Joseph T. Decker, Matthew S. Hall, Devak Nanua, Sophia M. Orbach, Jyotirmoy Roy, Amogh Angadi, Julianna Caton, Lauren Hesse, Jacqueline S. Jeruss, Lonnie D. Shea
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引用次数: 0
摘要
导言：基于自然杀伤（NK）细胞的疗法是治疗轻度癌症的一种前景广阔的新方法，然而新疗法的工程设计非常复杂，实体瘤的治疗进展缓慢。我们将单细胞 RNA 测序与 NK 细胞杀伤模型系统的活细胞成像相结合，将转录组数据与功能输出相关联。这些研究使用了一个 NK 细胞活性模型，即杀死 HeLa 宫颈癌细胞的 NK-92 细胞系。在与目标 HeLa 细胞共培养的过程中，通过显微镜观察 NK 细胞的杀伤活性，然后根据 NK 细胞的位置和 Annexin V 表达手动绘制杀伤活性图。利用 10× Genomics 平台对该培养体系中的 NK 细胞进行单细胞 RNA 测序，并利用 Viper 和 DoRothEA R 软件包推断转录因子的活性。结果NK细胞在与目标HeLa细胞培养10小时后具有不同的杀伤活性。对单细胞测序数据的分析发现，核因子卡巴B（NF-κB）、信号转导和转录激活因子1（STAT1）和MYC活性是我们的模型系统中NK细胞功能表型的潜在驱动因素。对转录因子活性的活细胞成像发现，NF-κB 活性与过去的杀伤活性显著相关。结论将转录因子活性的发光显微镜技术与单细胞 RNA 测序技术相结合，是为推断的转录组学数据分配功能表型的有效方法。
Dynamic Transcriptional Programs During Single NK Cell Killing: Connecting Form to Function in Cellular Immunotherapy
Introduction
Natural killer (NK) cell-based therapies are a promising new method for treating indolent cancer, however engineering new therapies is complex and progress towards therapy for solid tumors is slow. New methods for determining the underlying intracellular signaling driving the killing phenotype would significantly improve this progress.
Methods
We combined single-cell RNA sequencing with live cell imaging of a model system of NK cell killing to correlate transcriptomic data with functional output. A model of NK cell activity, the NK-92 cell line killing of HeLa cervical cancer cells, was used for these studies. NK cell killing activity was observed by microscopy during co-culture with target HeLa cells and killing activity subsequently manually mapped based on NK cell location and Annexin V expression. NK cells from this culture system were profiled by single-cell RNA sequencing using the 10× Genomics platform, and transcription factor activity inferred using the Viper and DoRothEA R packages. Luminescent microscopy of reporter constructs in the NK cells was then used to correlate activity of inferred transcriptional activity with killing activity.
Results
NK cells had heterogeneous killing activity during 10 h of culture with target HeLa cells. Analysis of the single cell sequencing data identified Nuclear Factor Kappa B (NF-κB), Signal Transducer and Activator of Transcription 1 (STAT1) and MYC activity as potential drivers of NK cell functional phenotype in our model system. Live cell imaging of the transcription factor activity found NF-κB activity was significantly correlated with past killing activity. No correlation was observed between STAT1 or MYC activity and NK cell killing.
Conclusions
Combining luminescent microscopy of transcription factor activity with single-cell RNA sequencing is an effective means of assigning functional phenotypes to inferred transcriptomics data.
期刊介绍：
The field of cellular and molecular bioengineering seeks to understand, so that we may ultimately control, the mechanical, chemical, and electrical processes of the cell. A key challenge in improving human health is to understand how cellular behavior arises from molecular-level interactions. CMBE, an official journal of the Biomedical Engineering Society, publishes original research and review papers in the following seven general areas:
Molecular: DNA-protein/RNA-protein interactions, protein folding and function, protein-protein and receptor-ligand interactions, lipids, polysaccharides, molecular motors, and the biophysics of macromolecules that function as therapeutics or engineered matrices, for example.
Cellular: Studies of how cells sense physicochemical events surrounding and within cells, and how cells transduce these events into biological responses. Specific cell processes of interest include cell growth, differentiation, migration, signal transduction, protein secretion and transport, gene expression and regulation, and cell-matrix interactions.
Mechanobiology: The mechanical properties of cells and biomolecules, cellular/molecular force generation and adhesion, the response of cells to their mechanical microenvironment, and mechanotransduction in response to various physical forces such as fluid shear stress.
Nanomedicine: The engineering of nanoparticles for advanced drug delivery and molecular imaging applications, with particular focus on the interaction of such particles with living cells. Also, the application of nanostructured materials to control the behavior of cells and biomolecules.
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