Scott R. Vandenberg, MD, PhD - US grants

A significant number of primary tumors in the CNS of children are primitive and embryonal. Their differentiation potential corresponds to the sequential stages of normal neurocytogenesis. They are also of importance because fetal neuroepithelial cells may be a selective target for neoplastic development. The therapeutic response is unpredictable due to poor understanding of their biologic behavior. Tumor arrest or regression has been associated with increased cellular maturation, and substances known to promote differentiation have been proposed as a therapeutic approach. Endogenous substances that may play a role in determining divergent neuroepithelial differentiation in human embryonal CNS tumors have not been studied in this regard. The applicant has studied a highly promising experimental model for neural differentiation in primitive CNS tumors i.e. the neuroepithelilal component of a transplantable mouse teratoma (OTT-6050) which 1) displays a spectrum of divergent neural differentiation corresponding to that of the human tumors 2) can be studied both in vivo and in vitro; 3) has a neural stem cell population capable or repetitive transplantation; and 4) expresses biochemical differentiation consistent with divergent neural cell populations. Serotonin, metabolic enzymes of the biogenic monoamine systems, and cyclic AMP are present in the tumor. Evidence from developing vertebrates and invertebrates suggests that biogenic monoamines may act as early neuroregulators. This research will study the role of monoamines and associated neuropeptides as modulators of early divergent differentiation in CNS tumors using the teratoma model in vivo and in vitro. Studies will focus in particular on serotinin and its membrane receptors because of its predominance among the teratoma monoamines. Morphologic techniques including histochemical fluorescence, immunocytochemistry and autoradiography will be combined with analytical binding kinetic, chromatographic and electrophoretic methods to determine the endogenous levels, synthesis, metabolic relationship with polyamines, interrelationships with monoamine-containing cells, and the time course in development of monoamine neuroregulators in primitive neural cells. An understanding of the mechanisms by which neoplastic neural tissue matures and which might partly be mediated by nonsynaptic neuroregulators may lead to better discrimination of, and thus more precise therapies applicable to, human embryonal CNS tumors.

The Tissue Core provides staff and technology dedicated to enhancing sample integrity and usability through use of optimized harvesting procedures; multi-modality preservation and processing; histopathologic- molecular morphologic characterization; and computerized inventory and web-based request and tracking systems. All aspects of sample identification, processing and storage are performed with strict compliance to the College of American Pathologists (CAP) guidelines. In order to maximize sharing and integration of SPORE projects, the Tissue Core collects and makes available data derived from all distributed samples. Specific Aims of SPORE Tissue Core: A. To acquire tissue samples from the operating room and SPORE Animal Research Core with optimized handling to maximize cell viability and/or minimize the warm-ischemic interval so as to meet the tissue accrual requirements for the Brain Tumor SPORE projects and trials. B. To perform quality control tests on archived tissue samples collected from the operating room and SPORE Animal Research Core, to ensure availability of adequate numbers of consistently handled specimens that will yield useable data for SPORE projects and clinical trials. C. To maintain a SPORE Tissue Core database containing demographic data, results from molecular analyses, and tissue distributions (internal and external) that will be linked to relational clinical databases maintained by the Biostatistics and Clinical Core. D. To provide routine and advanced tissue handling/processing and analytical techniques, including immunohistochemistry, chromogenic in situ hybridization (CISH), tissue microarray construction, laser capture micro-dissection, RNA extraction, and preparation of viable cells that will advance project hypothesis development and goal attainment.

Project Summary/Abstract The University of California, San Francisco (UCSF) is requesting funds for the purchase of a multiplexed ion beam imaging microscope (MIBIscope) from Ionpath, Inc. MIBI is a transformational new technology that enables highly multiplexed measurement of proteins in histological tissue sections, but it is currently unavailable at UCSF. The MIBIscope builds on the success of mass cytometry (i.e., CyTOF), a technology that uses metal-tagged antibodies and mass spectrometry to routinely quantify over 40 proteins in single cells. However, rather than measuring the abundance of these metal tags on cells in liquid suspension, MIBI quantifies these reporter ions directly off tissue on a microscope slide with 250nm resolution. This technology thus provides the investigator with spatial information about where cells reside in tissue, how they are organized into neighborhoods, and even where molecules of interest are localized within cells. The MIBI platform is the only technology that enables imaging of up to 54 antibodies simultaneously at 250nm resolution. The rationale for this proposal is the current lack of MIBI technology at UCSF and the significant number of research programs and specific projects that would significantly benefit from its availability. There are 33 scientific projects by 26 investigators from 14 different UCSF departments providing justification for funding. Consistent with the anticipated use by a diverse number of research groups, the MIBIscope will serve as a shared resource with a projected usage of nearly 5000 hours per year. A broad diversity of projects, ranging from basic science to clinical trial correlative studies, would utilize the MIBI technology. UCSF has recently invested $2M in a renovation project that supported the implementation of mass cytometry in a BSL2 facility for both pre-clinical and clinical studies. As a result, the infrastructure for conjugating antibodies to heavy metal isotopes is already fully functional and established as a service at UCSF. This will greatly facilitate the implementation of MIBI technology by providing a resource for the generation of custom reagents. UCSF investigators have also developed a novel recombinant antibody screening and production platform that will be uitilized for the development of new MIBI reagents. Moreover, technical staff at UCSF have developed the expertise required to effectively manage mass cytometry studies in a shared instrument setting, and these individuals are also prepared and eager to facilitate the implementation of MIBI technology. UCSF investigators serving on the proposed advisory board have extensive expertise in quantitative image analysis and single-cell analysis, which will ensure that researchers utilizing MIBI will have support as they analyze and interpret their resulting data. Users will have access to continued training, support, education courses, and monthly user group meetings, following the model established for mass cytometry at UCSF.

Biorepository and Tissue Biomarker Technology Shared Resource: Summary/Abstract The goal of the Biospecimen and Tissue Biomarker Technology Shared Resource (BTBMT) is to support the life cycle of high quality biospecimens for cancer research. The BTBMT is a result of the strategic reconfiguration of three previous Shared Resources: the Tissue Core, the Immunohistochemistry (IHC) & Molecular Pathology Core, and the Mouse Pathology Core.