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Most spatial transcriptomics technologies are limited by their resolution, with spot sizes larger than that of a single cell. Although joint analysis with single-cell RNA sequencing can alleviate this problem, current methods are limited to assessing discrete cell types, revealing the proportion of cell types inside each spot. To identify continuous variation of the transcriptome within cells of the same type, we developed Deconvolution of Spatial Transcriptomics profiles using Variational Inference (DestVI). Using simulations, we demonstrate that DestVI outperforms existing methods for estimating gene expression for every cell type inside every spot. Applied to a study of infected lymph nodes and of a mouse tumor model, DestVI provides high-resolution, accurate spatial characterization of the cellular organization of these tissues and identifies cell-type-specific changes in gene expression between different tissue regions or between conditions. DestVI is available as part of the open-source software package scvi-tools ( https://scvi-tools.org ).

The major cause of melanoma mortality is metastasis to distant organs, including lungs and brain. Reciprocal interactions of metastasizing tumor cells with stromal cells in secondary sites play a critical role in all stages of tumorigenesis and metastasis. Changes in the metastatic microenvironment were shown to precede clinically relevant metastases, and may occur prior to the arrival of disseminated tumor cells to the distant organ, thus creating a hospitable "premetastatic niche." Exosomes secreted by tumor cells were demonstrated to play an important role in the preparation of a hospitable metastatic niche. However, the functional role of melanoma-derived exosomes on metastatic niche formation, and the downstream pathways activated in stromal cells at the metastatic niche are largely unresolved. Here we show that extracellular vesicles (EVs) secreted by metastatic melanoma cells that spontaneously metastasize to lungs and to brain, activate proinflammatory signaling in lung fibroblasts and in astrocytes. Interestingly, unlike paracrine signaling by melanoma cells, EVs secreted by metastatic melanoma cells instigated a proinflammatory gene signature in lung fibroblasts but did not activate wound-healing functions, suggesting that tumor cell-secreted EVs activate distinct CAF characteristics and tumor-promoting functions. Moreover, melanoma-secreted EVs also activated proinflammatory signaling in astrocytes, indicating that EV-mediated reprogramming of stromal cells is a general mechanism of modulating the metastatic niche in multiple distant organs. Thus, our study demonstrates that melanoma-derived EVs reprogram tumor-promoting functions in stromal cells in a distinct manner, implicating a central role for tumor-derived EV signaling in promoting the formation of an inflammatory metastatic niche.

International audience; Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are prevalent liver conditions that underlie the development of life-threatening cirrhosis, liver failure and liver cancer. Chronic necro-inflammation is a critical factor in development of NASH, yet the cellular and molecular mechanisms of immune dysregulation in this disease are poorly understood. Here, using single-cell transcriptomic analysis, we comprehensively profiled the immune composition of the mouse liver during NASH. We identified a significant pathology-associated increase in hepatic conventional dendritic cells (cDCs) and further defined their source as NASH-induced boost in cycling of cDC progenitors in the bone marrow. Analysis of blood and liver from patients on the NAFLD/NASH spectrum showed that type 1 cDCs (cDC1) were more abundant and activated in disease. Sequencing of physically interacting cDC-T cell pairs from liver-draining lymph nodes revealed that cDCs in NASH promote inflammatory T cell reprogramming, previously associated with NASH worsening. Finally, depletion of cDC1 in XCR1DTA mice or using anti-XCL1-blocking antibody attenuated liver pathology in NASH mouse models. Overall, our study provides a comprehensive characterization of cDC biology in NASH and identifies XCR1+ cDC1 as an important driver of liver pathology.

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are prevalent liver conditions that underlie the development of life-threatening cirrhosis, liver failure and liver cancer. Chronic necro-inflammation is a critical factor in development of NASH, yet the cellular and molecular mechanisms of immune dysregulation in this disease are poorly understood. Here, using single-cell transcriptomic analysis, we comprehensively profiled the immune composition of the mouse liver during NASH. We identified a significant pathology-associated increase in hepatic conventional dendritic cells (cDCs) and further defined their source as NASH-induced boost in cycling of cDC progenitors in the bone marrow. Analysis of blood and liver from patients on the NAFLD/NASH spectrum showed that type 1 cDCs (cDC1) were more abundant and activated in disease. Sequencing of physically interacting cDC-T cell pairs from liver-draining lymph nodes revealed that cDCs in NASH promote inflammatory T cell reprogramming, previously associated with NASH worsening. Finally, depletion of cDC1 in XCR1

Recent progress in single-cell genomics urges its application in drug development, particularly of cancer immunotherapies. Current immunotherapy pipelines are focused on functional outcome and simple cellular and molecular readouts. A thorough mechanistic understanding of the cells and pathways targeted by immunotherapy agents is lacking, which limits the success rate of clinical trials. A large leap forward can be made if the immunotherapy target cells and pathways are characterized at high resolution before and after treatment, in clinical cohorts and model systems. This will enable rapid development of effective immunotherapies and data-driven design of synergistic drug combinations. In this Perspective, we discuss how emerging single-cell genomic technologies can serve as an engine for target identification and drug development. Amit and colleagues discuss where single-cell genomic technologies can be applied both in trial design and in the clinical trial stage to improve the development of immunotherapies.

Splicing expands, reshapes, and regulates the transcriptome of eukaryotic organisms. Despite its importance, key questions remain unanswered, including the following: Can splicing evolve when organisms adapt to new challenges? How does evolution optimize inefficiency of introns’ splicing and of the splicing machinery? To explore these questions, we evolved yeast cells that were engineered to contain an inefficiently spliced intron inside a gene whose protein product was under selection for an increased expression level. We identified a combination of mutations in Cis (within the gene of interest) and in Trans (in mRNA-maturation machinery). Surprisingly, the mutations in Cis resided outside of known intronic functional sites and improved the intron’s splicing efficiency potentially by easing tight mRNA structures. One of these mutations hampered a protein’s domain that was not under selection, demonstrating the evolutionary flexibility of multi-domain proteins as one domain functionality was improved at the expense of the other domain. The Trans adaptations resided in two proteins, Npl3 and Gbp2, that bind pre-mRNAs and are central to their maturation. Interestingly, these mutations either increased or decreased the affinity of these proteins to mRNA, presumably allowing faster spliceosome recruitment or increased time before degradation of the pre-mRNAs, respectively. Altogether, our work reveals various mechanistic pathways toward optimizations of intron splicing to ultimately adapt gene expression patterns to novel demands., An experimental evolution study involving an inefficiently spliced intron reveals that the splicing machinery, introns, and RNA quality control factors evolve in Cis and in Trans when cells optimize their transcriptome to new challenges.

Tumor immune cell compositions play a major role in response to immunotherapy, but the heterogeneity and dynamics of immune infiltrates in human cancer lesions remain poorly characterized. Here, we identify conserved intratumoral CD4 and CD8 T cell behaviors in scRNA-seq data from 25 melanoma patients. We discover a large population of CD8 T cells showing continuous progression from an early effector "transitional" into a dysfunctional T cell state. CD8 T cells that express a complete cytotoxic gene set are rare, and TCR sharing data suggest their independence from the transitional and dysfunctional cell states. Notably, we demonstrate that dysfunctional T cells are the major intratumoral proliferating immune cell compartment and that the intensity of the dysfunctional signature is associated with tumor reactivity. Our data demonstrate that CD8 T cells previously defined as exhausted are in fact a highly proliferating, clonal, and dynamically differentiating cell population within the human tumor microenvironment.

The yeast Saccharomyces cerevisiae is ideal for systematic studies relying on collections of modified strains (libraries). Despite the significance of yeast libraries and the immense variety of available tags and regulatory elements, only a few such libraries exist as their construction is extremely expensive and laborious. To overcome these limitations we developed a SWAp-Tag method (SWAT), in which one parental library can be modified easily and efficiently to give rise to an endless variety of libraries of choice. We showcase the versatility of the SWAT approach by constructing and investigating a library of ~1,800 strains carrying a SWAT-GFP module at the amino termini of endomembrane proteins and then using it to create two new libraries (mCherry or seamless GFP). Our work demonstrates how the SWAT method enables fast and effortless creation of yeast libraries, opening the door for endless new ways to systematically study cell biology.

The dynamics of haematopoietic stem cell differentiation and the hierarchy of oligopotent stem cells in the bone marrow remain controversial. Here we dissect haematopoietic progenitor populations at single cell resolution, deriving an unbiased reference model of transcriptional states in normal and perturbed murine bone marrow. We define the signature of the naive haematopoietic stem cell and find a continuum of core progenitor states. Core cell populations mix transcription of pre-myeloid and pre-lymphoid programs, but do not mix erythroid or megakaryocyte programs with other fates. CRISP-seq perturbation analysis confirms our models and reveals that Cebpa regulates entry into all myeloid fates, while Irf8 and PU.1 deficiency block later differentiation towards monocyte or granulocyte fates. Our transcriptional map defines a reference network model for blood progenitors and their differentiation trajectories during normal and perturbed haematopoiesis. Using a multi-tier scRNA-seq and CRISP-seq approach, Giladi et al. define a transcriptional signature for the naive haematopoietic stem cell state, and follow progenitor plasticity and fate commitment under the influence of cytokines and growth factors.

Can splicing be used by cells to adapt to new environmental challenges? While various adaptation mechanisms for regulating gene expression have been revealed for transcription and translation, the role of splicing and how it evolves to optimize gene-expression patterns has not been thoroughly investigated. To tackle this question, we employed a lab-evolution experimental approach that challenged yeast cells to increase expression levels of a gene that carries an inefficiently-spliced intron. We followed the evolution of multiple lines and found independent routes by which cells adapted. Surprisingly, we did not observe an intron loss event, a mechanism believed to be common in intron evolution. Instead, we identified mutations in cis that improved the intron’s splicing efficiency and increased the overall expression level of the entire gene. One of these cis-acting mutations occurred in an adjacent exon and hampered the functionality of the gene that was not under selection - demonstrating that adaptation of splicing efficiency may sometimes come at the expense of protein activity. Additionally, we observed adaptations in trans, which increased the cellular availability of the splicing machinery. These adaptations were achieved either by elevated expression levels of the splicing apparatus or, unexpectedly, by reduced expression levels of other intron-containing genes that are the natural consumers of this process. Ultimately, our work reveals novel molecular means by which the splicing machinery is changed by natural selection to optimize gene-expression patterns of cells.

Peroxisomes are cellular organelles with vital functions in lipid, amino acid and redox metabolism. The cellular formation and dynamics of peroxisomes are governed by PEX genes; however, the regulation of peroxisome abundance is still poorly understood. Here, we use a high-content microscopy screen in Saccharomyces cerevisiae to identify new regulators of peroxisome size and abundance. Our screen led to the identification of a previously uncharacterized gene, which we term PEX35, which affects peroxisome abundance. PEX35 encodes a peroxisomal membrane protein, a remote homolog to several curvature-generating human proteins. We systematically characterized the genetic and physical interactome as well as the metabolome of mutants in PEX35, and we found that Pex35 functionally interacts with the vesicle-budding-inducer Arf1. Our results highlight the functional interaction between peroxisomes and the secretory pathway.

In multicellular organisms, dedicated regulatory circuits control cell type diversity and responses. The crosstalk and redundancies within these circuits and substantial cellular heterogeneity pose a major research challenge. Here, we present CRISP-seq, an integrated method for massively parallel single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-pooled screens. We show that profiling the genomic perturbation and transcriptome in the same cell enables us to simultaneously elucidate the function of multiple factors and their interactions. We applied CRISP-seq to probe regulatory circuits of innate immunity. By sampling tens of thousands of perturbed cells in vitro and in mice, we identified interactions and redundancies between developmental and signaling-dependent factors. These include opposing effects of Cebpb and Irf8 in regulating the monocyte/macrophage versus dendritic cell lineages and differential functions for Rela and Stat1/2 in monocyte versus dendritic cell responses to pathogens. This study establishes CRISP-seq as a broadly applicable, comprehensive, and unbiased approach for elucidating mammalian regulatory circuits.

BackgroundThe robustness of ChIP-seq datasets is highly dependent upon the antibodies used. Currently, polyclonal antibodies are the standard despite several limitations: they are non-renewable, vary in performance between lots, and need to be validated with each new lot. In contrast, monoclonal antibody lots are renewable and provide consistent performance. To increase ChIP-seq standardization, we investigated whether monoclonal antibodies could replace polyclonal antibodies. We compared monoclonal antibodies that target five key histone modifications (H3K4me1, H3K4me3, H3K9me3, H3K27ac and H3K27me3) to their polyclonal counterparts in both human and mouse cells.ResultsOverall performance was highly similar for four monoclonal/polyclonal pairs, including when we used two distinct lots of the same monoclonal antibody. In contrast, the binding patterns for H3K27ac differed substantially between polyclonal and monoclonal antibodies. However, this was most likely due to the distinct immunogen used rather than the clonality of the antibody.ConclusionsAltogether, we found that monoclonal antibodies as a class perform as well as polyclonal antibodies for the detection of histone post-translational modifications in both human and mouse. Accordingly, we recommend the use of monoclonal antibodies in ChIP-seq experiments.

To optimally perform the diversity of metabolic functions that occur within peroxisomes, cells must dynamically regulate peroxisome size, number and content in response to the cell state and the environment. Except for transcriptional regulation little is known about the mechanisms used to perform this complicated feat. Focusing on the yeast Saccharomyces cerevisiae, we used complementary high-content screens to follow changes in localization of most proteins during growth in oleate. We found extensive changes in cellular architecture and identified several proteins that colocalized with peroxisomes that had not previously been considered peroxisomal proteins. One of the newly identified peroxisomal proteins, Ymr018w, is a protein with an unknown function that is similar to the yeast and human peroxisomal targeting receptor Pex5. We demonstrate that Ymr018w is a new peroxisomal-targeting receptor that targets a subset of matrix proteins to peroxisomes. We, therefore, renamed Ymr018w, Pex9, and suggest that Pex9 is a condition-specific targeting receptor that enables the dynamic rewiring of peroxisomes in response to metabolic needs. Moreover, we suggest that Pex5-like receptors might also exist in vertebrates.

Regulation of the localization of mRNAs and local translation are universal features in eukaryotes and contribute to cellular asymmetry and differentiation. In Saccharomyces cerevisiae, localization of mRNAs that encode membrane proteins requires the She protein machinery, including the RNA-binding protein She2p, as well as movement of the cortical endoplasmic reticulum (cER) to the yeast bud. In a screen for ER-specific proteins necessary for the directional transport of WSC2 and EAR1 mRNAs, we have identified enzymes that are involved in phospholipid metabolism. Loss of the phospholipid methyltransferase Cho2p, which showed the strongest impact on mRNA localization, disturbs mRNA localization, as well as ER morphology and segregation, owing to an increase in the amount of cellular phosphatidylethanolamine (PtdEtn). Mislocalized mRNPs containing She2p colocalize with aggregated cER structures, suggestive of the entrapment of mRNA and She2p by the elevated PtdEtn level. This was confirmed by the elevated binding of She2p to PtdEtn-containing liposomes. These findings underscore the importance of ER membrane integrity in mRNA transport.

Protein levels are a dominant factor shaping natural and synthetic biological systems. Although proper functioning of metabolic pathways relies on precise control of enzyme levels, the experimental ability to balance the levels of many genes in parallel is a major outstanding challenge. Here, we introduce a rapid and modular method to span the expression space of several proteins in parallel. By combinatorially pairing genes with a compact set of ribosome-binding sites, we modulate protein abundance by several orders of magnitude. We demonstrate our strategy by using a synthetic operon containing fluorescent proteins to span a 3D color space. Using the same approach, we modulate a recombinant carotenoid biosynthesis pathway in Escherichia coli to reveal a diversity of phenotypes, each characterized by a distinct carotenoid accumulation profile. In a single combinatorial assembly, we achieve a yield of the industrially valuable compound astaxanthin 4-fold higher than previously reported. The methodology presented here provides an efficient tool for exploring a high-dimensional expression space to locate desirable phenotypes.

Multiple myeloma, a plasma cell malignancy, is the second most common blood cancer. Despite extensive research, disease heterogeneity is poorly characterized, hampering efforts for early diagnosis and improved treatments. Here, we apply single cell RNA sequencing to study the heterogeneity of 40 individuals along the multiple myeloma progression spectrum, including 11 healthy controls, demonstrating high interindividual variability that can be explained by expression of known multiple myeloma drivers and additional putative factors. We identify extensive subclonal structures for 10 of 29 individuals with multiple myeloma. In asymptomatic individuals with early disease and in those with minimal residual disease post-treatment, we detect rare tumor plasma cells with molecular characteristics similar to those of active myeloma, with possible implications for personalized therapies. Single cell analysis of rare circulating tumor cells allows for accurate liquid biopsy and detection of malignant plasma cells, which reflect bone marrow disease. Our work establishes single cell RNA sequencing for dissecting blood malignancies and devising detailed molecular characterization of tumor cells in symptomatic and asymptomatic patients. Single cell dissection of plasma cell heterogeneity in myeloma patients reveals new insights into disease that may inform early diagnosis and clinical management.

To optimally perform the diversity of metabolic functions that occur within peroxisomes, cells must dynamically regulate peroxisome size, number and content in response to cell state and the environment. Except for transcriptional regulation little is known about the mechanisms used to perform this complicated fete. Here we used complementary high content screens to follow changes in most of the yeast, Saccharomyces cerevisiae, proteins during growth in oleate. We found extensive changes in cellular architecture and identified several proteins co-localizing with peroxisomes that have not previously been considered peroxisomal proteins. One of the newly identified peroxisomal proteins, Ymr018w, is a protein with an unknown function that is similar to the yeast and human peroxisomal targeting receptor Pex5. We demonstrate that Ymr018w is a novel peroxisomal targeting receptor that targets a subset of matrix proteins to peroxisomes. We have therefore renamed Ymr018w, Pex9, and suggest that Pex9 is a condition-specific targeting receptor that enables the dynamic rewiring of peroxisomes in response to metabolic needs. Moreover, we suggest that Pex5-like receptors may also exist in vertebrates.

Summary Cell function and activity are regulated through integration of signaling, epigenetic, transcriptional, and metabolic pathways. Here, we introduce INs-seq, an integrated technology for massively parallel recording of single-cell RNA sequencing (scRNA-seq) and intracellular protein activity. We demonstrate the broad utility of INs-seq for discovering new immune subsets by profiling different intracellular signatures of immune signaling, transcription factor combinations, and metabolic activity. Comprehensive mapping of Arginase 1-expressing cells within tumor models, a metabolic immune signature of suppressive activity, discovers novel Arg1+ Trem2+ regulatory myeloid (Mreg) cells and identifies markers, metabolic activity, and pathways associated with these cells. Genetic ablation of Trem2 in mice inhibits accumulation of intra-tumoral Mreg cells, leading to a marked decrease in dysfunctional CD8+ T cells and reduced tumor growth. This study establishes INs-seq as a broadly applicable technology for elucidating integrated transcriptional and intra-cellular maps and identifies the molecular signature of myeloid suppressive cells in tumors.