In vivo perturb-seq of cancer and microenvironment cells dissects oncologic drivers and radiotherapy responses in glioblastoma

IF 10.1 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Genome Biology Pub Date : 2024-10-07 DOI:10.1186/s13059-024-03404-6
S. John Liu, Christopher Zou, Joanna Pak, Alexandra Morse, Dillon Pang, Timothy Casey-Clyde, Ashir A. Borah, David Wu, Kyounghee Seo, Thomas O’Loughlin, Daniel A. Lim, Tomoko Ozawa, Mitchel S. Berger, Roarke A. Kamber, William A. Weiss, David R. Raleigh, Luke A. Gilbert
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Abstract

Genetic perturbation screens with single-cell readouts have enabled rich phenotyping of gene function and regulatory networks. These approaches have been challenging in vivo, especially in adult disease models such as cancer, which include mixtures of malignant and microenvironment cells. Glioblastoma (GBM) is a fatal cancer, and methods of systematically interrogating gene function and therapeutic targets in vivo, especially in combination with standard of care treatment such as radiotherapy, are lacking. Here, we iteratively develop a multiplex in vivo perturb-seq CRISPRi platform for single-cell genetic screens in cancer and tumor microenvironment cells that leverages intracranial convection enhanced delivery of sgRNA libraries into mouse models of GBM. Our platform enables potent silencing of drivers of in vivo growth and tumor maintenance as well as genes that sensitize GBM to radiotherapy. We find radiotherapy rewires transcriptional responses to genetic perturbations in an in vivo-dependent manner, revealing heterogenous patterns of treatment sensitization or resistance in GBM. Furthermore, we demonstrate targeting of genes that function in the tumor microenvironment, enabling alterations of ligand-receptor interactions between immune and stromal cells following in vivo CRISPRi perturbations that can affect tumor cell phagocytosis. In sum, we demonstrate the utility of multiplexed perturb-seq for in vivo single-cell dissection of adult cancer and normal tissue biology across multiple cell types in the context of therapeutic intervention, a platform with potential for broad application.
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癌细胞和微环境细胞的体内扰动-序列分析剖析胶质母细胞瘤的肿瘤学驱动因素和放疗反应
通过单细胞读数的遗传扰动筛选,可以对基因功能和调控网络进行丰富的表型分析。这些方法在体内具有挑战性,尤其是在癌症等成人疾病模型中,因为这些模型包括恶性细胞和微环境细胞的混合物。胶质母细胞瘤(GBM)是一种致命的癌症,目前还缺乏在体内系统检测基因功能和治疗靶点的方法,尤其是在结合放疗等标准治疗的情况下。在这里,我们迭代开发了一种多重体内 perturb-seq CRISPRi 平台,用于在癌细胞和肿瘤微环境细胞中进行单细胞基因筛选,该平台利用颅内对流增强技术将 sgRNA 文库输送到 GBM 小鼠模型中。我们的平台能有效抑制体内生长和肿瘤维持的驱动因子以及使 GBM 对放疗敏感的基因。我们发现,放疗以体内依赖的方式重新改写了基因扰动的转录反应,揭示了 GBM 治疗敏感或耐药的不同模式。此外,我们还证明了肿瘤微环境中功能基因的靶向性,使免疫细胞和基质细胞之间的配体-受体相互作用在体内CRISPRi扰动后发生改变,从而影响肿瘤细胞的吞噬功能。总之,我们证明了多路复用 perturb-seq 在治疗干预背景下对成人癌症和正常组织生物学进行体内单细胞剖析的实用性,这是一个具有广泛应用潜力的平台。
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来源期刊
Genome Biology
Genome Biology Biochemistry, Genetics and Molecular Biology-Genetics
CiteScore
21.00
自引率
3.30%
发文量
241
审稿时长
2 months
期刊介绍: Genome Biology stands as a premier platform for exceptional research across all domains of biology and biomedicine, explored through a genomic and post-genomic lens. With an impressive impact factor of 12.3 (2022),* the journal secures its position as the 3rd-ranked research journal in the Genetics and Heredity category and the 2nd-ranked research journal in the Biotechnology and Applied Microbiology category by Thomson Reuters. Notably, Genome Biology holds the distinction of being the highest-ranked open-access journal in this category. Our dedicated team of highly trained in-house Editors collaborates closely with our esteemed Editorial Board of international experts, ensuring the journal remains on the forefront of scientific advances and community standards. Regular engagement with researchers at conferences and institute visits underscores our commitment to staying abreast of the latest developments in the field.
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