Innate immune responses rely on inducible gene expression programmes which, in contrast to steady-state transcription, are highly dependent on cohesin. Here we address transcriptional parameters underlying this cohesin-dependence by single-molecule RNA-FISH and single-cell RNA-sequencing. We show that inducible innate immune genes are regulated predominantly by an increase in the probability of active transcription, and that probabilities of enhancer and promoter transcription are coordinated. Cohesin has no major impact on the fraction of transcribed inducible enhancers, or the number of mature mRNAs produced per transcribing cell. Cohesin is, however, required for coupling the probabilities of enhancer and promoter transcription. Enhancer-promoter coupling may not be explained by spatial proximity alone, and at the model locus Il12b can be disrupted by selective inhibition of the cohesinopathy-associated BET bromodomain BD2. Our data identify discrete steps in enhancer-mediated inducible gene expression that differ in cohesin-dependence, and suggest that cohesin and BD2 may act on shared pathways.
Ignacio Campillo-Marcosa, Damiana Alvarez-Errico, Regina A. Alandes, Elisabetta Mereu, Manel Esteller
Single-Cell Technologies and Analyses in Hematopoiesis and Hematological Malignancies
Experimental Hematology(2021), doi.org/10.1016/j.exphem.2021.05.0019 May 2021, .
In recent years, single-cell technologies have emerged as breakthrough techniques that enable the characterization of hematopoietic cell populations of normal and malignant tissue samples and will be combined in the near future with bulk technologies, currently used in clinical practice, in order to improve diagnosis, prognosis and search for novel molecular targets. These single-cell methods have the advantage of not masking cell-to-cell variation features and involve the study of genetic, epigenetic, transcriptional and proteomic landscapes from a single-cell perspective. Latest advances in this field have enabled the development of novel strategies that significantly increase both sensitivity and high-throughput. In this review, we emphasize emerging techniques aimed at assessing individual or multi-omic parameters at single-cell resolution, and analyze how these technologies have helped us understand hematopoietic variability and identify unknown and/or rare subpopulations. We also summarize the impact of these single-cell profiling strategies on the characterization of cell diversity within the tumor, and the clonal evolution of multiple hematological malignancies in samples from untreated and treated patients, which provide valuable information for diagnosis, prognosis and future treatments, and explain why current therapies may fail. However, despite these improvements, new challenges lie ahead.
Iacono G, Mereu E, Guillaumet-Adkins A, Corominas R, Cuscó I, Rodríguez-Esteban G, Gut M, Pérez-Jurado LA, Gut I, Heyn H
BigSCale: An Analytical Framework for Big-Scale Single-Cell Data
Genome Res. 2018 Jun;28(6):878-890 , .
Single-cell RNA sequencing (scRNA-seq) has significantly deepened our insights into complex tissues, with the latest techniques capable of processing tens of thousands of cells simultaneously. Analyzing increasing numbers of cells, however, generates extremely large data sets, extending processing time and challenging computing resources. Current scRNA-seq analysis tools are not designed to interrogate large data sets and often lack sensitivity to identify marker genes. With bigSCale, we provide a scalable analytical framework to analyze millions of cells, which addresses the challenges associated with large data sets. To handle the noise and sparsity of scRNA-seq data, bigSCale uses large sample sizes to estimate an accurate numerical model of noise. The framework further includes modules for differential expression analysis, cell clustering, and marker identification. A directed convolution strategy allows processing of extremely large data sets, while preserving transcript information from individual cells. We evaluated the performance of bigSCale using both a biological model of aberrant gene expression in patient-derived neuronal progenitor cells and simulated data sets, which underlines the speed and accuracy in differential expression analysis. To test its applicability for large data sets, we applied bigSCale to assess 1.3 million cells from the mouse developing forebrain. Its directed down-sampling strategy accumulates information from single cells into index cell transcriptomes, thereby defining cellular clusters with improved resolution. Accordingly, index cell clusters identified rare populations, such as reelin (Reln)-positive Cajal-Retzius neurons, for which we report previously unrecognized heterogeneity associated with distinct differentiation stages, spatial organization, and cellular function. Together, bigSCale presents a solution to address future challenges of large single-cell data sets.
A. Martinez-Marti, E. Felip, J. Matito, Mereu E, A. Navarro, S. Cedres, N. Pardo, A. Martinez de Castro, J. Remon, J. M. Miquel, A. Guillaumet-Adkins, E. Nadal, G. Rodriguez-Esteban, O. Arques, R. Fasani, P. Nuciforo, H. Heyn, A. Villanueva, H. G. Palmer, A. Vivancos.
Dual MET and ERBB inhibition overcomes intratumor plasticity in osimertinib-resistant-advanced non-small-cell lung cancer (NSCLC).
Ann Oncol . 2017 Oct 1;28(10):2451-2457 , .
Background: Third-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) such as osimertinib are the last line of targeted treatment of metastatic non-small-cell lung cancer (NSCLC) EGFR-mutant harboring T790M. Different mechanisms of acquired resistance to third-generation EGFR-TKIs have been proposed. It is therefore crucial to identify new and effective strategies to overcome successive acquired mechanisms of resistance.
Methods: For Amplicon-seq analysis, samples from the index patient (primary and metastasis lesions at different timepoints) as well as the patient-derived orthotopic xenograft tumors corresponding to the different treatment arms were used. All samples were formalin-fixed paraffin-embedded, selected and evaluated by a pathologist. For droplet digital PCR, 20 patients diagnosed with NSCLC at baseline or progression to different lines of TKI therapies were selected. Formalin-fixed paraffin-embedded blocks corresponding to either primary tumor or metastasis specimens were used for analysis. For single-cell analysis, orthotopically grown metastases were dissected from the brain of an athymic nu/nu mouse and cryopreserved at -80°C.
Results: In a brain metastasis lesion from a NSCLC patient presenting an EGFR T790M mutation, we detected MET gene amplification after prolonged treatment with osimertinib. Importantly, the combination of capmatinib (c-MET inhibitor) and afatinib (ErbB-1/2/4 inhibitor) completely suppressed tumor growth in mice orthotopically injected with cells derived from this brain metastasis. In those mice treated with capmatinib or afatinib as monotherapy, we observed the emergence of KRAS G12C clones. Single-cell gene expression analyses also revealed intratumor heterogeneity, indicating the presence of a KRAS-driven subclone. We also detected low-frequent KRAS G12C alleles in patients treated with various EGFR-TKIs.
Conclusion: Acquired resistance to subsequent EGFR-TKI treatment lines in EGFR-mutant lung cancer patients may induce genetic plasticity. We assess the biological insights of tumor heterogeneity in an osimertinib-resistant tumor with acquired MET-amplification and propose new treatment strategies in this situation.
Guillaumet-Adkins A*, Rodríguez-Esteban G*, Mereu E, endez-Lago M, Jaitin DA, Villanueva A, Vidal A, Martinez-Marti A, Felip E, Vivancos A, Keren-Shaul H, Heath S, Gut M, Amit I, Gut I, Heyn H
Single-cell transcriptome conservation in cryopreserved cells and tissues
Genome Biol . 2017 Mar 1;18(1):45 , .
A variety of single-cell RNA preparation procedures have been described. So far, protocols require fresh material, which hinders complex study designs. We describe a sample preservation method that maintains transcripts in viable single cells, allowing one to disconnect time and place of sampling from subsequent processing steps. We sequence single-cell transcriptomes from >1000 fresh and cryopreserved cells using 3'-end and full-length RNA preparation methods. Our results confirm that the conservation process did not alter transcriptional profiles. This substantially broadens the scope of applications in single-cell transcriptomics and could lead to a paradigm shift in future study designs.
Christian H. Holland, Jovan Tanevski, Javier Perales-Pat on, Jan Gleixner, Manu P. Kumar, Mereu E, Brian A. Joughin, Oliver Stegle, Douglas A. Lauffenburger, Holger Heyn, Bence, Szalai, Julio Saez-Rodriguez
Robustness and applicability of functional genomics tools on scRNA-seq data.
Genome Biol. 2020 Feb 12;21(1):36 , .
Background: Many functional analysis tools have been developed to extract functional and mechanistic insight from bulk transcriptome data. With the advent of single-cell RNA sequencing (scRNA-seq), it is in principle possible to do such an analysis for single cells. However, scRNA-seq data has characteristics such as drop-out events and low library sizes. It is thus not clear if functional TF and pathway analysis tools established for bulk sequencing can be applied to scRNA-seq in a meaningful way.
Results: To address this question, we perform benchmark studies on simulated and real scRNA-seq data. We include the bulk-RNA tools PROGENy, GO enrichment, and DoRothEA that estimate pathway and transcription factor (TF) activities, respectively, and compare them against the tools SCENIC/AUCell and metaVIPER, designed for scRNA-seq. For the in silico study, we simulate single cells from TF/pathway perturbation bulk RNA-seq experiments. We complement the simulated data with real scRNA-seq data upon CRISPR-mediated knock-out. Our benchmarks on simulated and real data reveal comparable performance to the original bulk data. Additionally, we show that the TF and pathway activities preserve cell type-specific variability by analyzing a mixture sample sequenced with 13 scRNA-seq protocols. We also provide the benchmark data for further use by the community.
Conclusions: Our analyses suggest that bulk-based functional analysis tools that use manually curated footprint gene sets can be applied to scRNA-seq data, partially outperforming dedicated single-cell tools. Furthermore, we find that the performance of functional analysis tools is more sensitive to the gene sets than to the statistic used.
Mereu E, Lafzi A, Moutinho C, Ziegenhain C, MacCarthy DJ, Alvarez A, Batlle E, Sagar, Grün D, Lau JK, Boutet SC, Sanada C, Ooi A, Jones RC, Kaihara K, Brampton C, Talaga Y, Sasagawa Y, Tanaka K, Hayashi T, Nikaido I, Fischer C, Sauer S, Trefzer T, Conrad C, Adiconis X, Nguyen LT, Regev A, Levin JZ, Parekh S, Janjic A, Wange LE, Bagnoli JW, Enard W, Gut M, Sandberg R, Gut I, Stegle O, Heyn H.
Benchmarking single-cell RNA- sequencing protocols for cell atlas projects.
Nat Biotechnol . 2020 Jun;38(6):747-755 , .
Single-cell RNA sequencing (scRNA-seq) is the leading technique for characterizing the transcriptomes of individual cells in a sample. The latest protocols are scalable to thousands of cells and are being used to compile cell atlases of tissues, organs and organisms. However, the protocols differ substantially with respect to their RNA capture efficiency, bias, scale and costs, and their relative advantages for different applications are unclear. In the present study, we generated benchmark datasets to systematically evaluate protocols in terms of their power to comprehensively describe cell types and states. We performed a multicenter study comparing 13 commonly used scRNA-seq and single-nucleus RNA-seq protocols applied to a heterogeneous reference sample resource. Comparative analysis revealed marked differences in protocol performance. The protocols differed in library complexity and their ability to detect cell-type markers, impacting their predictive value and suitability for integration into reference cell atlases. These results provide guidance both for individual researchers and for consortium projects such as the Human Cell Atlas.
Morral C, Stanisavljevic J, Hernando-Momblona X, Mereu E, Alvarez-Varela A, Cortina C., Stork D, Slebe F, Turon G, Whissell G, Sevillano M, Merlos-Suarez A, Casanova-Martì A, Moutinho C, W. Lowe S, E. Dow L, Villanueva A, Sancho E, Heyn H, Batlle E
Zonation of Ribosomal DNA Transcription Defines a Stem Cell Hierarchy in Colorectal Cancer.
Cell Stem Cell. 2020 Jun 4;26(6):845-861.e12 , .
Colorectal cancers (CRCs) are composed of an amalgam of cells with distinct genotypes and phenotypes. Here, we reveal a previously unappreciated heterogeneity in the biosynthetic capacities of CRC cells. We discover that the majority of ribosomal DNA transcription and protein synthesis in CRCs occurs in a limited subset of tumor cells that localize in defined niches. The rest of the tumor cells undergo an irreversible loss of their biosynthetic capacities as a consequence of differentiation. Cancer cells within the biosynthetic domains are characterized by elevated levels of the RNA polymerase I subunit A (POLR1A). Genetic ablation of POLR1A-high cell population imposes an irreversible growth arrest on CRCs. We show that elevated biosynthesis defines stemness in both LGR5+ and LGR5- tumor cells. Therefore, a common architecture in CRCs is a simple cell hierarchy based on the differential capacity to transcribe ribosomal DNA and synthesize proteins.
SPOTlight: Seeded NMF regression to Deconvolute Spatial Transcriptomics Spots with Single-Cell Transcriptomes.
Nucleic Acids Research . 2021 Feb 5 , .
The integration of orthogonal data modalities greatly supports the interpretation of transcriptomic landscapes in complex tissues. In particular, spatially resolved gene expression profiles are key to understand tissue organization and function. However, spatial transcriptomics (ST) profiling techniques lack single-cell resolution and require a combination with single-cell RNA sequencing (scRNA-seq) information to deconvolute the spatially indexed datasets. Leveraging the strengths of both data types, we developed SPOTlight, a computational tool that enables the integration of ST with scRNA-seq data to infer the location of cell types and states within a complex tissue. SPOTlight is centered around a seeded non-negative matrix factorization (NMF) regression, initialized using cell-type marker genes, and non-negative least squares (NNLS) to subsequently deconvolute ST capture locations (spots). Using synthetic spots, simulating varying reference quantities and qualities, we confirmed high prediction accuracy also with shallowly sequenced or small-sized scRNA-seq reference datasets. We trained the NMF regression model with sample-matched or external datasets, resulting in accurate and sensitive spatial predictions. SPOTlight deconvolution of the mouse brain correctly mapped subtle neuronal cell states of the cortical layers and the defined architecture of the hippocampus. In human pancreatic cancer, we successfully segmented patient sections into healthy and cancerous areas, and further fine-mapped normal and neoplastic cell states. Trained on an external pancreatic tumor immune reference, we charted the localization of clinical-relevant and tumor-specific immune cell states. Using SPOTlight to detect regional enrichment of immune cells and their co-localization with tumor and adjacent stroma provides an illustrative example in its flexible application spectrum and future potential in digital pathology.