Nanoparticles may provide unique advantages for targeted ultrasound imaging due to their novel physical and surface properties. In this study, we examined a nanoparticle agent developed from a biodegradable polymer, polylactic acid (PLA). The nanoparticles (mean diameter = 250 nm) were surface conjugated to an anti- Her2 antibody (i.e., Herceptin) for specific binding to breast cancer cells that overexpress Her2 receptors. We examined the targeting specificity and the resultant ultrasound enhancement in Her2-positive and negative cells. Flow cytometry and confocal imaging were used to assess the nanoparticle-cell binding. Her2-positive cells demonstrated substantial staining after incubation with nanoparticle/antibody conjugates, while minimal staining was found in Her2-negative cells, indicating receptor-specific binding of the conjugated PLA nanoparticles. In high-resolution ultrasound B-mode images (Vevo 660 micro-ultrasound, VisualSopnics), the average gray scale of the Her2-positive cells was consistently and significantly higher after nanoparticle treatment (133 ± 4 in treated cells versus 109 ± 4 in control, p < 0.001, n = 5), while no difference was detected in the cells that did not overexpress the receptors (117±3 in treated cells versus 118±5 in control). In conclusion, the feasibility of using targeted nanoparticles to enhance ultrasonic images was demonstrated in vitro. This may be a promising approach to target cancer biomarkers for site-specific ultrasound imaging.

	Detecting Vascular Changes In Tumour Xenografts Using Micro-Ultrasound And Micro-Ct Following Treatment With VEGFR-2 Blocking Antibodies. ALISON M. Y. CHEUNG,^* ALLISON S. BROWN,^* VIVIENE CUCEVIC,^* MARCIA ROY,^* ANDREW NEEDLES,^* VICTOR YANG,^* DANIEL J. HICKLIN,^‡ ROBERT S. KERBEL,^† and F. STUART FOSTER^* ^*Imaging Research and †Molecular and Cell Biology, Sunnybrook and Women’s College Health Sciences Centre, Toronto, ON, Canada; and ^‡ImClone Systems, New York, NY, USA Ultrasound in Med. & Biol. 2007 Aug;33(8):1259-68. Epub 2007 Apr 30 Blockade of vascular endothelial growth factor (VEGF) binding to its receptors on endothelial cells has been shown preclinically to induce tumour growth inhibition. Using ultrasound biomicroscopy (UBM) or micro-ultrasound imaging and micro-computed tomography (micro-CT) analysis, we have examined the effects of DC101, a highly specific vascular endothelial growth factor receptor-2 (VEGFR-2)-targeting antibody, in inducing growth inhibition and functional vascular changes in established melanoma (MeWo) xenografts in mice. Postprocessing of UBM imaging loops for speckle variance was introduced to estimate the level of functional blood flow in tumours. Perfused tumour area visualized by speckle variance revealed decreased blood flow within 48 h after DC101 injection (control versus DC101: 1.90 _ 0.25% versus 1.01 _ 0.11%, p < 0.01) and following a 3-wk DC101 therapy (control versus DC101: 0.76 _ 0.14% versus 0.45 _ 0.05%, p _ 0.04), suggesting that VEGFR-2 blockade mediates both early and long-term effects on tumour blood flow. The growth of xenografts was significantly inhibited after treating with DC101 for 3 wk compared with controls. In addition to UBM, we examined the tumour vasculature in three-dimension (3D) using contrast-enhanced Micro-CT imaging, which displayed a reduction in the number of tumour vessels following extended VEGFR-2 blockade (vascular density of control versus DC101: 48.4 _ 5.4% versus 20.6 _ 1.8%). Lastly, decreased microvessel density (MVD) was noted in DC101-treated xenografts (3 wk) by performing immunohistochemical staining of endothelial marker CD34. Our study investigates tumour response to DC101 using complementing micro-ultrasound and micro-CT imaging tools.

	Ultrasound biomicroscopy permits in vivo characterization of zebrafish liver tumors. Goessling W, North TE, Zon LI. Stem Cell Program, Hematology/Oncology, Children's Hospital and Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts Nat Methods. 2007 Jul;4(7):551-3. Epub 2007 Jun 17 Zebrafish are a valuable vertebrate model to study carcinogenesis, but noninvasive imaging is challenging because adult fish are not transparent. Here we show that tumors could be readily detected in vivo using high-resolution microscopic ultrasound (Vevo 770 micro-ultrasound, VisualSonics Inc.) in zebrafish. We successfully obtained tissue perfusion calculations and cellular aspirates, and analyzed tumor progression and response to treatment. Ultrasound biomicroscopy allows longitudinal studies of tumor development and real-time assessment of therapeutic effects in zebrafish. NOTE: This paper not only uses the Vevo 770 for 2D and 3D imaging and sizing of tumors in vivo in zebrafish, but MicroMarker contrast agents are also used in the protocols to successfully and quantitatively measure tumor vascularity and perfusion in these zebrafish models of liver cancer.

	Therapeutic effect of CS-706, a specific cyclooxygenase-2 inhibitor, on gallbladder carcinoma in BK5.ErbB-2 mice. Kiguchi K, Ruffino L, Kawamoto T, Franco E, Kurakata S, Fujiwara K, Hanai M, Rumi M, Digiovanni J. Department of Carcinogenesis, The University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, TX Mol Cancer Ther. 2007 Jun;6(6):1709-17. Biliary tract cancer is still challenging to treat and manage due to its poor sensitivity to conventional therapies and the inability to prevent or detect the early tumor formation. The most well known risk factor for gallbladder cancer is the presence of chronic inflammation, usually related to gallstones. It has been suggested that cyclooxygenase-2 (COX-2) plays a variety of roles in the gastrointestinal tract, including pathogenic processes such as neoplasia. Recently, we have generated transgenic mice that overexpress rat ErbB-2 under the control of bovine keratin 5 promoter (BK5.ErbB-2 mice). Homozygous BK5.ErbB-2 mice develop adenocarcinoma of gallbladder with an approximately 90% incidence. In addition to the activation of ErbB-2 and epidermal growth factor receptor, mRNA and protein levels of COX-2 were up-regulated in the gallbladder carcinomas that developed in these transgenic mice. The aim of this study was to examine the effects of a COX-2 inhibitor, CS-706, on the development of gallbladder carcinomas using the BK5.ErbB-2 mouse model. Ultrasound image analysis (Vevo 660 micro-ultrasound, VisualSonics) as well as histologic evaluation revealed a significant therapeutic effect of CS-706 on the gallbladder tumors, either as reversion to a milder phenotype or inhibition of tumor progression. The antitumor effect was associated with inhibition of prostaglandin E(2) synthesis. CS-706 treatment also down-regulated the activation of ErbB-2 and epidermal growth factor receptor, resulting in decreased levels of phosphorylated Akt and COX-2 in gallbladder cancers of BK5.ErbB-2 mice. Based on our results, targeting COX-2 could provide a potentially new and effective therapy alone or in combination with other therapeutic agents for patients with biliary tract cancer. NOTE: The Vevo 660 micro-ultrasound was used to non-invasively monitor and measure gallbladder tumors in treated and untreated mice.

	Vitamin E Analogs, a Novel Group of. Jiri Neuzil, Marco Tomasetti, Yan Zhao, Lan-Feng Dong, Marc Birringer, Xiu-Fang Wang, Pauline Low, Kun Wu, Brian A. Salvatore, and Steven J. Ralph Apoptosis Research Group, School of Medical Science, Griffith University, Southport, Queensland, Australia (J.N., L.F.D., X.F.W.); Molecular Therapy Group, Institute of Molecular Genetics, Czech Academy of Sciences, Prague, Czech Republic (J.N.); Department of Nutrition and Food, Harbin Medical University, Harbin, Heilongjiang Province, China (Y.Z., K.W.); Department of Molecular Pathology and Innovative Therapies, Polytechnic University of Marche, Ancona, Italy (M.T.); Institute of Human Nutrition, Friedrich Schiller University, Jena, Germany (M.B.); Genomic Research Centre, School of Medical Science, Griffith University, Southport, Queensland, Australia (P.L., S.J.R.); and Department of Chemistry and Physics, Louisiana State University, Shreveport, Louisiana (B.A.S.) Molecular Pharmacology. 2007 May;71(5):1185-99. Epub 2007 Jan 12 The search for a selective and efficient anticancer agent for treating all neoplastic disease has yet to deliver a universally suitable compound(s). The majority of established anticancer drugs either are nonselective or lose their efficacy because of the constant mutational changes of malignant cells. Until recently, a largely neglected target for potential anticancer agents was the mitochondrion, showing a considerable promise for future clinical applications. Vitamin E (VE) analogs, epitomized by alpha-tocopheryl succinate, belong to the group of "mitocans" (mitochondrially targeted anticancer drugs). They are selective for malignant cells, cause destabilization of their mitochondria, and suppress cancer in preclinical models. This review focuses on our current understanding of VE analogs in the context of their proapoptotic/anticancer efficacy and suggests that their effect on mitochondria may be amplified by modulation of alternative pathways operating in parallel. We show here that the analogs of VE that cause apoptosis (which translates into their anticancer efficacy) generally do not possess antioxidant (redox) activity and are prototypical of the mitocan group of anticancer compounds. Therefore, by analogy to Oscar Wilde's play The Importance of Being Earnest, we use the motto in the title "the importance of being redox-silent" to emphasize an essentially novel paradigm for cancer therapy, in which redox-silence is a prerequisite property for most of the anticancer activities described in this communication.

	Design, calibration and evaluation of a robotic needle-positioning system for small animal imaging applications. Waspe AC, Cakiroglu HJ, Lacefield JC, Fenster A. Biomedical Engineering Graduate Program, University of Western Ontario, London, Ontario Phys Med Biol. 2007 Apr 7;52(7):1863-78. Epub 2007 Mar 9. A needle-positioning robot has been developed for image-guided interventions in small animal research models. The device is designed to position a needle with an error less or equal to 100 microm. The robot has two rotational axes (pitch and roll) to control needle orientation, and one linear axis to perform needle insertion. The three axes intersect at a single point to create a remote centre of motion (RCM) that acts as a fulcrum for the orientation of the needle. The RCM corresponds to the skin-entry point of the needle into the animal. The robot was calibrated to ensure that the three axes intersected at a single point defining an RCM and that the needle tip was positioned at the RCM. Needle-positioning accuracy and precision were quantified in Cartesian coordinates at ten target locations in the plane of each rotational axis. The measured needle-positioning accuracy in free space was 54 +/- 12 microm for the pitch axis plane and 91 +/- 21 microm for the roll axis plane. The measured needle-positioning precision was 15 and 17 microm for the pitch and roll axes planes, respectively. The robot's ability to insert a needle into a tumour in a euthanized mouse was demonstrated. NOTE: A Vevo 770 micro-ultrasound system was used to show the guidance of this robotic injection system in a tumor in vivo and to show the successful placement of a target bead.

	A Peptide Conjugate of Vitamin E Succinate Targets Breast Cancer Cells with High ErbB2 Expression. Wang XF, Birringer M, Dong LF, Veprek P, Low P, Swettenham E, Stantic M, Yuan LH, Zobalova R, Wu K, Ledvina M, Ralph SJ, Neuzil J. Apoptosis Research Group and Genomics Research Centre, School of Medical Science, Griffith University, Southport, Queensland, Australia. Cancer Res. 2007 Apr 1;67(7):3337-44. Overexpression of erbB2 is associated with resistance to apoptosis. We explored whether high level of erbB2 expression by cancer cells allows their targeting using an erbB2-binding peptide (LTVSPWY) attached to the proapoptotic alpha-tocopheryl succinate (alpha-TOS). Treating erbB2-low or erbB2-high cells with alpha-TOS induced similar levels of apoptosis, whereas alpha-TOS-LTVSPWY induced greater levels of apoptosis in erbB2-high cells. alpha-TOS rapidly accumulated in erbB2-high cells exposed to alpha-TOS-LTVSPWY. The extent of apoptosis induced in erbB2-high cells by alpha-TOS-LTVSPWY was suppressed by erbB2 RNA interference as well as by inhibition of either endocytotic or lysosomal function. alpha-TOS-LTVSPWY reduced erbB2-high breast carcinomas in FVB/N c-neu transgenic mice. We conclude that a conjugate of a peptide targeting alpha-TOS to erbB2-overexpressing cancer cells induces rapid apoptosis and efficiently suppresses erbB2-positive breast tumors. Note: Tumor size was quantified by ultrasound imaging using the Vevo 770 instrument (Visualsonics, Toronto, Ontario, Canada) allowing 40 micron resolution of individual scans.

	Functional Neoangiogenesis Imaging of Genetically Engineered Mouse Prostate Cancer Using Three-Dimensional Power Doppler Ultrasound. Jim W. Xuan,¹, Michael Bygrave⁶, Hongyi Jiang¹, Fatma Valiyeva¹, Joy Dunmore-Buyze⁶, David W. Holdsworth^2,6, Jonathan I. Izawa¹, Glenn Bauman³, Madeleine Moussa⁴, Scott F. Winter⁷, Norman M. Greenberg⁷, Joseph L. Chin¹, Maria Drangova^2,6, Aaron Fenster^2,3,6 and James C. Lacefield^2,5,6 Departments of ¹ Surgery, ² Medical Biophysics, ³ Oncology, ⁴ Pathology, and ⁵ Electrical and Computer Engineering, University of Western Ontario and ⁶ Imaging Research Laboratories, Robarts Research Institute, London, Ontario, Canada; and ⁷ Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington Cancer Research. 2007 Mar 15; 67, 2830-2839 We report the first application of high-frequency three-dimensional power Doppler ultrasound imaging (Vevo 770, VisualSonics) in a genetically engineered mouse (GEM) prostate cancer model. We show that the technology sensitively and specifically depicts functional neoangiogenic blood flow because little or no flow is measurable in normal prostate tissue or tumors smaller than 2–3 mm diameter, the neoangiogenesis "switch-on" size. Vascular structures depicted by power Doppler were verified using Microfil-enhanced micro-computed tomography (micro-CT) and by correlation with microvessel distributions measured by immunohistochemistry and enhanced vascularity visualized by confocal microscopy in two GEM models [transgenic adenocarcinoma of the mouse prostate (TRAMP) and PSP94 gene-directed transgenic mouse adenocarcinoma of the prostate (PSP-TGMAP)]. Four distinct phases of neoangiogenesis in cancer development were observed, specifically, (a) an early latent phase; (b) establishment of a peripheral capsular vascular structure as a neoangiogenesis initiation site; (c) a peak in tumor vascularity that occurs before aggressive tumor growth; and (d) rapid tumor growth accompanied by decreasing vascularity. Microsurgical interventions mimicking local delivery of antiangiogenesis drugs were done by ligating arteries upstream from feeder vessels branching to the prostate. Microsurgery produced an immediate reduction of tumor blood flow, and flow remained low from 1 h to 2 weeks or longer after treatment. Power Doppler, in conjunction with micro-CT, showed that the tumors recruit secondary blood supplies from nearby vessels, which likely accounts for the continued growth of the tumors after surgery. The microsurgical model represents an advanced angiogenic prostate cancer stage in GEM mice corresponding to clinically defined hormone-refractory prostate cancer. Three-dimensional power Doppler imaging is completely noninvasive and will facilitate basic and preclinical research on neoangiogenesis in live animal models. [Cancer Res 2007;67(6):2830–9]

	Ultrasonic characterization of whole cells and isolated nuclei. Taggart LR, Baddour RE, Giles A, Czarnota GJ, Kolios MC. Ontario Cancer Institute/Princess Margaret Hospital, University Health Network, Toronto, ON, Canada Ultrasound Med Biol. 2007 Mar;33(3):389-401 High frequency ultrasound imaging (20 to 60 MHz, VS40, VisualSonics) is increasingly being used in small animal imaging, molecular imaging and for the detection of structural changes during cell and tissue death. Ultrasonic tissue characterization techniques were used to measure the speed of sound, attenuation coefficient and integrated backscatter coefficient for (a) acute myeloid leukemia cells and corresponding isolated nuclei, (b) human epithelial kidney cells and corresponding isolated nuclei, (c) multinucleated human epithelial kidney cells and d) human breast cancer cells. The speed of sound for cells varied from 1522 to 1535 m/s, while values for nuclei were lower, ranging from 1493 to 1514 m/s. The attenuation coefficient slopes ranged from 0.0798 to 0.1073 dB mm(-1) MHz(-1) for cells and 0.0408 to 0.0530 dB mm(-1) MHz(-1) for nuclei. Integrated backscatter coefficient values for cells and isolated nuclei showed much greater variation and increased from 1.71 x 10(-4) Sr(-1) mm(-1) for the smallest nuclei to 26.47 x 10(-4) Sr(-1) mm(-1) for the cells with the largest nuclei. The findings suggest that integrated backscatter coefficient values, but not attenuation or speed of sound, are correlated with the size of the nuclei.

	Blockade of Hedgehog Signaling Inhibits Pancreatic Cancer Invasion and Metastases: A New Paradigm for Combination Therapy in Solid Cancers. Georg Feldmann¹,⁵, Surajit Dhara1,⁵, Volker Fendrich², Djahida Bedja³, Robert Beaty¹,⁵, Michael Mullendore¹,⁵, Collins Karikari1,⁵, Hector Alvarez1,⁵, Christine Iacobuzio-Donahue¹,⁵, Antonio Jimeno⁴, Kathleen L. Gabrielson³, William Matsui⁴ and Anirban Maitra1,⁴,⁵ Departments of ¹ Pathology, ² Surgery, ³ Comparative Medicine, and ⁴ Oncology, ⁵ The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland Cancer Research. 2007 Mar 1;67(5):2187-96 In the context of pancreatic cancer, metastasis remains the most critical determinant of resectability, and hence survival. The objective of this study was to determine whether Hedgehog (Hh) signaling plays a role in pancreatic cancer invasion and metastasis because this is likely to have profound clinical implications. In pancreatic cancer cell lines, Hh inhibition with cyclopamine resulted in down-regulation of snail and up-regulation of E-cadherin, consistent with inhibition of epithelial-to-mesenchymal transition, and was mirrored by a striking reduction of in vitro invasive capacity (P < 0.0001). Conversely, Gli1 overexpression in immortalized human pancreatic ductal epithelial cells led to a markedly invasive phenotype (P < 0.0001) and near total down-regulation of E-cadherin. In an orthotopic xenograft model, cyclopamine profoundly inhibited metastatic spread; only one of seven cyclopamine-treated mice developed pulmonary micrometastases versus seven of seven mice with multiple macrometastases in control animals. Combination of gemcitabine and cyclopamine completely abrogated metastases while also significantly reducing the size of "primary" tumors. Gli1 levels were up-regulated in tissue samples of metastatic human pancreatic cancer samples compared with matched primary tumors. Aldehyde dehydrogenase (ALDH) overexpression is characteristic for both hematopoietic progenitors and leukemic stem cells; cyclopamine preferentially reduced "ALDH-high" cells by approximately 3-fold (P = 0.048). We confirm pharmacologic Hh pathway inhibition as a valid therapeutic strategy for pancreatic cancer and show for the first time its particular efficacy against metastatic spread. By targeting specific cellular subpopulations likely involved in tumor initiation at metastatic sites, Hh inhibitors may provide a new paradigm for therapy of disseminated malignancies, particularly when used in combination with conventional antimetabolites that reduce "bulk" tumor size.

	Secoisolariciresinol Diglucoside: Relevance to Angiogenesis and Cardioprotection against Ischemia-Reperfusion Injury. Suresh Varma Penumathsa, Srikanth Koneru, Mahesh Thirunavukkarasu, Lijun Zhan, Kailash Prasad, and Nilanjana Maulik Molecular Cardiology and Angiogenesis Laboratory, Department of Surgery, University of Connecticut Health Center, Farmington, Connecticut (S.V.P., S.K., M.T., L.Z., N.M.); and Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada (K.P.) The Journal of Pharmacology and experimental therapeutics. 2007 Feb;320(2):951-9. Epub 2006 Nov 28 Therapeutic angiogenesis represents a novel approach for the prevention and treatment of ischemic heart disease. This study examined a novel method of stimulating myocardial angiogenesis using secoisolariciresinol diglucoside (SDG), a plant lignan isolated from flaxseed. SDG has been shown to decrease serum cholesterol and reduce the extent of atherosclerosis. In the present study, the angiogenic properties of SDG were investigated in three different models. First, in the in vitro model, human coronary arteriolar endothelial cells (HCAEC) treated with SDG (50 and 100 microM) showed a significant increase in tubular morphogenesis compared with control. Western blot analysis indicated an increased expression of vascular endothelial growth factor (VEGF), kinase insert domain-containing receptor (KDR), Flt-1, angiopoietin-1 (Ang-1), Tie-1, and phosphorylated endothelial nitric oxide synthase (p-eNOS) in the SDG-treated cells. Second, in the ex vivo ischemia/reperfusion model, SDG-treated rats (20 mg/kg b.wt./day for 2 weeks orally) showed an increased level of aortic flow and functional recovery after 2 h of reperfusion following 30 min of ischemia compared with the control group [dP/dt (mm Hg/s) of 2110 +/- 35 versus 1752 +/- 62]. SDG reduced infarct size compared with the control group by 32% (38 versus 26%) and also decreased cardiomyocyte apoptosis. Increased protein expression of VEGF, Ang-1, and p-eNOS was also observed in the SDG-treated group. Third, in the in vivo myocardial infarction model, SDG increased capillary density and myocardial function as evidenced by increased fractional shortening and ejection fraction. In conclusion, these results suggest that SDG has potent angiogenic and antiapoptotic properties that may contribute to its cardioprotective effect in ischemic models.

	Therapy-Induced Acute Recruitment of Circulating Endothelial Progenitor Cells to Tumors. Shaked Y, Ciarrocchi A, Franco M, Lee CR, Man S, Cheung AM, Hicklin DJ, Chaplin D, Foster FS, Benezra R, Kerbel RS. Department of Molecular and Cellular Biology Research, Sunnybrook Health Sciences Centre, Toronto Science. 2006 Sep 22;313(5794):1785-7 The contribution of bone marrow–derived circulating endothelial progenitor cells (CEPs) to tumor angiogenesis has been controversial, primarily because of their low numbers in blood vessels of untreated tumors. We show that treatment of tumor-bearing mice with vascular disrupting agents (VDAs) leads to an acute mobilization of CEPs, which home to the viable tumor rim that characteristically remains after such therapy. Disruption of this CEP spike by antiangiogenic drugs or by genetic manipulation resulted in marked reductions in tumor rim size and blood flow as well as enhanced VDA antitumor activity. Longitudinal measurement of blood flow within the tumors were measured nsuiing high-frequency micro-ultrasound (Vevo 660, VisualSonics). These findings also provide a mechanistic rationale for the enhanced efficacy of VDAs when combined with antiangiogenic drugs.

	The Use of Targeted Mouse Models for Preclinical Testing of Novel Cancer Therapeutics. Kenneth P. Olive and David A. Tuveson University of Vermont, Cardiovascular Research Institute Burlington, VT, USA. Clinical Cancer Research2006 Sept 15; Vol. 12, 5277-5287 The use of genetically engineered cancer-prone mice as relevant surrogates for patients during the development of pertinent clinical applications is an unproven expectation that awaits direct demonstration. Despite the generally disappointing findings using tumor xenografts and certain early transgenic cancer models to predict therapeutic efficacy in patients, the dramatic progress of mouse models in recent years engenders optimism that the newest generation of mouse models will provide a higher standard of predictive utility in the process of drug development. Note: To image the KC and KPC models, we have chosen to pursue high-resolution ultrasound as a noninvasive means of imaging. Due to the small size of a mouse, extremely high resolution may be achieved from a 35 MH ztransducer (Vevo 660, VisualSonics,Inc.), while still maintaining a deep enough field of view to image the entire abdominal cavity. This system offers the advantage of short session time (f15 min/mouse), high-resolution with the ability to reconstruct and quantitate tumor volumes, and small instrument size to enable the placement of the ultrasound unit directly in our animal room. Using ultrasound, we have been able to image and quantify tumors over time following the detection of lesions as small as 1 mm in diameter.

	Rapamycin improves lymphoproliferative disease in murine autoimmune lymphoproliferative syndrome (ALPS). David T. Teachey, Dana A. Obzut, Kelly Axsom, John K. Choi, Kelly C. Goldsmith, Junior Hall, Jessica Hulitt, Catherine S. Manno, John M. Maris, Nicholas Rhodin, Kathleen E. Sullivan, Valerie I. Brown, and Stephan A. Grupp From the Divisions of Oncology, Hematology, and Immunology in the Department of Pediatrics, and the Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine Blood. 2006 Sept 15; Vol.108; No.6; 1965-1971. Epub June 6, 2006 Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of abnormal lymphocyte survival caused by defective Fas-mediated apoptosis, leading to lymphadenopathy, hepatosplenomegaly, and an increased number of double-negative T cells (DNTs). Treatment options for patients with ALPS are limited. Rapamycin has been shown to induce apoptosis in normal and malignant lymphocytes. Since ALPS is caused by defective lymphocyte apoptosis, we hypothesized that rapamycin would be effective in treating ALPS. We tested this hypothesis using rapamycin in murine models of ALPS. We followed treatment response with serial assessment of DNTs by flow cytometry in blood and lymphoid tissue, by serial monitoring of lymph node and spleen size with ultrasonography (Vevo 660 micro-ultrasound, VisualSonics), and by enzyme-linked immunosorbent assay (ELISA) for anti–double-stranded DNA (dsDNA) antibodies. Three-dimensional ultrasound measurements in the mice correlated to actual tissue measurements at death (r = .9648). We found a dramatic and statistically significant decrease in DNTs, lymphadenopathy, splenomegaly, and autoantibodies after only 4 weeks when comparing rapamycin-treated mice with controls. Rapamycin induced apoptosis through the intrinsic mitochondrial pathway. We compared rapamycin to mycophenolate mofetil, a second-line agent used to treat ALPS, and found rapamycin's control of lymphoproliferation was superior. We conclude that rapamycin is an effective treatment for murine ALPS and should be explored as treatment for affected humans.

	Characterization of Digital Waveforms Using Thermodynamic Analogs: Detection of Contrast-Targeted Tissue In Vivo. Michael S. Hughes, Member, IEEE, Jon N. Marsh, Member, IEEE, Hyuing Zhang, Adam K. Woodson, John S. Allen, Elizabeth K. Lacy, Cordelia Carradine, Gregory M. Lanza, and Samuel A. Wickline Washington University School of Medicine MO IEEE transactions on ultrasonics, ferroelectrics, and frequency control. 2006 Sep;53(9):1609-16 We describe characterization of backscatter from tumor tissue targeted with a nanoparticle-based ultrasound contrast agent in vivo using analogs of thermodynamic quantities. We apply these waveform characteristics to detection of tumor neovasculature in tumors implanted in athymic nude mice, which were imaged using a research ultrasound scanner over a 2-hour period after injection of alpha upsilon beta3-targeted perfluorocarbon nanoparticles. Images were constructed from backscattered ultrasound using two different approaches: fundamental B-mode imaging and a signal receiver based on a thermodynamic analog (H(C)). The study shows that the thermodynamic analog is capable of detecting differences in backscattered signals that are not apparent with the B-mode approach.

	Oncolytic Viral Therapy By Bladder Instillation Using An E1a, E1b Double-Restricted Adenovirus In An Orthotopic Bladder Cancer Model. Hua Wang, Makoto Satoh, Hisashi Abe, Makoto Sunamura, Takuya Moriya, Shigeto Ishidoya, Seiichi Saito, Hirofumi Hamada, And Yoichi Arai From the Division of Urology and Division of Gastroenterological Surgery, Department of Surgery, Tohoku University Graduate School of Medicine; Department of Pathology, Tohoku University Hospital, Sendia, Japan; and Department of Molecular Medicine, Sapporo Medical University, Sapporo, Japan Urology, 2006 Sep;68(3):674-81. Epub 2006 Sep 18 Objectives. To investigate the therapeutic effect of AxdAdB-3, a double-restricted oncolytic adenovirus harboring a mutant E1A and an E1B-55KD deletion, on human bladder cancer cell lines and the SCID mouse model of orthotopic bladder cancer. Methods. The cytopathic effects of AxdAdB-3 were evaluated in several cell lines (YTS-1, YTS-3, T24, J82, 5637) derived from human bladder or ureteral cancer and in a normal bladder mucosa-derived cell line (HCV29) with AxCAlacZ (control) or AxE1AdB (E1B-55KD-defective adenovirus) or dl922-947 (E1A-mutated adenovirus). The efficacy of bladder instillation therapy with AxdAdB-3 for orthotopic bladder cancer of SCID mice was investigated. The oncolytic effects were monitored by ultrasound examination. Results. AxdAdB-3 caused the oncolysis of bladder cancer cell lines in vitro, and it was more cytopathic than AxE1AdB or dl922-947 in the cancer cell lines. AxdAdB-3 was not cytotoxic against HCV29. Direct instillation of AxdAdB-3 into the bladder of the orthotopic model inhibited tumor growth, leading to significantly prolonged survival. Conclusions. Oncolytic viral therapy delivered by instillation of AxdAdB-3 is a promising tool for treating bladder cancer. NOTE: Bladder tumor progression in the mice was traced by noninvasive abdominal ultrasound micro-imaging, using a Vevo 660 high-resolution imaging system (VisualSonics, Toronto, Ontario, Canada).

	Volume measurement variability in three-dimensional high-frequency ultrasound images of murine liver metastases. Wirtzfeld LA, Graham KC, Groom AC, Macdonald IC, Chambers AF, Fenster A, Lacefield JC. Biomedical Engineering Graduate Program, University of Western Ontario, Imaging Research Laboratories, Robarts Research Institute, London, Ontario. Phys Med Biol. 2006 May 21;51(10):2367-81. Epub 2006 Apr 26. The identification and quantification of tumour volume measurement variability is imperative for proper study design of longitudinal non-invasive imaging of pre-clinical mouse models of cancer. Measurement variability will dictate the minimum detectable volume change, which in turn influences the scheduling of imaging sessions and the interpretation of observed changes in tumour volume. In this paper, variability is quantified for tumour volume measurements from 3D high-frequency ultrasound images (Vevo 660, VisualSonics) of murine liver metastases. Experimental B16F1 liver metastases were analysed in different size ranges including less than 1 mm(3), 1-4 mm(3), 4-8 mm(3) and 8-70 mm(3). The intra- and inter-observer repeatability was high over a large range of tumour volumes, but the coefficients of variation (COV) varied over the volume ranges. The minimum and maximum intra-observer COV were 4% and 14% for the 1-4 mm(3) and <1 mm(3) tumours, respectively. For tumour volumes measured by segmenting parallel planes, the maximum inter-slice distance that maintained acceptable measurement variability increased from 100 to 600 microm as tumour volume increased. Comparison of free breathing versus ventilated animals demonstrated that respiratory motion did not significantly change the measured volume. These results enable design of more efficient imaging studies by using the measured variability to estimate the time required to observe a significant change in tumour volume.

	Nanosecond pulsed electric fields cause melanomas to self-destruct. Richard Nuccitelli^a, ^bUwe Pliquett^a, Xinhua Chen^a, Wentia Ford^a, R. James Swansona, Stephen J. Beebe^{a, c}, Juergen F. Kolb^a and Karl H. Schoenbach^a ^aFrank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, bBioElectroMed Corp., Norfolk, VA ^cEastern Virginia Medical School, Norfolk, VA Received 11 February 2006. Available online 10 March 2006. Biochem Biophys Res Commun. 2006 May 5;343(2):351-60. Epub 2006 Mar 10 We have discovered a new, drug-free therapy for treating solid skin tumors. Pulsed electric fields greater than 20 kV/cm with rise times of 30 ns and durations of 300 ns penetrate into the interior of tumor cells and cause tumor cell nuclei to rapidly shrink and tumor blood flow to stop. Melanomas shrink by 90% within two weeks following a cumulative field exposure time of 120 µs. A second treatment at this time can result in complete remission. This new technique provides a highly localized targeting of tumor cells with only minor effects on overlying skin. Each pulse deposits 0.2 J and 100 pulses increase the temperature of the treated region by only 3 °C, ten degrees lower than the minimum temperature for hyperthermia effects. This paper describes this study and includes the use of a Vevo 770 (VisualSonics) and it’s 2D and 3D Power Doppler capabilities to non-invasively monitor tumor progression, regression and 3D vasculature structure and flow in a mouse model of melanoma.

	Phospholipase C-{delta}1 Is a Critical Target for Tumor Necrosis Factor Receptor-Mediated Protection against Adriamycin-Induced Cardiac Injury. Lien YC, Noel T, Liu H, Stromberg AJ, Chen KC, St Clair DK. Graduate Center for Toxicology, Department of Statistics, University of Kentucky, Lexington, Kentucky 40536, USA. Cancer Res. 2006 Apr 15;66(8):4329-38. The clinical application of adriamycin, an exceptionally good chemotherapeutic agent, is limited by its dose-related cardiomyopathy. Our recent study showed that tumor necrosis factor-alpha (TNF-alpha) receptors mediated cytoprotective signaling against adriamycin-induced mitochondrial injury and cardiomyocyte apoptosis. In the present study, we investigated the potential targets of TNF receptor-mediated cytoprotective signaling by global genome microarray analysis using wild-type and TNF receptor-deficient mice. Microarray analysis revealed that adriamycin treatment induced the down-regulation of several mitochondrial functions and energy production-related genes in double TNF receptor-deficient mice, notably, phospholipase C-delta1, a protein involved in fatty acid metabolism and calcium regulation. The role of phospholipase C-delta1 in TNF receptor-mediated cardioprotection against adriamycin-induced injury was evaluated by measuring changes in cardiac function using high-frequency ultrasound biomicroscopy (Vevo 660, VisualSonics). Selective inhibition of phospholipase C activity in wild-type mice by its inhibitor, U73122, exacerbated adriamycin-induced cardiac dysfunction. Inhibition of phospholipase C-delta1 resulted in the significant decrease of left ventricular ejection fraction and fractional shortening, and the decreased levels were similar to those observed in adriamycin-treated double TNF receptor-deficient mice. The data derived from the global genome analysis identified phospholipase C-delta1 as an important target for TNF receptors and revealed the critical role of TNF receptor signaling in the protection against adriamycin-induced cardiotoxicity.

	Targeted anti-vascular endothelial growth factor receptor-2 therapy leads to short-term and long-term impairment of vascular function and increase in tumor hypoxia. Franco M, Man S, Chen L, Emmenegger U, Shaked Y, Cheung AM, Brown AS, Hicklin DJ, Foster FS, Kerbel RS. Molecular and Cellular Biology Research, Sunnybrook and Women's College Health Sciences Centre, 2075 Bayview Avenue, Toronto, Ontario, Canada M4N 3M5. Cancer Research. 2006 Apr 1;66(7):3639-48. Because antiangiogenic therapies inhibit the growth of new tumor-associated blood vessels, as well as prune newly formed vasculature, they would be expected to reduce the supply of oxygen and thus increase tumor hypoxia. However, it is not clear if antiangiogenic treatments lead only to consistent and sustained increases in hypoxia, or transient decreases in tumor hypoxia along with periods of increased hypoxia. We undertook a detailed analysis of an orthotopically transplanted human breast carcinoma (MDA-MB-231) over a 3-week treatment period using DC101, an anti-vascular endothelial growth factor receptor 2 antibody. We observed consistent reductions in microvascular density, blood flow (measured by high-frequency micro-ultrasound, (Vevo 660, VisualSonics), and perfusion. These effects resulted in an increase in the hypoxic tumor fraction, measured with an exogenous marker, pimonidazole, concurrent with an elevation in hypoxia-inducible factor-1alpha expression, an endogenous marker. The increase in tumor hypoxia was evident within 5 days and remained so throughout the entire course of treatment. Vascular perfusion and flow were impaired at days 2, 5, 7, 8, 14, and 21 after the first injection, but not at 4 hours. A modest increase in the vessel maturation index was detected after the 3-week treatment period, but this was not accompanied by an improvement in vascular function. These results suggest that sustained hypoxia and impairment of vascular function can be two consistent consequences of antiangiogenic drug treatment. The implications of the results are discussed, particularly with respect to how they relate to different theories for the counterintuitive chemosensitizing effects of antiangiogenic drugs, even when hypoxia is increased.

	Validation of Ultrasonography to Evaluate Murine Orthotopic Oral Cavity Tumors. Pezold JC, Zinn K, Talbert MA, Desmond R, Rosenthal EL. Department of Surgery, Division of Otolaryngology - Head and Neck Surgery, University of Alabama at Birmingham, Birmingham, Ala., USA. ORL J Otorhinolaryngol Relat Spec.2006 Apr; 68(3):159-63. Epub 2006 Feb 6 Background: The murine orthotopic oral cavity tumor model allows evaluation of tumor growth and invasion. Currently, serial measurements of tissue growth are difficult to obtain since invasive procedures or animal sacrifice is necessary to evaluate tumor size. High-resolution ultrasound (Vevo 660, VisualSonics) was evaluated as a noninvasive method to monitor tumor size in vivo. Methods: Sixteen immunodeficient mice, age 9 weeks, were injected transcervically with a human squamous cell carcinoma cell line into the tongue, and tumor volume was assessed by high-frequency ultrasound at 11 days. The animals were subsequently sacrificed and the tumors processed for histology. Tumor size was then calculated by caliper measurement in two dimensions. Results: Tumor dimensions obtained using ultrasound were found to significantly correlate with the histologic measurements (Spearman coefficient 0.90, p < 0.0001). Tumor dimensions were on average larger using ultrasound versus histologic measurements, although this was not significantly different than zero (95% confidence interval -13.96 to 62.37 mm(2)). Conclusions: High-resolution ultrasound accurately measures tumor volume in the murine orthotopic oral cavity tumor model without sacrifice.

	Fibroblast-derived MT1-MMP promotes tumor progression in vitro and in vivo. Wenyue Zhang, Lynn M Matrisian, Kenn Holmeck, Catherine C Vick and Eben L Rosenthal Department of Surgery, Division of Otolaryngology - Head and Neck Surgery, University of Alabama at Birmingham, Birmingham, Ala., USA. BMC Cancer2006 March 6; 6:52 Background: Identification of fibroblast derived factors in tumor progression has the potential to provide novel molecular targets for modulating tumor cell growth and metastasis. Multiple matrix metalloproteases (MMPs) are expressed by both mesenchymal and epithelial cells within head and neck squamous cell carcinomas (HNSCCs), but the relative importance of these enzymes and the cell source is the subject of controversy. Methods: The invasive potential of HNSCC tumor cells were assessed in vitro atop type I collagen gels in coculture with wild-type (WT), MMP-2 null, MMP-9 null or MT1-MMP null fibroblasts. A floor of mouth mouse model of HNSCC was used to assess in vivo growth after co-injection of FaDu tumor cells with MMP null fibroblasts. In vivio imaging and measurements of the tumors were performed with a high-frequency micro-ultrasound (Vevo 660, VisualSonics). Results: Here we report changes in tumor phenotype when FaDu HNSCCs cells are cocultured with WT, MMP-2 null, MMP-9 null or MT1-MMP null fibroblasts in vitro and in vivo. WT, MMP-2 null and MMP-9 null fibroblasts, but not MT1-MMP null fibroblasts, spontaneously invaded into type I collagen gels. WT fibroblasts stimulated FaDu tumor cell invasion in coculture. This invasive phenotype was unaffected by combination with MMP-9 null fibroblasts, reduced with MMP-2 null fibroblasts (50%) and abrogated in MT1-MMP null fibroblasts. Co-injection of FaDu tumor cells with fibroblasts in an orthotopic oral cavity SCID mouse model demonstrated a reduction of tumor volume using MMP-9 and MMP-2 null fibroblasts (48% and 49%, respectively) compared to WT fibroblasts. Consistent with in vitro studies, MT1-MMP null fibroblasts when co-injected with FaDu cells resulted in a 90% reduction in tumor volume compared to FaDu cells injected with WT fibroblasts. Conclusion: These data suggest a role for fibroblast-derived MMP-2 and MT1-MMP in HNSCC tumor invasion in vitro and tumor growth in vivo.

	Establishment of a serum tumor marker for preclinical trials of mouse prostate cancer models. Huizen IV, Wu G, Moussa M, Chin JL, Fenster A, Lacefield JC, Sakai H, Greenberg NM, Xuan JW Departments of Surgery, Pathology and Imaging Research Laboratories, Robarts Research Institute, London, Ontario. Clin Cancer Res. 2005 Nov 1;11(21):7911-9 Current prostate cancer research in both basic and preclinical trial studies employ genetically engineered mouse models. However, unlike in human prostate cancer patients, rodents have no counterpart of prostatic-specific antigen (PSA) for monitoring prostate cancer initiation and progression. In this study, we established a mouse serum tumor marker from a mouse homologue of human prostate secretory protein of 94 amino acids (PSP94). Immunohistochemistry studies on different histologic grades from both transgenic and knock-in mouse prostate cancer models showed the down-regulation of tissue PSP94 expression (P < 0.001), the same as for PSA and PSP94 in humans. The presence of mouse serum PSP94 was shown by affinity column and immunoprecipitation purification using a polyclonal mouse PSP94 antibody. A competitive ELISA protocol was established to quantify serum PSP94 levels with a sensitivity of 1 ng/mL. Quantified serum levels of mouse PSP94 ranged from 49.84 ng/mL in wild-type mice to 113.86, 400.45, and 930.90 ng/mL in mouse prostatic intraepithelial neoplasia with microinvasion, well differentiated, moderately differentiated, and poorly differentiated prostate cancer genetically engineered prostate cancer mice, respectively (P < 0.01, n = 68). This increase in serum PSP94 is also well correlated with age and tumor weight. Through longitudinal monitoring of serum PSP94 levels of castrated mice (androgen ablation therapy), we found a correlation between responsiveness/refractory prostate tissues and serum PSP94 levels. The utility of mouse serum PSP94 as a marker in hormone therapy was further confirmed by three-dimensional micro-ultrasound imaging (Vevo 660 high-resolution imaging system, VisualSonics). The establishment of the first rodent prostate cancer serum biomarker will greatly facilitate both basic and preclinical research on human prostate cancer.

	The use of three-dimensional ultrasound micro-imaging to monitor prostate tumor development in a transgenic prostate cancer mouse model. Wu G, Wang L, Yu L, Wang H, Xuan JW. Department of Urology, Xi-jing Hospital, the Fourth Military Medical University, Xi'an. wuguojun@fmmu.edu.cn Tohoku J Exp Med. 2005 Nov;207(3):181-9. Longitudinal studies of mouse cancer models required large cohorts since autopsy was the only reliable method to evaluate treatment efficacy. This paper reports the use of high-resolution three-dimensional ultrasound micro-imaging (Vevo 660 high-resolution micro-ultrasound system from VisualSonics) to monitor prostate tumor development in genetically engineered mice. Twenty-nine genetically engineered prostate cancer mice, including castrated and uncastrated mice, were imaged by three-dimensional ultrasound. Qualitative comparisons of three-dimensional ultrasound images with histology sections of prostate tumors demonstrate the ability of ultrasound to accurately depict the size and shape of malignant masses in live mice. The correlation coefficient of tumor diameter measurements performed in vivo with three-dimensional micro-ultrasound and at autopsy was 0.997. Prospective tumor detection sensitivity and specificity were 91.7% and 100%. Representative exponential growth curves constructed via longitudinal ultrasound imaging indicated diameter doubling times from 10 to 37 days for four prostate tumors during an initial period of rapid progression. Three-dimensional micro-ultrasound will likely become the micro-imaging modality most readily adopted for mouse pre-clinical trial studies.

	Ultrasonic Molecular Imaging of Primordial Angiogenic Vessels in Rabbit and Mouse Models With v3-integrin Targeted Nanoparticles Using Information-Theoretic Signal Detection: Results at High Frequency and in the Clinical Diagnostic Frequency Range. M. Hughes, J. N. Marsh, Jeffrey Arbeit, Robert Neumann,R. W. Fuhrhop, G. M. Lanza, and S. A. Wickline Washington University School of Medicine IEEE Ultrasonics Symposium. 2005 Sept 18-21; Vol: 1, PP. 617- 620 The objective of this study was to assess the feasibility of image-based identification of nanoparticle targeted angiogenic neovasculature using backscattered ultrasound in several frequency ranges (7 to 15 and ~20-35MHz). We employed a liquid-perfluorocarbon nanoparticle contrast agent to target the expression of avß3 in tumors implanted two different animal models. Ten K14-HPV16 transgenic mice were treated with either normal saline (n=5) or 0.3 mg/kg i.v. of avß3-targeted nanoparticles (n=5) and imaged dynamically for two hours using a research ultrasound imager (VisualSonics, Vevo 660, 30MHz probe) modified to store digitized RF waveforms. Data were analyzed for all mice at all times post-injection using conventional grayscale and C H (an information-theoretic quantity). These data demonstrate the ability and complementarity of information-theoretic receivers in conjunction with targeted nanoparticles to elucidate the presence of avß3-integrins in primordial neovasculature, particularly in acoustically unfavorable environments.

	Chemopreventive and Therapeutic Efficacy of Orally Active Tyrosine Kinase Inhibitors in a Transgenic Mouse Model of Gallbladder Carcinoma. Kaoru Kiguchi¹, Lynnsie Ruffino¹, Toru Kawamoto¹,², Tetsuo Ajiki¹ and John DiGiovanni¹ ¹Department of Carcinogenesis, The University of Texas, M.D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas and ² Department of Surgery, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan Clinical Cancer Research2005 Aug 1; Vol. 11, 5572-5580 Biliary tract cancer (BTC) is the second most common primary hepatobiliary cancer after hepatocellular cancer. At the time of diagnosis, most BTC are at an advanced stage and are unresectable. There is presently no effective curative treatment of the advanced disease nor is there any effective clinical therapy that will prevent the development of BTC. All of these factors render gallbladder cancer nearly incurable with a poor survival rate. The aim of our study was to provide a better understanding of the mechanisms involved in the development of gallbladder carcinoma as the advancement of more effective treatment options would significantly improve prognosis. In the present study, we examined the effect of gefitinib, a selective epidermal growth factor receptor/tyrosine kinase inhibitor (EGFR/TKI), on the development of gallbladder carcinoma in BK5.erbB2 mice. In addition, we examined the effect of another quinazoline derivative, GW2974, which is able to block the activation of both the EGFR and erbB2, in this model. Animals were treated with either 400 ppm gefitinib or 200 ppm GW2974 as a supplement in the diet using either a chemopreventive or therapeutic protocol. The results show that both compounds were potent chemopreventive and therapeutic agents in this mouse model of human BTC. The results also suggest that activation of the EGFR plays an important role in development of BTC in this model and that targeting both the EGFR and erbB2 may be an effective strategy for treatment of this disease.

	A new three-dimensional ultrasound microimaging technology for preclinical studies using a transgenic prostate cancer mouse model. Wirtzfeld LA, Wu G, Bygrave M, Yamasaki Y, Sakai H, Moussa M, Izawa JI, Downey DB, Greenberg NM, Fenster A, Xuan JW, Lacefield JC. Biomedical Engineering Graduate Program and Departments of Surgery, Pathology, Diagnostic Radiology and Nuclear Medicine, Medical Biophysics, and Electrical and Computer Engineering, University of Western Ontario. Cancer Res. 2005 Jul 15;65(14):6337-45. Prostate cancer is the most common cancer in adult men in North America. Preclinical studies of prostate cancer employ genetically engineered mouse models, because prostate cancer does not occur naturally in rodents. Widespread application of these models has been limited because autopsy was the only reliable method to evaluate treatment efficacy in longitudinal studies. This article reports the first use of three-dimensional ultrasound microimaging (Vevo 660, VisualSonics) for measuring tumor progression in a genetically engineered mouse model, the 94-amino acid prostate secretory protein gene-directed transgenic prostate cancer model. Qualitative comparisons of three-dimensional ultrasound images with serial histology sections of prostate tumors show the ability of ultrasound to accurately depict the size and shape of malignant masses in live mice. Ultrasound imaging identified tumors ranging from 2.4 to 14 mm maximum diameter. The correlation coefficient of tumor diameter measurements done in vivo with three-dimensional ultrasound and at autopsy was 0.998. Prospective tumor detection sensitivity and specificity were both >90% when diagnoses were based on repeated ultrasound examinations done on separate days. Representative exponential growth curves constructed via longitudinal ultrasound imaging indicated volume doubling times of 5 and 13 days for two prostate tumors. Compared with other microimaging and molecular imaging modalities, the application of three-dimensional ultrasound imaging to prostate cancer in mice showed advantages, such as high spatial resolution and contrast in soft tissue, fast and uncomplicated protocols, and portable and economical equipment that will likely enable ultrasound to become a new microimaging modality for mouse preclinical trial studies. PMID: 16024636 [PubMed - in process]

	Three-dimensional High-Frequency Ultrasound Imaging for Longitudinal Evaluation of Liver Metastases in Preclinical Models. Graham KC, Wirtzfeld LA, Mackenzie LT, Postenka CO, Groom AC, Macdonald IC, Fenster A, Lacefield JC, Chambers AF. Department of Medical Biophysics, The University of Western Ontario, London, Canada Cancer Research. 2005 Jun 15;65(12):5231-7. Liver metastasis is a clinically significant contributor to the mortality associated with melanoma, colon, and breast cancer. Preclinical mouse models are essential to the study of liver metastasis, yet their utility has been limited by the inability to study this dynamic process in a noninvasive and longitudinal manner. This study shows that three-dimensional high-frequency ultrasound (Vevo 660 high-resolution Imaging System from VisualSonics) can be used to non-invasively track the growth of liver metastases and evaluate potential chemotherapeutics in experimental liver metastasis models. Liver metastases produced by mesenteric vein injection of B16F1 (murine melanoma), PAP2 (murine H-ras-transformed fibroblast), HT-29 (human colon carcinoma), and MDA-MB-435/HAL (human breast carcinoma) cells were identified and tracked longitudinally. Tumor size and location were verified by histologic evaluation. Tumor volumes were calculated from the three-dimensional volumetric data, with individual liver metastases showing exponential growth. The importance of volumetric imaging to reduce uncertainty in tumor volume measurement was shown by comparing three-dimensional segmented volumes with volumes estimated from diameter measurements and the assumption of an ellipsoid shape. The utility of high-frequency ultrasound imaging in the evaluation of therapeutic interventions was established with a doxorubicin treatment trial. These results show that three-dimensional high-frequency ultrasound imaging may be particularly well suited for the quantitative assessment of metastatic progression and the evaluation of chemotherapeutics in preclinical liver metastasis models.

	Three-dimensional ultrasound biomicroscopy for xenograft growth analysis. Cheung AM, Brown AS, Hastie LA, Cucevic V, Roy M, Lacefield JC, Fenster A, Foster FS. Imaging Research, Sunnybrook and Women's College Health Sciences Center, Toronto, ONT, Canada. Ultrasound Med Biol. 2005 Jun;31(6):865-870. We reported the use of high-frequency ultrasound biomicroscopy (UBM) in the quantitative analysis of early tumor growth in mice bearing melanoma xenografts in a noninvasive longitudinal assay. Initially, measurements of tumor width, depth and length were obtained using on-screen UBM calipers in real time and tumor volume was calculated with the standard ellipsoid formula w d l pi/6. We were able to detect initiating minute tumor nodules, with the lower limit of detection at approximately 0.01 mm(3) in volume. Successive parallel cross-sectional UBM images (33 mum step) encompassing the complete length of these tumors were also obtained and reconstructed into 3-D representations. Subsequent segmentational volumetric analysis provided a measure of tumor volume. Volume measurements using the two techniques were highly correlated when all 33 xenografts were studied (r = 0.9813, p < 0.0001) and a lower degree of correlation was measured with a subset of early small tumors (r = 0.7973, n = 16, p = 0.0004). Further analysis demonstrated that 3-D segmentational volumetric analysis yielded volume estimates that were often smaller than the caliper-and-formula calculation for most early developing xenografts. Thus, 3-D UBM imaging and segmentation is expected to be especially valuable for small tumors that were observed to grow in irregular shapes other than ellipsoids. (E-mail: ).

	High-frequency Doppler ultrasound monitors the effects of antivascular therapy on tumor blood flow.. Goertz DE, Yu JL, Kerbel RS, Burns PN, Foster FS. Sunnybrook and Women's College Health Sciences Centre, Department of Medical Biophysics, University of Toronto, Toronto, Ontario, M4N 3M5 Canada Cancer Research. 2002 Nov 15;62(22):6371-5. The effect of antivascular therapy on blood flow in superficial tumors was monitored using novel high frequency Doppler (HFD) ultrasound techniques. Human melanoma cells (MeWo) were injected orthotopically into the skin of athymic nude mice. Volumetric HFD imaging of established melanomas detected a significant reduction in blood flow 4 h after injection of the tumor vascular targeting agent ZD6126 followed by a recovery of flow by 24 h after injection. Measurements of tumor perfusion in situ by Hoechst 33342 staining correlated with the ultrasound results. This study demonstrates the feasibility of HFD as a noninvasive, quantitative tool for following longitudinally the effects of antivascular therapy on blood flow in superficial tumors.

	Ultrasound Imaging of Apoptosis: DNA-Damage Effects Visualized. Gregory J. Czarnota, Michael C. Kolios, John W. Hunt, Michael D. Sherar Department of Radiation Oncology, University of Toronto, Ryerson University, Toronto, Canada Methods in Molecular Biology. 2002 Apr 30; vol. 203, pp. 257-277. > A new method for detection of DNA damage that leads to the physiological process of apoptosis is discussed. (page 257) > Studies suggest data to date indicate the capability of high frequency ultrasound to detect cells undergoing apoptosis is based on the interactions of high frequency ultrasound waves with the chromosomal nuclear material in cells, which undergoes structural changes of condensation and subsequent fragmentation with the process of programmed cell death. (page 257) > Apoptosis has been induced in cells and tissues where the backscatter signals associated with apoptosis were analyzed. (pages 263-271) > Future developments pertaining to improving the depth of penetration of the ultrasound signal should assist greatly in the application of this technology in human clinical use. (page 275)

	Ultrasound backscatter microscope analysis of mouse melanoma progression. Turnbull DH, Ramsay JA, Shivji GS, Bloomfield TS, From L, Sauder DN, Foster FS. Skirball Institute of Biomolecular Medicine, New York University Medical Center, NY 10016, USA. Ultrasound Med Biol. 1996 Oct; 22(7):845-53. The incidence and mortality rate of cutaneous melanoma continue to increase throughout the world, making the study of melanoma biology an important area of current research. While recent breakthroughs in transgenic mouse technology have led to promising mouse skin models of melanoma, there is presently no technique available for quantitatively studying subsurface melanoma progression, in vivo. We demonstrate the first application of an imaging method called ultrasound backscatter microscopy (UBM) for imaging early murine melanomas with spatial resolution of 30 microns axial and 60 microns lateral. Murine B16 F10 melanomas have been imaged from their earliest detection, over several days, until they are 2 to 5 mm in diameter. Melanoma dimensions measured by UBM were found to be in excellent agreement with those determined histopathologically on the excised tumours. The relative rms errors in UBM-determined melanoma height and width were found to be 8.7% and 4.2%, respectively. The mean rate of increase in tumour height of early murine melanoma was found to be 0.37 +/- 0.06 mm/day. Computer-generated volumetric renderings of melanomas have been produced from three-dimensional image data, allowing quantitative comparisons of tumour volumes to be made. Using a priori assumptions of ellipsoid tumour shape, the relative error in UBM-determined volume was shown to be less than 17%. These results should be of considerable interest to investigators studying melanoma biology using mouse skin models, and have implications in the use of high frequency ultrasound imaging for the clinical assessment of cutaneous melanoma.

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