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 eighteen different laboratories (approximately 50 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, colorectal, gastric, glioblastoma, and thyroid cancer progression and metastasis. In the Laboratory of Genitourinary Cancer Pathogenesis (LGCP), 19 scientists including, postdoctoral fellows, post baccalaureate fellows, and summer students routinely use the Core Facility in their research efforts. 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. Dr Adam Sowalsky's lab is comparing the genomic and transcriptomic profiles of high grade prostate cancers (that have progressed to Gleason score 7 or higher) to indolent cancers (Gleason score 6 prostate cancers from an active surveillance cohort). Researchers from the Sowalsky lab use whole slide scanning to process hundreds of immunohistochemically stained radical prostatectomy slides inorder to identify the regions of interest which require further microdissection and analysis. The VanderWeele Lab in the LGCP is looking at the phenotypic responses of prostate cancer cells to AR inhibition to gain insight into resistance mechanisms. They are using whole slide scanning and widefield microscopy to examine IF and IHC stained tissue arrays to look at the interaction between PI3K pathway signaling and AR activity in patient derived xenografts and organoid cultures. 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 Tofilin Lab is using the Core slide scanning microscope in their study of glioblastoma (GBM) stem-like cells (GSCs). They are able to obtain high quality images to survey the entire brain from a number of replicate mice at a resolution that allows them to make quantitative measures of the labeled cells in discrete brain regions. The study would have taken a prohibitive amount of time to acquire on any other microscope they had access to. Postdoctoral fellows in Dr. Zheng-Gang Liu's 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. 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 one recent publication in 2018 demonstrating that activation of platelets by CD97 leads to a process wherein lysophosaphatidic acid (LPA) is released, giving the tumor cell more invasive properties as a result of binding to a CD97-LPA receptor complex. Concurrently, tumor-activated platelets secrete ATP, a molecule which helps tumor cells breach the endothelial barrier of healthy tissue. These invasive processes give the tumor cell an increased ability to metastasize.
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