Laboratory of Cancer Biology and Genetics Microscopy Core Facility

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

The LCBG Microscopy Core houses a number of instruments including a Carl Zeiss LSM 980 laser scanning confocal microscope with an Airyscan 2 super resolution detector. The Core also has a Zeiss AxioObserver.Z1 fluorescent microscope with a motorized scanning stage, and a Nikon TI2 fluorescent microscope with a Z Piezo stage with incubation. Finally, the facility has two Carl Zeiss AxioScan Z.1 slide scanning microscope with bright-field and fluorescence imaging capabilities. Researchers from over 50 different laboratories (approximately 80 scientists) in the NCI are currently using the LCBG Microscopy Core. The research focus of the scientists using the Core includes ovarian, breast, prostate, colorectal, gastric, glioblastoma, and thyroid cancer progression and metastasis. In the LCBG, 20 scientists including, postdoctoral fellows, post baccalaureate fellows, and summer students routinely use the Core in their research efforts. Below are summaries of research from 5 of the labs utilizing the Core. Dr. Weigert's lab focuses on the role of membrane remodeling during cancer with a particular focus on head and neck squamous cell carcinoma. They use intravital microscopy in live mice to longitudinally follow tumor initiation and progression within the same animal at a cellular and subcellular level. Notably, they identified a specific step during tumor progression in which cancerous lesions either spontaneously regress or further develop to malignant stages. To provide detailed molecular information on this key step, the Weigert lab coupled intravital microscopy with correlative spatial multi-omics and multiplex immunofluorescent staining. The LCBG Microscopy Core has been instrumental to this work, imaging hundreds of multiplex fluorescent slides on the Core's Axioscan slide scanning microscopes and developing a robust pipeline for quantitative analysis. Specifically, the data generated will be used in 3 manuscripts under preparation that unravel respectively how immune-evasion is triggered by re-wiring metabolism, immune-signaling, and glycan landscape.Dr. Liu's lab has collaborated the LCBG Microscopy Core to study necroptosis and the related proteins, ZBP, MLKL, and Tissue Factor (TF). Dr. Liu's laboratory has focused on how glucose deprivation leads to necroptosis. Using the Core for confocal fluorescence microscopy, they showed that glucose deprivation promotes mitochondrial DNA release, which then interacts with cellular ZBP, triggering necroptosis. A follow-up study examined how ZBP1 undergoes a phase transition that facilitates the recognition of viral Z-NAs and the activation of downstream signal transduction. This phase transition involves the formation of condensates that are characterized by liquid-liquid phase separation properties. This study is using the Core's confocal to follow the phase transition events that lead to activation of necroptosis. In addition, Dr. Liu's lab studies the affects necroptosis on the immune microenvironment using the Core for fluorescent multiplex high throughput microscopy. Most recently, Dr. Liu's group is investigating the thrombosis associated with necroptosis through the activation of tissue factor and the downstream clotting cascade. The Liu group as used the Axioscan slide scanning microscope for multiplex fluorescent staining to determine clotting in tissues (liver, kidney and lung) from different mouse models of necroptosis and inflammation. Dr. Sowalsky's lab is trying to identify and characterize the mechanisms driving the development of aggressive prostate cancer. His lab is developing strategies for the earlier detection of aggressive prostate cancer and approaches for preventing or delaying its progression. To accomplish this goal, they are performing in-depth genomic analyses of prostate cancer specimens from patients, taken at baseline, with full clinical annotation of their outcome following treatment. These studies include active surveillance, neoadjuvant androgen deprivation therapy, and first-line radiation therapy (+/- ADT). tissue. A critical component to their analyses is the ability to discern areas of interest for microdissection followed by next gen sequencing. This is accomplished using IHC stains for multiple markers of prostate cancer, followed by imaging of the slides, before finally undergoing review by board certified pathologists. The LCBG Microscopy Core has been key to this work, imaging thousands of slides on the core Axioscan slide scanner. Dr. Tofilon's lab is focused on understanding Glioblastoma stem like cells (GSCs) radioresistance in different microenvironments throughout the brain. For this purpose, intracranial injection of (GSCs) in the Striatum of nude mice was performed and they showed that GSCs recapitulates the migration nature of GBM and migrate to the corpus callosum and the olfactory bulb. As a method to study GSCs radioresistance in different microenvironments, they used the halogenated thymidine analog CldU (5-chloro-2'-deoxyuridine) which is incorporated into DNA during the S phase and allowed identification of proliferating cells in orthotopic brain tumor xenografts. Charlotte Degorre and Ian Sutton used the Axioscan slide scanning microscope to show the differences in the incorporation of the CldU in the different brain locations via immunohistochemistry. Moreover, the confocal microscope was also used to study the mechanism of radioresistance by analyzing (1) the DNA damage repair proteins P-H2AX and 53BP1 and (2) the contact between the GSCs and different types of mouse neurons using markers such as MAP2, TH and GABA. The Axioscan slide scanning microscope is also used to analyze protein expression, such as proteasome related protein, in different parts of the brain in order to confirm spatial transcriptomic data. All those project benefits from the high throughput and automation of the Axioscan to analyze a large number of samples as well as the high resolution and the 3D reconstruction capacity of the confocal microscope. Dr. Takeda's laboratory focuses on regulatory elements as critical somatic drivers of prostate cancer disease progression. Based on observations from epigenomic profiling of primary patient samples, they developed in vitro laboratory models amenable to mechanistic studies involving genome-scale functional genomics. Using this approach, they discovered a distal enhancer of the AR that is activated and amplified in 80-90% of CRPC. They subsequently demonstrated that duplication of the enhancer in an in vitro model is sufficient to drive a castration-resistant phenotype. Understanding the mechanism of AR enhancer activation may provide new targets for therapeutic intervention. Towards this goal, the Takeda lab used a genomic editing CRIPSR screening strategy to perform mutagenesis studies to identify critical cis-regulatory elements at nucleotide resolution. Critical to validating the CRISPR screening results requires measuring hits on a single cell level given heterogeneity of editing. The LCBG Microscopy Core has previously developed a single molecule RNA fluorescence in situ hybridization method (smRNA FISH). Given the technical expertise required for the staining protocol, imaging, and data analysis, it was unlikely the lab would have pursued smRNA FISH, or it would have taken significantly more time and resources to develop a protocol as the laboratory does not routinely perform microscopy. The Takeda lab successfully collaborated with the LCBG Microscopy Core to perform smRNA FISH for all of their validation studies. This has significantly accelerated completion of their projects and will be included in a publication under preparation