Rapolas Zilionis

Single-cell barcoding and sequencing using droplet microfluidics

Single-cell RNA sequencing has recently emerged as a powerful tool for mapping cellular heterogeneity in diseased and healthy tissues, yet high-throughput methods are needed for capturing the unbiased diversity of cells. Droplet microfluidics is among the most promising candidates for capturing and processing thousands of individual cells for whole-transcriptome or genomic analysis in a massively parallel manner with minimal reagent use. We recently established a method called inDrops, which has the capability to index >15,000 cells in an hour. A suspension of cells is first encapsulated into nanoliter droplets with hydrogel beads (HBs) bearing barcoding DNA primers. Cells are then lysed and mRNA is barcoded (indexed) by a reverse transcription (RT) reaction. Here we provide details for (i) establishing an inDrops platform (1 d); (ii) performing hydrogel bead synthesis (4 d); (iii) encapsulating and barcoding cells (1 d); and (iv) RNA-seq library preparation (2 d). inDrops is a robust and scalable platform, and it is unique in its ability to capture and profile >75% of cells in even very small samples, on a scale of thousands or tens of thousands of cells.

Osteoblasts remotely supply lung tumors with cancer-promoting SiglecFhigh neutrophils

A bona fide portrayal of tumor growth Bone has a well-established role in advanced cancer. It provides a supportive microenvironment for the growth of metastatic cells that escape the primary tumor, which ultimately leads to loss of bone mass. Engblom et al. show that bone may also contribute to early-stage tumorigenesis through a mechanism that leads to an increase in bone mass (see the Perspective by Zhang and Lyden). In mouse models of lung adenocarcinoma, primary tumor cells remotely activated bone-resident cells called osteoblasts, which have a bone-building function. The activated osteoblasts in turn triggered production of a certain type of neutrophil that infiltrates the primary tumor and promotes its growth. Patients with early-stage lung cancer were also found to have an increase in bone density, consistent with the findings in mice. Science, this issue p. eaal5081; see also p. 1127 Systemic cross-talk between tumor and bone can boost the growth of early-stage lung cancer in mice. INTRODUCTION Myeloid cells have emerged as key regulators of cancer growth because of their abundance in the tumor stroma in a broad range of cancers, their association with clinical outcome, and their ability to modulate tumor progression. Most tumor-infiltrating myeloid cells derive from circulating precursors, which are produced in distant tissues, and some tumors amplify myeloid cell activity by skewing hematopoiesis toward the myeloid lineage or increasing myeloid cell populations in the periphery. For example, patients across diverse cancer types can present with elevated levels of myeloid progenitor cells in peripheral blood. Additionally, increased numbers of circulating myeloid cells, such as neutrophils, often correlate with poorer clinical outcome. It is therefore important to consider host changes that occur away from the tumor stroma to more fully understand the biological processes underlying tumor growth. RATIONALE The bone marrow is a tissue of particular interest as it is the main production site for hematopoietic cells corresponding to all circulating blood lineages in the adult. The marrow contains resident cell components, such as osteoblasts, which not only participate in bone maintenance but also regulate hematopoiesis and immune cell fate. However, our understanding of bone dynamics in the context of cancer (growing at sites distant from the local bone microenvironment) and related immune responses remains limited. To address this knowledge gap, we explored whether a common solid cancer—lung adenocarcinoma—remotely affects bone tissue and how this might shape tumor-associated hematopoietic responses and tumor growth. RESULTS We show in different mouse models and in cancer patients (n = 70) that lung adenocarcinomas increase bone stromal activity even in the absence of local metastasis. Animal studies further reveal that the cancer-induced bone phenotype involves bone-resident osteocalcin-expressing (Ocn+) osteoblastic cells. Ocn+ cells affect distant tumor progression because experimentally reducing the number of these cells limits lung tumor growth. Also, Ocn+ cells are required for full-fledged tumor infiltration by a distinct subset of neutrophils that are defined by their high expression of the lectin SiglecF (sialic acid–binding immunoglobulin-like lectin F). Compared to other neutrophils, SiglecFhigh cells express genes associated with cancer-promoting processes, including angiogenesis, myeloid cell differentiation and recruitment, extracellular matrix remodeling, suppression of T cell responses, and tumor cell proliferation and growth. Additionally, SiglecFhigh neutrophils have increased reactive oxygen species production, promote macrophage differentiation, and boost tumor progression in vivo. We further report that the soluble receptor for advanced glycation end products (sRAGE) is up-regulated in the circulation of tumor-bearing mice and fosters osteoblastic activity and osteoblast-dependent neutrophil maturation in vitro. CONCLUSION This study identifies systemic cross-talk between lung tumors and bones: Lung tumors can remotely activate Ocn+ osteoblastic cells in bones even in the absence of local metastasis. In turn, these Ocn+ cells supply tumors with SiglecFhigh neutrophils, which foster cancer progression. The findings bear scientific and therapeutic importance because they reveal contributions of the host systemic environment to tumor growth and they position Ocn+ cells, SiglecFhigh neutrophils, and sRAGE as candidate clinical biomarkers and possible intervention points for anticancer therapy. Systemic cross-talk between lung tumors and bones. Lung adenocarcinomas can remotely activate Ocn+ osteoblastic cells in bones even in the absence of local metastasis. In turn, these osteoblasts supply tumors with SiglecFhigh neutrophils, which exhibit cancer-promoting functions (left). By contrast, the bone marrow in steady state only produces SiglecFlow neutrophils (right). Bone marrow–derived myeloid cells can accumulate within tumors and foster cancer outgrowth. Local immune-neoplastic interactions have been intensively investigated, but the contribution of the systemic host environment to tumor growth remains poorly understood. Here, we show in mice and cancer patients (n = 70) that lung adenocarcinomas increase bone stromal activity in the absence of bone metastasis. Animal studies reveal that the cancer-induced bone phenotype involves bone-resident osteocalcin-expressing (Ocn+) osteoblastic cells. These cells promote cancer by remotely supplying a distinct subset of tumor-infiltrating SiglecFhigh neutrophils, which exhibit cancer-promoting properties. Experimentally reducing Ocn+ cell numbers suppresses the neutrophil response and lung tumor outgrowth. These observations posit osteoblasts as remote regulators of lung cancer and identify SiglecFhigh neutrophils as myeloid cell effectors of the osteoblast-driven protumoral response.

Population snapshots predict early haematopoietic and erythroid hierarchies

The formation of red blood cells begins with the differentiation of multipotent haematopoietic progenitors. Reconstructing the steps of this differentiation represents a general challenge in stem-cell biology. Here we used single-cell transcriptomics, fate assays and a theory that allows the prediction of cell fates from population snapshots to demonstrate that mouse haematopoietic progenitors differentiate through a continuous, hierarchical structure into seven blood lineages. We uncovered coupling between the erythroid and the basophil or mast cell fates, a global haematopoietic response to erythroid stress and novel growth factor receptors that regulate erythropoiesis. We defined a flow cytometry sorting strategy to purify early stages of erythroid differentiation, completely isolating classically defined burst-forming and colony-forming progenitors. We also found that the cell cycle is progressively remodelled during erythroid development and during a sharp transcriptional switch that ends the colony-forming progenitor stage and activates terminal differentiation. Our work showcases the utility of linking transcriptomic data to predictive fate models, and provides insights into lineage development in vivo.

Single-cell gene expression reveals a landscape of regulatory T cell phenotypes shaped by the TCR

CD4+ T regulatory cells (Treg) are central to immune homeostasis, their phenotypic heterogeneity reflecting the diverse environments and target cells that they regulate. To understand this heterogeneity, we combined single-cell RNA-seq, activation reporter and T cell receptor (TCR) analysis to profile thousands of Treg or conventional CD4+FoxP3– T cells (Tconv) from mouse lymphoid organs and human blood. Treg and Tconv pools showed areas of overlap, as resting ‘furtive’ Tregs with overall similarity to Tconvs or as a convergence of activated states. All Tregs expressed a small core of FoxP3-dependent transcripts, onto which additional programs were added less uniformly. Among suppressive functions, Il2ra and Ctla4 were quasiconstant, inhibitory cytokines being more sparsely distributed. TCR signal intensity did not affect resting/activated Treg proportions but molded activated Treg programs. The main lines of Treg heterogeneity in mice were strikingly conserved in human blood. These results reveal unexpected TCR-shaped states of activation, providing a framework to synthesize previous observations of Treg heterogeneity.Regulatory T (Treg) cells have distinct transcriptional programs underpinning their suppressive functions. Benoist and colleagues use single-cell RNA-seq to describe the transcriptional landscape of Treg cells and the effects of T cell–receptor signaling.

DNA Nanoparticles for Improved Protein Synthesis In Vitro.

Abstract The amplification and digital quantification of single DNA molecules are important in biomedicine and diagnostics. Beyond quantifying DNA molecules in a sample, the ability to express proteins from the amplified DNA would open even broader applications in synthetic biology, directed evolution, and proteomics. Herein, a microfluidic approach is reported for the production of condensed DNA nanoparticles that can serve as efficient templates for in vitro protein synthesis. Using phi29 DNA polymerase and a multiple displacement amplification reaction, single DNA molecules were converted into DNA nanoparticles containing up to about 104 clonal gene copies of the starting template. DNA nanoparticle formation was triggered by accumulation of inorganic pyrophosphate (produced during DNA synthesis) and magnesium ions from the buffer. Transcription–translation reactions performed in vitro showed that individual DNA nanoparticles can serve as efficient templates for protein synthesis in vitro.

Emergence of the erythroid lineage from multipotent hematopoiesis

Red cell formation begins with the hematopoietic stem cell, but the manner by which it gives rise to erythroid progenitors, and their subsequent developmental path, remain unclear. Here we combined single-cell transcriptomics of murine hematopoietic tissues with fate potential assays to infer a continuous yet hierarchical structure for the hematopoietic network. We define the erythroid differentiation trajectory as it emerges from multipotency and diverges from 6 other blood lineages. With the aid of a new flow-cytometric sorting strategy, we validated predicted cell fate potentials at the single cell level, revealing a coupling between erythroid and basophil/mast cell fates. We uncovered novel growth factor receptor regulators of the erythroid trajectory, including the proinflammatory IL-17RA, found to be a strong erythroid stimulator; and identified a global hematopoietic response to stress erythropoiesis. We further identified transcriptional and high-purity FACS gates for the complete isolation of all classically-defined erythroid burst-forming (BFU-e) and colony-forming progenitors (CFU-e), finding that they express a dedicated transcriptional program, distinct from that of terminally-differentiating erythroblasts. Intriguingly, profound remodeling of the cell cycle is intimately entwined with CFU-e developmental progression and with a sharp transcriptional switch that extinguishes the CFU-e stage and activates terminal differentiation. Underlying these results, our work showcases the utility of theoretic approaches linking transcriptomic data to predictive fate models, providing key insights into lineage development in vivo.

Molecular diversification of regulatory T cells in nonlymphoid tissues

Different tissue microenvironments differentially refine Treg cell transcriptional regulatory modules already primed in lymphoid organs. Mapping Treg regulomes Technological advances are allowing immunologists to study rare populations of immune cells that take residence in various tissues including adipose tissue, skin, and the lung. Here, DiSpirito et al. have generated transcriptomes and chromatin accessibility maps of mouse regulatory T cells (Tregs) that reside in visceral adipose tissue, muscle, and the colon and compared them with the profiles generated from splenic Tregs. They have used these data sets to define transcriptional networks that are shared by all these populations and to identify networks that are unique to one or more tissue-resident Treg populations. Foxp3+CD4+ regulatory T cells (Tregs) accumulate in certain nonlymphoid tissues, where they control diverse aspects of organ homeostasis. Populations of tissue Tregs, as they have been termed, have transcriptomes distinct from those of their counterparts in lymphoid organs and other nonlymphoid tissues. We examined the diversification of Tregs in visceral adipose tissue, skeletal muscle, and the colon vis-à-vis lymphoid organs from the same individuals. The unique transcriptomes of the various tissue Treg populations resulted from layering of tissue-restricted open chromatin regions over regions already open in the spleen, the latter tagged by super-enhancers and particular histone marks. The binding motifs for a small number of transcription factor (TF) families were repeatedly enriched within the accessible chromatin stretches of Tregs in the three nonlymphoid tissues. However, a bioinformatically and experimentally validated transcriptional network, constructed by integrating chromatin accessibility and single-cell transcriptomic data, predicted reliance on different TF family members in the different tissues. The network analysis also revealed that tissue-restricted and broadly acting TFs were integrated into feed-forward loops to enforce tissue-specific gene expression in nonlymphoid-tissue Tregs. Overall, this study provides a framework for understanding the epigenetic dynamics of T cells operating in nonlymphoid tissues, which should inform strategies for specifically targeting them.

Publisher Correction: Single-cell analysis of experience-dependent transcriptomic states in the mouse visual cortex

In the version of this article initially published, the x-axis labels in Fig. 3c read Vglut, Gad1/2, Aldh1l1 and Pecam1; they should have read Vglut+, Gad1/2+, Aldh1l1+ and Pecam1+. In Fig. 4, the range values were missing from the color scales; they are, from left to right, 4–15, 0–15, 4–15 and 0–15 in Fig. 4a and 4–15, 4–15 and 4–8 in Fig. 4h. In the third paragraph of the main text, the phrase reading “Previous approaches have analyzed a limited number of inhibitory cell types, thus masking the full diversity of excitatory populations” should have read “Previous approaches have analyzed a limited number of inhibitory cell types and masked the full diversity of excitatory populations.” In the second paragraph of Results section “Diversity of experience-regulated ERGs,” the phrase reading “thus suggesting considerable divergence within the gene expression program responding to early stimuli” should have read “thus suggesting considerable divergence within the early stimulus-responsive gene expression program.” In the fourth paragraph of Results section “Excitatory neuronal LRGs,” the sentence reading “The anatomical organization of these cell types into sublayers, coupled with divergent transcriptional responses to a sensory stimulus, suggested previously unappreciated functional subdivisions located within the laminae of the mouse visual cortex and resembling the cytoarchitecture in higher mammals” should have read “The anatomical organization of these cell types into sublayers, coupled with divergent transcriptional responses to a sensory stimulus, suggests previously unappreciated functional subdivisions located within the laminae of the mouse visual cortex, resembling the cytoarchitecture in higher mammals.” In the last sentence of the Results, “sensory-responsive genes” should have read “sensory-stimulus-responsive genes.” The errors have been corrected in the HTML and PDF versions of the article.

Publisher Correction: Single-cell gene expression reveals a landscape of regulatory T cell phenotypes shaped by the TCR

In the version of this article initially published, the Supplementary Note was missing. The Supplementary Note has now been provided online and is cited in the Methods section of the article. The error has been corrected in the HTML and PDF version of the article.