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2022, Cell. DOI: 10.1016/j.cell.2022.02.015
Spatial CRISPR genomics identifies regulators of the tumor microenvironment
Maxime Dhainaut , Samuel A. Rose , Guray Akturk , Aleksandra Wroblewska , Sebastian R. Nielsen , Eun Sook Park , Mark Buckup , Vladimir Roudko , Luisanna Pia , Robert Sweeney , Jessica Le Berichel , C. Matthias Wilk , Anela Bektesevic , Brian H. Lee , Nina Bhardwaj , Adeeb H. Rahman , Alessia Baccarini , Sacha Gnjatic , Dana Pe’er , Miriam Merad , Brian D. Brown
Abstract:
While CRISPR screens are helping uncover genes regulating many cell-intrinsic processes, existing approaches are suboptimal for identifying extracellular gene functions, particularly in the tissue context. Here, we developed an approach for spatial functional genomics called Perturb-map. We applied Perturb-map to knock out dozens of genes in parallel in a mouse model of lung cancer and simultaneously assessed how each knockout influenced tumor growth, histopathology, and immune composition. Moreover, we paired Perturb-map and spatial transcriptomics for unbiased analysis of CRISPR-edited tumors. We found that in Tgfbr2 knockout tumors, the tumor microenvironment (TME) was converted to a fibro-mucinous state, and T cells excluded, concomitant with upregulated TGFβ and TGFβ-mediated fibroblast activation, indicating that TGFβ-receptor loss on cancer cells increased TGFβ bioavailability and its immunosuppressive effects on the TME. These studies establish Perturb-map for functional genomics within the tissue at single-cell resolution with spatial architecture preserved and provide insight into how TGFβ responsiveness of cancer cells can affect the TME.
2023-05-30 09:12:00
#paper doi:10.1016/j.cell.2022.02.015 Spatial CRISPR genomics identifies regulators of the tumor microenvironment 这篇文章主要包含两个方面: 1.空间CRISPR技术的开发:“Perturb-map”技术,原位CRISPR+多重成像+空间转录组学,该技术是基础作者2018年cell发表的三联体蛋白条形码Pro-Codes单细胞CRISPR技术。三联体蛋白提高并行敲除基因的数目,另外,nPC荧光蛋白标签定位在细胞核中,通过图像分割软件能很好的分割单细胞。 2.空间CRISPR技术的应用:作者通过空间CRISPR技术研究每个基因敲除后如何影响肿瘤生长、组织病理学和免疫组成。并行敲除32个跟免疫治疗相关的靶基因(包括细胞因子,免疫配体和分泌因子),研究肿瘤细胞对免疫细胞招募和排斥的影响。癌细胞的基因如何控制免疫微环境,这有助于开发新的癌症免疫疗法。 文章对于4T1乳腺和KP肺部肿瘤进行研究,他观察到肺癌具有克隆型分布,每个KP肿瘤病变都由单个KP癌细胞形成。敲除单个基因的细胞扎堆分布,也非常适合后续的免疫分析。文章主要对肺癌研究,但是为什么肺癌细胞会克隆型分布,文章没有具体说明。 3.空间CRISPR技术的延伸:很多癌症如4T1乳腺,单基因敲除的肿瘤细胞分布高度不均匀,也不适合空间CRISPR技术的研究。文章2022年发表,后续没有找到相关的研究课题和文章引用。感觉还是有一定的局限性。
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