2021 |
Klein, Allon Moshe Megason, Sean G [⬀] |
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. |
Mapping the Signaling Landscape of Vertebrate Development At Single Cell Resolution
Abstract (Project Summary) A major goal of developmental biology is to understand the detailed molecular progression of all embryonic cell lineages, from pluripotency to adulthood, and how signalling pathways control lineage choices at every step of differentiation. Such an understanding addresses several fundamental questions in developmental biology, while having practical implications for re-programming cells in disease, and for in vitro differentiation for cell therapy. Recently, we developed droplet microfluidic single cell RNA-Sequencing (scRNA-Seq) technology, which allows profiling the transcriptome of tens of thousands of single cells at low cost, and we additionally developed a method to combine droplet scRNA-Seq with lineage tracing, and the computational methods required to reconstruct time series of differentiation from scRNA-Seq and lineage data. In preliminary work, we applied these tools to generate a comprehensive map of cell state trajectories in zebrafish development through the first 24 hours post fertilization. In this proposal, we will extend our map of zebrafish development, combining scRNA-Seq with clonal analysis to track every cell state in the developing zebrafish embryo up to 7 days post-fertilization. We will then use staged, acute perturbations of seven major signaling pathways, followed by scRNA-Seq, to define which signaling pathways control the flow of cells down different trajectories throughout development, as well as their context dependent and invariant gene targets. Focusing deeply on neural tube patterning, we will then dissect the transcription factor networks that integrate signaling pathways, by CRISPR/Cas9 perturbation coupled to scRNA-Seq. This proposal builds on a multi-year collaboration between two labs with strong and synergistic expertise -- the Megason lab in the use of zebrafish for developmental systems biology and the Klein lab in single cell genomics and analysis.
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2021 |
Klein, Allon Moshe Pittet, Mikael |
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. |
Single Cell Genome-Wide Myeloid Response Profiling in Immunotherapy
Project summary Tumor microenvironments are home to diverse immune cell types but current immunotherapeutic approaches are focused largely on activating cytolytic T cells. The potential of other, myeloid lineage cells in fighting cancer are hitherto much less explored. For example, we have a limited understanding of the complexity of myeloid cell subtypes, we cannot fully discriminate between tumor-promoting and tumor-suppressing cells, and we lack information about defined myeloid cell-associated molecular pathways that could be harnessed for therapy. Here, we will use cutting-edge, unbiased single cell profiling to reveal unappreciated immunoregulatory myeloid cell types, alongside in vitro and in vivo perturbations, to reveal unappreciated tumor-infiltrating myeloid (TIM) cell populations and define their functional role in lung cancer. To this end, we will first determine human TIM states, their correlation with clinical parameters, and whether these states are conserved between human and mouse lung adenocarcinoma. We will specifically test the hypotheses that i) yet-unappreciated myeloid cell states in tumors and peripheral blood of human patients correlate with patient survival (and possibly other clinical parameters), and ii) genetically engineered mouse tumor models of lung adenocarcinoma host conserved human TIM states and justify further animal studies of the function of these states. Second, we will focus on so-called GN2 and GN3 neutrophil subsets, considering our initial data showing their existence in both human and mouse lung tumors and their relevance to cancer progression (Science, 2017 in Press and our unpublished data included in this application) and that neutrophils are emerging as strong predictors of survival for diverse solid tumors and most notably lung cancer. We will specifically test the hypotheses that i) GN2 and GN3 neutrophils have distinct tumor-promoting functions, and ii) these subsets use specific molecular signaling pathways to foster lung cancer progression. To achieve our goals, we will combine efforts of two labs with complementary expertise: tumor immunobiology and myeloid cells (Pittet), and single cell RNA sequencing (scRNA-Seq) and theory/bioinformatics (Klein). We have further teamed up with clinicians to obtain both blood samples and fresh tumor biopsies from lung cancer patients (our preliminary data are also included in this application). Overall, the approaches and resources developed here could have major implications for developing new and more efficient immunotherapies. Also, by targeting the immune system beyond T cells, we will exploit the diversity of non-redundant immune components as a way to overcome limitations of current treatment options.
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