Single-cell multiomics sequencing and analyses of human colorectal cancer

Bian, Shuhui; Hou, Yu; Zhou, Xin; Li, Xianlong; Yong, Jun; Wang, Yicheng; Wang, Wendong; Jia, Yan; Hu, Boqiang; Guo, Hongshan; Wang, Jilian; Gao, Shuai; Mao, Yunuo; Dong, Ji; Zhu, Ping; Xiu, Dianrong; Yan, Liying; Wen, Lu; Qiao, Jie; Tang, Fuchou; Fu, Wei

doi:10.1126/science.aao3791

Cited by 281 publications

(337 citation statements)

References 29 publications

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“…This paper describes interfacial nanoinjection (INJ), a simple microfluidic droplet‐handling system developed for multistep nanoliter assays on standard 96‐ or 384‐well plates, and a simple pipeline that couples INJ and fluorescence‐activated cell sorting (FACS) for single‐cell analyses with high versatility, high throughput, and low cost. Recent research advances have promoted the study of biological systems with respect to their physiological phenotypes, genomics, transcriptomics, epigenomics, proteomics, multiomics, and biodiversity at the single‐cell level. Single‐cell analysis allows the fundamental understanding of cell‐to‐cell heterogeneity and provides unprecedented resolution in complex biological systems, especially those from rare cell types or uncultivated microbes .…”

Section: Introductionmentioning

confidence: 99%

Interfacial Nanoinjection‐Based Nanoliter Single‐Cell Analysis

Yun

Zheng

et al. 2019

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Single‐cell analysis offers unprecedented resolution for the investigation of cellular heterogeneity and the capture of rare cells from large populations. Here, described is a simple method named interfacial nanoinjection (INJ), which can miniaturize various single‐cell assays to be performed in nanoliter water‐in‐oil droplets on standard microwell plates. The INJ droplet handler can adjust droplet volumes for multistep reactions on demand with high precision and excellent monodispersity, and consequently enables a wide range of single‐cell assays. Importantly, INJ can be coupled with fluorescence‐activated cell sorting (FACS), which is currently the most effective and accurate single‐cell sorting and isolation method. FACS‐INJ pipelines for high‐throughput plate well‐based single‐cell analyses, including single‐cell proliferation, drug‐resistance testing, polymerase chain reaction (PCR), reverse‐transcription PCR, and whole‐genome sequencing are introduced. This FACS‐INJ pipeline is compatible with a wide range of samples and can be extended to various single‐cell analysis applications in microbiology, cell biology, and biomedical diagnostics.

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Section: Introductionmentioning

confidence: 99%

Interfacial Nanoinjection‐Based Nanoliter Single‐Cell Analysis

Yun

Zheng

et al. 2019

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“…scRNA-seq reports in the past years have provided useful information in this respect. Different types of CAFs have been reported in breast and colorectal tumors, which is likely to be associated with different cell origins [34][35][36]. Additionally, each group of CAFs has special functions in the recruitment of immune cells and in the induction of the epithelial-mesenchymal transition (EMT) in tumor cells [24,29,34,36].…”

Section: Tumor Microenvironmentmentioning

confidence: 99%

“…Abbreviation: EMT, epithelial-mesenchymal transition. See [24,25,29,30,[34][35][36]40,43,[48][49][50].…”

Section: Key Figurementioning

confidence: 99%

Tumor Functional Heterogeneity Unraveled by scRNA-seq Technologies

2020

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Effective cancer treatment has been precluded by the presence of various forms of intratumoral complexity that drive treatment resistance and metastasis. Recent single-cell sequencing technologies are significantly facilitating the characterization of tumor internal architecture during disease progression. New applications and advances occurring at a fast pace predict an imminent broad application of these technologies in many research areas. As occurred with next-generation sequencing (NGS) technologies, once applied to clinical samples across tumor types, single-cell sequencing technologies could trigger an exponential increase in knowledge of the molecular pathways involved in cancer progression and contribute to the improvement of cancer treatment. Multifaceted Heterogeneity and Its Impact on Cancer ProgressionTumors comprise various cell populations in constant evolution. Some of this complex heterogeneity derives from genetic diversification and Darwinian selection of tumor cells as they adapt to variable environments. Next-generation sequencing (NGS; see Glossary) used for the past decade had enough sensitivity to detect mutations present in minor cell populations and, combined with multisampling of human tumors (multisampling sequencing), fostered many studies that characterized intratumor heterogeneity in various cancers [1]. The level of intratumor heterogeneity is considered a main driver of therapy resistance and metastasis and is associated with poor prognosis [2].In addition, human cancers frequently have tumor cell populations with different transcriptional programs. This functional diversity is likely associated with the genetic heterogeneity described above but is also the result of many other factors. First, the presence of a hierarchical structure, where a group of quiescent stem-like cells fosters the growth of a tumor comprising cells in different differentiation states, was demonstrated in various tumor types [3]. Additionally, different transcriptional programs can be activated in tumor cells as a response to stochastic factors or to a variable tumor microenvironment. This functional diversity provides tumors with a plasticity that grants a high capacity for adaptation [4].Finally, human tumors comprise not only malignant/transformed cells but also a plethora of different cell types recruited from the surrounding tissue and the immune system. The tumor microenvironment shows also genetic and transcriptional diversity and plays important roles in tumor progression, metastasis, and treatment resistance [1,5].Fine characterization of these levels of tumor heterogeneity is essential to the successful treatment of cancer patients. The recent development of technologies based on sequencing individual cells (single-cell sequencing technologies) opens new ways for the characterization of tumor heterogeneity. At the genetic level, single-cell DNA-seq technologies offer higher sensitivity in the detection of minority clones, the reconstruction of clone structure, and the identification of concurrent ...

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“…Our findings underscore the importance of using single-cell profiles as a rich, comprehensive and interpretable phenotypic readout. With advances in other single cell profiling approaches (e.g., single-cell ATAC-Seq (Rubin et al, 2019)), single-cell multi-omics (Bian et al, 2018), and spatial genomics Rodriques et al, 2019), we expect in vivo Perturb-Seq to be coupled in the near future with diverse readouts to better define the function of disease-risk associated variants from molecular mechanisms to non-cell autonomous effects in tissues. Spatial transcriptomics in particular should be well suited for use with in vivo Perturb-Seq, and should help uncover non-cell autonomous effects.…”

Section: Discussionmentioning

confidence: 99%

In vivo Perturb-Seq reveals neuronal and glial abnormalities associated with Autism risk genes

Jin

Simmons

Guo

et al. 2019

Preprint

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The thousands of disease risk genes and loci identified through human genetic studies far outstrip our current capacity to systematically study their functions. New experimental approaches are needed for functional investigations of large panels of genes in a biologically relevant context. Here, we developed a scalable genetic screen approach, in vivo Perturb-Seq, and applied this method to the functional evaluation of 35 autism spectrum disorder (ASD) de novo loss-of-function risk genes. Using CRISPR-Cas9, we introduced frameshift mutations in these risk genes in pools, within the developing brain in utero, and then performed single-cell RNA-Seq in the postnatal brain. We identified cell type-specific gene signatures from both neuronal and glial cell classes that are affected by genetic perturbations, and pointed at elements of both convergent and divergent cellular effects across this cohort of ASD risk genes. In vivo Perturb-Seq pioneers a systems genetics approach to investigate at scale how diverse mutations affect cell types and states in the biologically relevant context of the developing organism.

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Single-cell multiomics sequencing and analyses of human colorectal cancer

Cited by 281 publications

References 29 publications

Interfacial Nanoinjection‐Based Nanoliter Single‐Cell Analysis

Interfacial Nanoinjection‐Based Nanoliter Single‐Cell Analysis

Tumor Functional Heterogeneity Unraveled by scRNA-seq Technologies

In vivo Perturb-Seq reveals neuronal and glial abnormalities associated with Autism risk genes

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