Single cell dissection of plasma cell heterogeneity in symptomatic and asymptomatic myeloma

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

Nuclear Retention of mRNA in Mammalian Tissues

mRNA is thought to predominantly reside in the cytoplasm, where it is translated and eventually degraded. Although nuclear retention of mRNA has a regulatory potential, it is considered extremely rare in mammals. Here, to explore the extent of mRNA retention in metabolic tissues, we combine deep sequencing of nuclear and cytoplasmic RNA fractions with single-molecule transcript imaging in mouse beta cells, liver, and gut. We identify a wide range of protein-coding genes for which the levels of spliced polyadenylated mRNA are higher in the nucleus than in the cytoplasm. These include genes such as the transcription factor ChREBP, Nlrp6, Glucokinase, and Glucagon receptor. We demonstrate that nuclear retention of mRNA can efficiently buffer cytoplasmic transcript levels from noise that emanates from transcriptional bursts. Our study challenges the view that transcripts predominantly reside in the cytoplasm and reveals a role of the nucleus in dampening gene expression noise

Spatial sorting enables comprehensive characterization of liver zonation

The mammalian liver is composed of repeating hexagonal units termed lobules. Spatially resolved single-cell transcriptomics revealed that about half of hepatocyte genes are differentially expressed across the lobule, yet technical limitations impeded reconstructing similar global spatial maps of other hepatocyte features. Here, we show how zonated surface markers can be used to sort hepatocytes from defined lobule zones with high spatial resolution. We apply transcriptomics, miRNA array measurements and mass spectrometry proteomics to reconstruct spatial atlases of multiple zonated features. We demonstrate that protein zonation largely overlaps with mRNA zonation, with the periportal HNF4α as an exception. We identify zonation of miRNAs such as miR-122, and inverse zonation of miRNAs and their hepatocyte target genes, highlighting potential regulation of protein levels through zonated mRNA degradation. Among the targets we find the pericentral Wnt receptors Fzd7 and Fzd8 and the periportal Wnt inhibitors Tcf7l1 and Ctnnbip1. Our approach facilitates reconstructing spatial atlases of multiple cellular features in the liver and other structured tissues

Space-time logic of liver gene expression at sub-lobular scale

The mammalian liver is a central hub for systemic metabolic homeostasis. Liver tissue is spatially structured, with hepatocytes operating in repeating lobules, and sub-lobule zones performing distinct functions. The liver is also subject to extensive temporal regulation, orchestrated by the interplay of the circadian clock, systemic signals and feeding rhythms. However, liver zonation has previously been analysed as a static phenomenon, and liver chronobiology has been analysed at tissue-level resolution. Here, we use single-cell RNA-seq to investigate the interplay between gene regulation in space and time. Using mixed-effect models of messenger RNA expression and smFISH validations, we find that many genes in the liver are both zonated and rhythmic, and most of them show multiplicative space-time effects. Such dually regulated genes cover not only key hepatic functions such as lipid, carbohydrate and amino acid metabolism, but also previously unassociated processes involving protein chaperones. Our data also suggest that rhythmic and localized expression of Wnt targets could be explained by rhythmically expressed Wnt ligands from non-parenchymal cells near the central vein. Core circadian clock genes are expressed in a non-zonated manner, indicating that the liver clock is robust to zonation. Together, our scRNA-seq analysis reveals how liver function is compartmentalized spatio-temporally at the sub-lobular scale

A single cell atlas of the human liver tumor microenvironment

Abstract Malignant cell growth is fueled by interactions between tumor cells and the stromal cells composing the tumor microenvironment. The human liver is a major site of tumors and metastases, but molecular identities and intercellular interactions of different cell types have not been resolved in these pathologies. Here, we apply single cell RNA‐sequencing and spatial analysis of malignant and adjacent non‐malignant liver tissues from five patients with cholangiocarcinoma or liver metastases. We find that stromal cells exhibit recurring, patient‐independent expression programs, and reconstruct a ligand–receptor map that highlights recurring tumor–stroma interactions. By combining transcriptomics of laser‐capture microdissected regions, we reconstruct a zonation atlas of hepatocytes in the non‐malignant sites and characterize the spatial distribution of each cell type across the tumor microenvironment. Our analysis provides a resource for understanding human liver malignancies and may expose potential points of interventions

Lgr5+ telocytes are a signaling source at the intestinal villus tip

The intestinal epithelium is a structured organ composed of crypts harboring Lgr5+ stem cells, and villi harboring differentiated cells. Spatial transcriptomics have demonstrated profound zonation of epithelial gene expression along the villus axis, but the mechanisms shaping this spatial variability are unknown. Here, we combine laser capture micro-dissection and single cell RNA sequencing to uncover spatially zonated populations of mesenchymal cells along the crypt-villus axis. These include villus tip telocytes (VTTs) that express Lgr5, a gene previously considered a specific crypt epithelial stem cell marker. VTTs are elongated cells that line the villus tip epithelium and signal through Bmp morphogens and the non-canonical Wnt5a ligand. Their ablation is associated with perturbed zonation of enterocyte genes induced at the villus tip. Our study provides a spatially-resolved cell atlas of the small intestinal stroma and exposes Lgr5+ villus tip telocytes as regulators of the epithelial spatial expression programs along the villus axis

Distal Fecal Wash Host Transcriptomics Identifies Inflammation Throughout the Colon and Terminal IleumSummary

Background & Aims: Noninvasive modalities for assessing active endoscopic and histologic inflammation in Crohn’s disease and ulcerative colitis patients are critically needed. Fecal wash host shed-cell transcriptomics has been shown to be a robust classifier of endoscopic and histologic inflammation in inflammatory bowel disease patients with distal colitis. Whether such fecal washes can inform on inflammatory processes occurring in more proximal intestinal segments is currently unknown. Methods: Fifty-nine inflammatory bowel disease patients and 50 controls were prospectively enrolled. Biopsy specimens and fecal washes from the distal colon, proximal colon, and terminal ileum were compared. Host transcriptomics were performed on the biopsy specimens and fecal washes obtained during colonoscopy at predefined locations throughout the colon and terminal ileum and results were associated with concurrent clinical, endoscopic, and histologic parameters. Results: We found that host transcriptomics of distal fecal washes robustly classify histologic inflammation in ileal and proximal colonic Crohn’s disease, even without distal colonic involvement (area under the receiver operating characteristic curve, 0.94 ± 0.09). We further found that fecal washes consist of modules of co-expressed genes of immune, stromal, and epithelial origin that are indicative of endoscopic disease severity. Fecal wash host transcriptomics also captures expression of gene modules previously associated with a lack of response to biological therapies. Conclusions: Our study establishes the accuracy of distal colonic fecal washes for identifying and scoring inflammatory processes throughout the entire ileal–colonic axis

Single-Cell Analysis of Diverse Pathogen Responses Defines a Molecular Roadmap for Generating Antigen-Specific Immunity

International audienceThe immune system generates pathogen-tailored responses. The precise innate immune cell types and pathways that direct robust adaptive immune responses have not been fully characterized. By using fluorescent pathogens combined with massively parallel single-cell RNA-seq, we comprehensively characterized the initial 48 h of the innate immune response to diverse pathogens. We found that across all pathogens tested, most of the lymph node cell types and states showed little pathogen specificity. In contrast, the rare antigen-positive cells displayed pathogen-specific transcriptional programs as early as 24 h after immunization. In addition, mycobacteria activated a specific NK-driven IFNγ response. Depletion of NK cells and IFNγ showed that IFNγ initiated a monocyte-specific signaling cascade, leading to the production of major chemokines and cytokines that promote Th1 development. Our systems immunology approach sheds light on early events in innate immune responses and may help further development of safe and efficient vaccines