2009 |
Zong, Hui |
R55Activity Code Description: Undocumented code - click on the grant title for more information. |
Modeling Glioma With Sporadic Co-Loh of P53 and Nf1 Using a Mouse Genetic Mosaic |
0.972 |
2010 — 2015 |
Zong, Hui |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Characterizing the Glioma Cell of Origin in Vivo Using Madm, a Mouse Genetic Mosa
DESCRIPTION (provided by applicant): Glioma is the most common type of malignant brain tumor. Despite widespread advances in cancer medicine, this devastating disease remains incurable. Recently, it was reported that gliomas contain a population of tumor stem cells that can form self renewable tumor spheres in culture and re- initiate gliomas after transplantation into immuno-suppressed mice. Targeting these tumor stem cells is an exciting prospect towards a glioma cure. However, critical information required to develop such therapeutic strategies, including the developmental origin of the glioma stem cells, remains unknown. To address the tumor cell of origin problem, it requires the use of glioma animal models that allow the analysis of early-stage pre-malignant tumor cells. Unfortunately, current mouse models cannot provide adequate in vivo resolution for such studies. Our laboratory has developed a new mouse glioma model based on a novel genetic mosaic system termed MADM (Mosaic Analysis with Double Markers, Zong et al Cell 2005). Using MADM, we can generate rare, green fluorescent protein (GFP)-labeled neural stem cells (NSCs) that are double null for two key tumor suppressor genes, p53 and Neurofibromatosis Type 1 (NF1), within an otherwise normal mouse. This approach allows us to analyze the entire course of gliomagenesis with single-cell resolution in vivo. Our preliminary findings show that, although the mutations are generated specifically in NSCs, resulting glioma cells manifest many cellular features of oligodendrocyte precursors (OPCs). Prior to malignancy, OPCs are the only cell lineage that drastically over-expands in the MADM mice. In the glioma tumor mass, OPCs are also the predominant cell type that maintains active cell divisions. When we purify these OPC-like glioma cells they manifest salient glioma stem cell features, including forming renewable tumor spheres, differentiating into multiple cell lineages, and reinitiating gliomas after being transplanted into immuno-suppressed mice. Based on these preliminary results, we will test the following hypothesis: 1) OPCs are the key cell type that initiates and renews gliomagenesis;2) mutant OPCs can de-differentiate to acquire stem cell properties;and 3) targeting OPCs or their stem-cell characteristics will be effective treatment strategies for gliomas. Our proposed studies will lead to valuable basic understanding of the developmental process of gliomas. The identification of tumor-initiating cells should provide a basis for designing rationale treatment strategies for the cure. Conceptually, our proposed work explores the uncharted territory of tumor initiation, and provides critical groundwork for the refinement of mouse models for mechanistic understanding of human cancers. PUBLIC HEALTH RELEVANCE: Studies proposed in this grant aim at the identification of the origin of cancer stem cells for glioma, a currently incurable disease. A thorough understanding of these cells should enable the design of rationale therapeutic strategies by targeting specific cells or genes to treat gliomas. As a consequence, the enhanced specificity should lead to more effective and less toxic drugs, which could eventually provide a cure for gliomas.
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2013 — 2014 |
Zong, Hui |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Highly Specific, Temporally Controllable Mouse Genetic Tools For Investigating In
DESCRIPTION (provided by applicant): Glial cells form intimate contact with neurons and help orchestrate brain wiring during development and facilitate neuronal functions in adulthood. Therefore, it is important to elucidate glial functions in vivo. However, highly specific and adaptable mouse lines for studying two major glial cell types, mature astrocytes and NG2 cells are still lacking. Here we propose to create mouse genetic tools that would allow one to probe into the function of these two types of glial cells in a temporally controllable fashion. The proposed Tet-OFF binary system consists of two groups of mice: 1) driver lines that express tTA in specific types of glial cells; 2) effector lines that express transgenes for cell ablation or transcriptomic profiling under the control of Tet Operon (TetO). The system would allow one to pick and choose individual driver and effector lines to study a specific problem in glial biology. When combined with existing Cre lines, intersectional genetics can provide unprecedented cell type-specificity to analyze glial functions in vivo. Although apparently straightforward, problems of leakiness, variegation/mosaicism, and silencing of transgene expression could derail such efforts. With careful preparation, our lab is now ready to take on this challenge. With our extensive experience with ES cell-based knock-in strategy gained from creating and optimizing a mouse genetic mosaic system, we will use carefully designed targeting strategies to ensure the faithfulness and completeness of transgene expression. As importantly, we have broadly consulted with leaders in the field of glial biology and mouse genetics, and will target these transgenes into the most promising genomic loci for faithful and specific transgene expression. We believe that genetic tools described in this grant will have a broad impact on studying the roles of glial cells for normal functions and diseases in the central nervous system. For example, RNA tagging in astrocytes or NG2 cells will allow one to study the transcriptional landscape of these cells throughout the normal development or under certain pathological conditions. Cell ablation experiments could help reveal the critical contributions of specific glial cell types in brain wiring during development and cognitive functions in adulthood. Applied to brain tumor research, cell ablation will help identify important cellular targets for effective therapeutic intervention. Lastly, principles learned from the proposed work will provide a firm foundation for the successful expansion of the system in the future to address a diverse range of exciting topics in neuroscience.
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2016 — 2020 |
Zong, Hui |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Deconstruct Tumor Microenvironment in Medulloblastoma
Tumor is a living organ. Effective cancer treatment must deal with not only the genetic alterations within tumor cells, but also the supportive tumor microenvironment (TME). Our lab studies the roles of TME in medulloblastoma, the most common malignant pediatric brain tumor. Although improved radiation and chemotherapy greatly boosted patient survival rates, traditional treatment often leads to devastating side effects in young children. Since aberrant sonic hedgehog (Shh) signaling in granule neuron precursors (GNPs) is a common cause of desmoplastic medulloblastoma, inhibitors of the Shh pathway that can effectively kill tumor cells have been developed. However, toxicity in patients and mutations that resist these inhibitors greatly reduced their clinical applicability. To identify additional mechanisms that could alleviate that problem, we set out to investigate mechanisms of tumor-TME interactions with mouse genetic models and found the prominent presence of astrocytes and tumor-associated microglia/macrophages (TAMs). We also screened a panel of growth factors with qRT-PCR, and found that IGF1 fits the bill as a TME factor since it is consistently elevated in the tumor mass, but is absent from tumor GNPs. Further studies showed that IGF1 greatly promoted proliferation of tumor GNPs in culture, and that the loss of IGF1R specifically in GNPs led to halted tumor progression. Using in situ hybridization, we pinpointed TAMs but not other cell types as the IGF1-secreting TME cell type. Co-culture of tumor cells with TAMs led to sustained proliferation, an effect abrogated by the IGF1-blocking agent. Finally, we found that IL-4 is produced by astrocytes in the TME, which is known to promote IGF1 expression in microglia/macrophages. In conclusion, we have identified a TME network that centered on IGF1 signaling to promote medulloblastoma progression. Based on our preliminary findings, we hypothesize that disrupting the TME-tumor crosstalk along the IGF1 axis should be an effective therapeutic strategy for medulloblastoma. In this grant application, we propose to test our hypothesis by directly inhibiting IGF1R signaling in tumor cells, by removing IGF1 from TAMs, and by cutting off the IL-4 signaling from astrocytes to TAMs thus reduce their IGF1 production. We have assembled a team of experts in mouse genetics, immunology, and human brain tumor pathology, and are confident that our studies will contribute to the development of highly effective, novel treatment strategies. In the long term, the principles that emerge from our studies should not only benefit medulloblastoma patients, but also provide a basis for innovative therapies for other neurological diseases.
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1 |
2018 — 2019 |
Zong, Hui |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Establish a Zebrafish Genetic Mosaic System For Disease Modeling and Developmental Biology
Many human diseases, such as developmental disorders, cancer and neurological diseases, are caused by genetic mosaicism, in which the disease-causing cells carry distinct gene mutations from the rest of the body. Therefore, genetic mosaic animal models are highly valuable for both basic and translational research. For basic research, genetic mosaic models reveal in vivo behaviors of mutant cells to facilitate our understanding of both gene functions in normal biological processes and genetic mechanisms of disease etiology. For translational research, genetic mosaic models can be used for preclinical testing of therapeutic strategies for their efficacy in preventing, treating, and even reversing diseases. While Cre-loxP based conditional knockout models as genetic mosaics have led to many groundbreaking discoveries, the resolution is often at the tissue- level, which creates significantly challenges for cellular resolution phenotypic analysis. To overcome this problem, previously we have established a genetic mosaic system in mouse, termed Mosaic Analysis with Double Markers (MADM, Zong 2005 Cell). From a colorless, heterozygous mouse, the MADM system generates sparse GFP+ mutant and RFP+ WT sibling cells through inter-chromosomal mitotic recombination. Sparse labeling and 100% color-genotype matching enable in vivo phenotypic analysis at the single-cell resolution. MADM was broadly adopted in many fields such as neurobiology (Hippenmeyer 2010 Neuron), developmental biology (Packard 2013 Developmental cell), and cancer biology (Liu 2011 Cell). While we have learned a lot of fascinating biology with the mouse MADM system, zebrafish as a model organism carries great advantages in terms of the transparency of its body and the ease to generate a large, genetically identical population. If one could establish a zebrafish equivalent of the mouse MADM system, live imaging could be readily used to gain dynamic information of in vivo gene functions and disease-progression mechanisms. As importantly, high-throughput screening could be readily set up to identify drug candidates that could stop or even reverse the disease progression in such a model with a multi-well, high-content imaging based platform. To establish the zebrafish MADM system, in Specific Aim 1, we will construct the MADM cassettes tailored for zebrafish and precisely target two MADM alleles at the pre-selected identical locus in the genome with the CRISPR-CAS9 system to establish the zebrafish MADM system; and in Specific Aim 2, we will perform basic characterization of the zebrafish MADM system after breeding a few Cre transgenes into the founder zebrafish, including the level of reporter gene expression, recombination efficiency in various tissues, etc. Upon the establishment of the zebrafish MADM, we will deposit it into public repository such as ZIRC for all labs in the field to use. We envision that the system will have a significant impact on fields including developmental biology, neuroscience, cancer biology, regenerative medicine, and much more.
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