The Core Facility houses a Carl Zeiss LSM 780 laser scanning module mounted on an Axio Observer Z1 motorized fluorescent microscope. It also has two Zeiss Axio Observer Z1 fluorescent microscopes with attached computers equipped with Zen software for image acquisition. In addition, the facility also houses an Olympus VivaView fluorescent incubator microscope capable of imaging cells growing in culture over several days or weeks. Finally, the facility has a Carl Zeiss AxioScan Z.1 slide scanning microscope with bright-field and fluorescence imaging capabilities for creating virtual images of whole slides. Researchers from seventeen different laboratories (approximately 53 users) in the NCI are currently using the LGCP Microscopy Core Facility. The research focus of the scientists using the facility includes ovarian, breast, prostate and thyroid cancer progression and metastasis, chemotherapy used in pediatric oncology, and AIDS therapy. In the Laboratory of Genitourinary Cancer Pathogenesis (LGCP), 25 scientists including staff scientists, postdoctoral fellows, post baccalaureate fellows, and summer students routinely use the core facility in their research efforts. Postdoctoral fellows in Dr. Zheng-Gang Lius laboratory are using the Core to investigate TNF-induced necroptosis. Confocal microscopy is being used by this group to investigate the role and cellular localization of the necroptosis related proteins MLKL and RARgamma. In Dr. Kathy Kelly's laboratory, postdoctoral and baccalaureate fellows are using microscopy to study the molecular mechanisms and identify differentiation markers and signaling molecules on both primary murine and human prostate cells in order to elucidate the signaling pathways involved in prostate cancer metastasis. They are using whole slide scanning to characterize normal and transformed prostate epithelial cells stained with progression markers and signaling molecules in 2 and 3 dimensional cultures. Confocal microscopy is also being used for observing the localization of these components in the spherical and tubular structures that grow in 3D culture. Scientists from several other branches or laboratories are currently using the core as an integral part of their research. Below are some examples of ongoing projects using the core facility. The Usdin Lab in the NIMH is investigating the effects of a pharmacogenetic manipulation in the mouse brain, mapping the induction of an immediate early gene. They are using the Core slide scanning microscope to obtain high quality images to survey the entire brain under two treatment conditions from a number of replicate animals and at a resolution that allows them to make quantitative measures of the number of labeled neurons in discrete brain regions. The study would have taken a prohibitive amount of time to acquire on any other microscope they had access to. The Kebebew Lab is trying to understand the role and the effects of the anti-cancer drugs on undifferentiated thyroid cancer cells growth. Using the Olympus Vivaview incubator microscope, they were able to make time lapse images of the mechanisms leading to cell death when treated with the proteasome inhibitor Carfilzomib and HDAC inhibitor CUC101. They also have an ongoing study to understand the mechanisms leading to cell cycle arrest in LOX depleted anaplastic thyroid cancer cells.The Tanner Lab is investigating mechanisms of metastasis using zebrafish models. While the conceptual framework describing cancer cell trafficking through the lymph and circulation systems to colonize distant organs is well accepted, actual visualization of this process has proven difficult. Dr. Tanner exploits the use of optically transparent, embryonic zebrafish injected with human cancer cells as a xenograft model for real-time visualization of the metastatic cascade. Dr. Tanner uses a variety of equipment within the Core to obtain high-resolution images in which cancer cell migration speeds, residence times, and interactions with host cells have been characterized to better understand the metastatic spread of cancer. Apart from collaborating on projects involving the microscopy core facility, teaching scientists various aspects of microscopy, and maintaining the equipment in the core, I am conducting research on the mechanisms of CD97 signal transduction and the role of CD97 in cancer progression and metastasis in collaboration with the branch Chief of LGCP, Kathy Kelly. The details of this project are described in Kathy Kelly's annual report. CD97, an adhesion class G protein coupled receptor, is expressed on inflammatory cells and several carcinomas. CD97 expression increases in parallel with malignant grade in thyroid, esophageal, gastric, colorectal, and prostate tumors. We have demonstrated that CD97 acts both as a cell-autonomous receptor on tumor cells and as a ligand for integrins alpha5 beta1 and alphav beta3 on endothelial cells. We showed that in its capacity as a receptor, CD97 signaling couples through the G-alpha 12/13 family of heterotrimeric G proteins resulting in increased Rho activity. An analysis of prostate and thyroid tumor cell lines has shown that abnormal overexpression of endogenously-expressed CD97 seems to lead to ligand-independent signaling. Depletion of endogenous CD97 in prostate tumor cell lines resulted in decreased metastasis to bone. We have two recent publications demonstrating that CD97 plays an active role in prostate and thyroid cancer progression probably via heterodimerization with the lysophosphatidic receptor.
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