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Prognostic model for lung adenocarcinoma based on experimental drug-resistant cell lines and clinical patients.
OBJECTIVE: Despite advances in EGFR-TKIs for lung adenocarcinoma (LUAD), resistance remains a major hurdle. This study aimed to develop a prognostic model integrating immune microenvironment features and in vitro resistance mechanisms to predict outcomes and guide therapy. MATERIALS AND METHODS: erlotinib-, gefitinib-, and osimertinib-resistant HCC827 cell lines were established by exposing them to increasing EGFR-TKIs concentrations. RNA-sequencing was conducted on non-resistant HCC827 and erlotinib/gefitinibresistant cell lines. From the erlotinib-resistant, gefitinib-resistant cell lines and The Cancer Genome Atlas Program-Lung adenocarcinoma (TCGA-LUAD) data, a prognostic risk score model was constructed via Least Absolute Shrinkage and Selection Operator-Cox Proportional Hazards Model (LASSO-COX). Furthermore, immune infiltration was assessed using Gene Set Variation Analysis (GSVA), and single-cell RNA-seq (GSE241934) resolved expression patterns in EGFR-mutant vs. wild-type tumors. In vitro validation included RT-PCR in Osimertinib resistant (OR)-HCC827 cells. RESULTS: A 3-gene (PPP1R3G, CREG2, LYPD3) RiskScore were developed. The RiskScore predicted poor survival and resistance across all EGFR-TKI generations, with osimertinib-resistant HCC827 cells showing significant upregulation of signature genes. High-risk patients exhibited immune-suppressive microenvironments (enriched regulatory T cells, depleted mast cells) and distinct scRNA-seq profiles. A nomogram (C-index = 0.7) integrated RiskScore with clinical factors for personalized prognosis. CONCLUSION: This model bridges in vitro resistance mechanisms with clinical immune landscapes, offering a tool to stratify patients for EGFR-TKIs, immunotherapies, or combinatorial strategies.
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A transcription factor regulatory atlas for activity inference and perturbation prediction.
Inferring transcription factor (TF) activity from transcriptomes and predicting transcriptome-wide responses to TF perturbations remain challenging, in part because available TF-mRNA resources often face a trade-off between precision and coverage and typically lack signed regulatory information. Here, we present TFActProfiler, a TF-mRNA resource and computational framework that learns signed, quantitative TF-mRNA regulatory coefficients by integrating heterogeneous prior evidence (ChIP-based, motif-based, and curated TF-mRNA annotations) with large-scale bulk and single-cell RNA-seq atlases. TFActProfiler contains 2 606 176 signed TF-mRNA interactions and improves TF activity inference in TF knockdown benchmarks relative to widely used regulon resources while retaining broad TF and target coverage. In addition, because the same learned regulatory coefficients can be used to model downstream transcriptional effects, TFActProfiler enables prediction of transcriptome-wide gene expression responses to TF knockdown without training on task-matched perturbation data. When perturbation datasets are available, TFActProfiler can be further refined to achieve performance comparable to state-of-the-art machine-learning baselines. By providing a direction-aware representation of TF-mRNA regulation for both activity inference and perturbation-response modeling, TFActProfiler supports systematic dissection of gene regulatory programs across diverse cellular contexts.
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scNetViz: from single cells to networks using Cytoscape.
Single-cell RNA-sequencing (scRNA-seq) has revolutionized molecular biology and medicine by enabling high-throughput studies of cellular heterogeneity in diverse tissues. Applying network biology approaches to scRNA-seq data can provide useful insights into genes driving heterogeneous cell-type compositions of tissues. Here, we present scNetViz- a Cytoscape app to aid biological interpretation of cell clusters in scRNA-seq data using network analysis. scNetViz calculates the differential expression of each gene across clusters and then creates a cluster-specific gene functional interaction network between the significantly differentially expressed genes for further analysis, such as pathway enrichment analysis. To automate a complete data analysis workflow, scNetViz integrates parts of the Scanpy software, which is a popular Python package for scRNA-seq data analysis, with Cytoscape apps such as stringApp, cyPlot, and enhancedGraphics. We describe our implementation of methods for accessing data from public single cell atlas projects, differential expression analysis, visualization, and automation. scNetViz enables users to analyze data from public atlases or their own experiments, which we illustrate with two use cases. Analysis can be performed via the Cytoscape GUI or CyREST programming interface using R (RCy3) or Python (py4cytoscape).
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Intratumoral Heterogeneity and Immune Microenvironment in Hepatoblastoma Revealed by Single-Cell RNA Sequencing.
Hepatoblastoma (HB) is a common paediatric liver malignancy characterised by significant intratumoral heterogeneity and a complex tumour microenvironment (TME). Using single-cell RNA sequencing (scRNA-seq), we analysed 43,592 cells from three tumour regions and adjacent normal tissue of an HB patient. Our study revealed distinct cellular compositions and varying degrees of malignancy across different tumour regions, with the T1 region showing the highest malignancy and overexpression of HMGB2 and TOP2A. Survival analysis demonstrated that high HMGB2 expression is associated with poor prognosis and increased recurrence, suggesting its potential as a prognostic marker. Additionally, we identified a diverse immune microenvironment enriched with regulatory T cells (Tregs) and CD8+ effector memory T cells (Tem), indicating potential immune evasion mechanisms. Notably, CTLA-4 and PD-1 were highly expressed in Tregs and Tem cells, highlighting their potential as immunotherapy targets. Myeloid cells, including Kupffer cells and dendritic cells, also exhibited distinct functional roles in different tumour regions. This study provides the first comprehensive single-cell atlas of HB, revealing critical insights into its intratumoral heterogeneity and immune microenvironment. Our findings not only advance the understanding of HB biology but also offer new directions for precision medicine, including the development of targeted therapies and immunotherapeutic strategies to improve patient outcomes.
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Hypoxia Contributes to Poor Prognosis in Primary IDH-wt GBM by Inducing Tumor Cells MES-Like Transformation Trend and Inhibiting Immune Cells Activity.
AIMS: To reveal the influence of hypoxia on tumor cells and immune cells in primary IDH-wt glioblastoma patients. METHODS: Single-cell RNA-seq data and bulk RNA-seq data were acquired from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases, respectively. Hypoxia status and subtypes of tumor cells were identified based on single-sample Gene Set Enrichment Analysis (ssGSEA). Regulon network analysis of different subtypes under different conditions was conducted by SCENIC. Within tumor microenvironment, biological process activity analysis and cell-cell communication network were conducted to uncover the inner links between each cell subtype under different hypoxia status. RESULTS: Different types of tumor cell in GBM possessed different hypoxia status, and MES-like subtype was under a more severe hypoxia condition than other subtypes. Hypoxia also induced MES-like signature gene expression within each tumor cell, which could stimulate tumor cell proliferation and invasion by regulating cell-cell communication. Additionally, hypoxia inhibited immune cell activity in the tumor microenvironment by inducing macrophage phenotype polarization and upregulating immune-inhibited cell-cell interaction within immune cells. Interactions between tumor cells and immune cells under hypoxia status also promoted tumor progression. CONCLUSIONS: Hypoxia was a poor prognostic marker for primary IDH-wt GBM patients. Meanwhile, it could induce tumor cells' MES-like transformation trend and inhibit antitumor function of immune cells.
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An Integrative Single-cell Transcriptomic Atlas of the Post-natal Mouse Mammary Gland Allows Discovery of New Developmental Trajectories in the Luminal Compartment.
The mammary gland is a highly dynamic organ which undergoes periods of expansion, differentiation and cell death in each reproductive cycle. Partly because of the dynamic nature of the gland, mammary epithelial cells (MECs) are extraordinarily heterogeneous. Single cell RNA-seq (scRNA-seq) analyses have contributed to understand the cellular and transcriptional heterogeneity of this complex tissue. Here, we integrate scRNA-seq data from three foundational reports that have explored the mammary gland cell populations throughout development at single-cell level using 10× Chromium Drop-Seq. We center our analysis on post-natal development of the mammary gland, from puberty to post-involution. The new integrated study corresponds to RNA sequences from 53,686 individual cells, which greatly outnumbers the three initial data sets. The large volume of information provides new insights, as a better resolution of the previously detected Procr+ stem-like cell subpopulation or the identification of a novel group of MECs expressing immune-like markers. Moreover, here we present new pseudo-temporal trajectories of MEC populations at two resolution levels, that is either considering all mammary cell subtypes or focusing specifically on the luminal lineages. Interestingly, the luminal-restricted analysis reveals distinct expression patterns of various genes that encode milk proteins, suggesting specific and non-redundant roles for each of them. In summary, our data show that the application of bioinformatic tools to integrate multiple scRNA-seq data-sets helps to describe and interpret the high level of plasticity involved in gene expression regulation throughout mammary gland post-natal development.
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Single-cell RNA-seq reveals invasive trajectory and determines cancer stem cell-related prognostic genes in pancreatic cancer.
Pancreatic duct adenocarcinoma (PDAC) is an aggressive and lethal malignancy. Pancreatic cancer stem cells (PCSCs) are assumed to contribute to the initiation and invasion of PDAC. In this study, we performed single-cell RNA sequencing (scRNA-seq) analysis of PDAC tumor samples from patients and control pancreas tissues to reveal the transformation process of cancer stem cell (CSC)-like ductal cells into ductal cells with invasive potential and we screened out CSC-related genes (CRGs). Subsequently, we applied LASSO and Cox regression models to identify five CRGs with potential prognostic values and constructed a risk prognostic model using the Cancer Genome Atlas datasets. The risk models were verified using Gene Expression Omnibus datasets. Patients in the high-risk group had a significantly poor overall survival (Pvalue<0.0001), as illustrated by the Kaplan-Meier survival curve, and the area under the curve confirmed the accuracy of predictions by our risk model. Tumor mutation burden variations were used to further explore the differences between the two risk cohorts. In addition, the Human Protein Atlas was used to investigate the protein expression of five hub CRGs. In brief, we utilized scRNA-seq to reveal the invasive trajectory of ductal cells and identified crucial CRGs in PDAC, which may help predict patient survival and provide potential clinical therapeutic targets against CSCs.
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Building a FAIR data ecosystem for incorporating single-cell transcriptomics data into agricultural genome to phenome research.
INTRODUCTION: The agriculture genomics community has numerous data submission standards available, but the standards for describing and storing single-cell (SC, e.g., scRNA- seq) data are comparatively underdeveloped. METHODS: To bridge this gap, we leveraged recent advancements in human genomics infrastructure, such as the integration of the Human Cell Atlas Data Portal with Terra, a secure, scalable, open-source platform for biomedical researchers to access data, run analysis tools, and collaborate. In parallel, the Single Cell Expression Atlas at EMBL-EBI offers a comprehensive data ingestion portal for high-throughput sequencing datasets, including plants, protists, and animals (including humans). Developing data tools connecting these resources would offer significant advantages to the agricultural genomics community. The FAANG data portal at EMBL-EBI emphasizes delivering rich metadata and highly accurate and reliable annotation of farmed animals but is not computationally linked to either of these resources. RESULTS: Herein, we describe a pilot-scale project that determines whether the current FAANG metadata standards for livestock can be used to ingest scRNA-seq datasets into Terra in a manner consistent with HCA Data Portal standards. Importantly, rich scRNA-seq metadata can now be brokered through the FAANG data portal using a semi-automated process, thereby avoiding the need for substantial expert curation. We have further extended the functionality of this tool so that validated and ingested SC files within the HCA Data Portal are transferred to Terra for further analysis. In addition, we verified data ingestion into Terra, hosted on Azure, and demonstrated the use of a workflow to analyze the first ingested porcine scRNA-seq dataset. Additionally, we have also developed prototype tools to visualize the output of scRNA-seq analyses on genome browsers to compare gene expression patterns across tissues and cell populations. This JBrowse tool now features distinct tracks, showcasing PBMC scRNA-seq alongside two bulk RNA-seq experiments. DISCUSSION: We intend to further build upon these existing tools to construct a scientist-friendly data resource and analytical ecosystem based on Findable, Accessible, Interoperable, and Reusable (FAIR) SC principles to facilitate SC-level genomic analysis through data ingestion, storage, retrieval, re-use, visualization, and comparative annotation across agricultural species.
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The offonome reveals on and off states of gene expression near the detection limit of RNA-seq.
RNA-seq, widely used for gene expression profiling, provides nucleotide level genome coverage and summary gene expression values. Generally, low-expressed genes are ignored due to their unfavorable signal-to-noise ratio, however, these genes may offer crucial information, such as detecting rare cells in bulk tissues. In this study, we applied an approach that transforms the expression levels of low-expressed genes into a robust dichotomized on/off state by leveraging similarities in transcript coverage shape. Applied to three human cancer cohorts from the Cancer Genome Atlas (TCGA), chosen based on tissue morphology and anatomic site, we identified genes, the "offonome" near the detection limit, consistently or occasionally off across samples. Genes in the offonome spectrum proved useful for supervised and unsupervised applications, including characterizing oncogenic pathways, and identifying rare populations of cells in bulk tissue. Interrogating the offonome is relevant to bulk tumor analyses like TCGA, potentially expediting gene investigation in low-input situations like single cell RNA-seq.
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Ultra-high-throughput single-cell RNA sequencing and perturbation screening with combinatorial fluidic indexing.
Cell atlas projects and high-throughput perturbation screens require single-cell sequencing at a scale that is challenging with current technology. To enable cost-effective single-cell sequencing for millions of individual cells, we developed 'single-cell combinatorial fluidic indexing' (scifi). The scifi-RNA-seq assay combines one-step combinatorial preindexing of entire transcriptomes inside permeabilized cells with subsequent single-cell RNA-seq using microfluidics. Preindexing allows us to load several cells per droplet and computationally demultiplex their individual expression profiles. Thereby, scifi-RNA-seq massively increases the throughput of droplet-based single-cell RNA-seq, and provides a straightforward way of multiplexing thousands of samples in a single experiment. Compared with multiround combinatorial indexing, scifi-RNA-seq provides an easy and efficient workflow. Compared to cell hashing methods, which flag and discard droplets containing more than one cell, scifi-RNA-seq resolves and retains individual transcriptomes from overloaded droplets. We benchmarked scifi-RNA-seq on various human and mouse cell lines, validated it for primary human T cells and applied it in a highly multiplexed CRISPR screen with single-cell transcriptome readout of T cell receptor activation.