Patterning and folding of intestinal villi by active mesenchymal dewetting.

Tissue folds are structural motifs critical to organ function. In the intestine, bending of a flat epithelium into a periodic pattern of folds gives rise to villi, finger-like protrusions that enable nutrient absorption. However, the molecular and mechanical processes driving villus morphogenesis remain unclear. Here, we identify an active mechanical mechanism that simultaneously patterns and folds the intestinal epithelium to initiate villus formation. At the cellular level, we find that PDGFRA+ subepithelial mesenchymal cells generate myosin II-dependent forces sufficient to produce patterned curvature in neighboring tissue interfaces. This symmetry-breaking process requires altered cell and extracellular matrix interactions that are enabled by matrix metalloproteinase-mediated tissue fluidization. Computational models, together with in vitro and in vivo experiments, revealed that these cellular features manifest at the tissue level as differences in interfacial tensions that promote mesenchymal aggregation and interface bending through a process analogous to the active dewetting of a thin liquid film.

Lineage tracing reveals the phylodynamics, plasticity, and paths of tumor evolution.

Tumor evolution is driven by the progressive acquisition of genetic and epigenetic alterations that enable uncontrolled growth and expansion to neighboring and distal tissues. The study of phylogenetic relationships between cancer cells provides key insights into these processes. Here, we introduced an evolving lineage-tracing system with a single-cell RNA-seq readout into a mouse model of Kras;Trp53(KP)-driven lung adenocarcinoma and tracked tumor evolution from single-transformed cells to metastatic tumors at unprecedented resolution. We found that the loss of the initial, stable alveolar-type2-like state was accompanied by a transient increase in plasticity. This was followed by the adoption of distinct transcriptional programs that enable rapid expansion and, ultimately, clonal sweep of stable subclones capable of metastasizing. Finally, tumors develop through stereotypical evolutionary trajectories, and perturbing additional tumor suppressors accelerates progression by creating novel trajectories. Our study elucidates the hierarchical nature of tumor evolution and, more broadly, enables in-depth studies of tumor progression.

A DNA-Based T Cell Receptor Reveals a Role for Receptor Clustering in Ligand Discrimination.

A T cell mounts an immune response by measuring the binding strength of its T cell receptor (TCR) for peptide-loaded MHCs (pMHC) on an antigen-presenting cell. How T cells convert the lifetime of the extracellular TCR-pMHC interaction into an intracellular signal remains unknown. Here, we developed a synthetic signaling system in which the extracellular domains of the TCR and pMHC were replaced with short hybridizing strands of DNA. Remarkably, T cells can discriminate between DNA ligands differing by a single base pair. Single-molecule imaging reveals that signaling is initiated when single ligand-bound receptors are converted into clusters, a time-dependent process requiring ligands with longer bound times. A computation model reveals that receptor clustering serves a kinetic proofreading function, enabling ligands with longer bound times to have disproportionally greater signaling outputs. These results suggest that spatial reorganization of receptors plays an important role in ligand discrimination in T cell signaling.

A Mechanogenetic Toolkit for Interrogating Cell Signaling in Space and Time.

Tools capable of imaging and perturbing mechanical signaling pathways with fine spatiotemporal resolution have been elusive, despite their importance in diverse cellular processes. The challenge in developing a mechanogenetic toolkit (i.e., selective and quantitative activation of genetically encoded mechanoreceptors) stems from the fact that many mechanically activated processes are localized in space and time yet additionally require mechanical loading to become activated. To address this challenge, we synthesized magnetoplasmonic nanoparticles that can image, localize, and mechanically load targeted proteins with high spatiotemporal resolution. We demonstrate their utility by investigating the cell-surface activation of two mechanoreceptors: Notch and E-cadherin. By measuring cellular responses to a spectrum of spatial, chemical, temporal, and mechanical inputs at the single-molecule and single-cell levels, we reveal how spatial segregation and mechanical force cooperate to direct receptor activation dynamics. This generalizable technique can be used to control and understand diverse mechanosensitive processes in cell signaling. VIDEO ABSTRACT.

Loss of PPARγ activity characterizes early protumorigenic stromal reprogramming and dictates the therapeutic window of opportunity.

Although robustly expressed in the disease-free (DF) breast stroma, CD36 is consistently absent from the stroma surrounding invasive breast cancers (IBCs). In this study, we primarily observed CD36 expression in adipocytes and intralobular capillaries within the DF breast. Larger vessels concentrated in interlobular regions lacked CD36 and were instead marked by the expression of CD31. When evaluated in perilesional capillaries surrounding ductal carcinoma in situ, a nonobligate IBC precursor, CD36 loss was more commonly observed in lesions associated with subsequent IBC. Peroxisome proliferator-activated receptor γ (PPARγ) governs the expression of CD36 and genes involved in differentiation, metabolism, angiogenesis, and inflammation. Coincident with CD36 loss, we observed a dramatic suppression of PPARγ and its target genes in capillary endothelial cells (ECs) and pericytes, which typically surround and support the stability of the capillary endothelium. Factors present in conditioned media from malignant cells repressed PPARγ and its target genes not only in cultured ECs and pericytes but also in adipocytes, which require PPARγ for proper differentiation. In addition, we identified a role for PPARγ in opposing the transition of pericytes toward a tumor-supportive myofibroblast phenotype. In mouse xenograft models, early intervention with rosiglitazone, a PPARγ agonist, demonstrated significant antitumor effects; however, following the development of a palpable tumor, the antitumor effects of rosiglitazone were negated by the repression of PPARγ in the mouse stroma. In summary, PPARγ activity in healthy tissues places several stromal cell types in an antitumorigenic state, directly inhibiting EC proliferation, maintaining adipocyte differentiation, and suppressing the transition of pericytes into tumor-supportive myofibroblasts.

Bisphenol A replacement chemicals, BPF and BPS, induce protumorigenic changes in human mammary gland organoid morphology and proteome.

SignificanceBisphenol A (BPA), found in many plastic products, has weak estrogenic effects that can be harmful to human health. Thus, structurally related replacements-bisphenol S (BPS) and bisphenol F (BPF)-are coming into wider use with very few data about their biological activities. Here, we compared the effects of BPA, BPS, and BPF on human mammary organoids established from normal breast tissue. BPS disrupted organoid architecture and induced supernumerary branching. At a proteomic level, the bisphenols altered the abundance of common targets and those that were unique to each compound. The latter included proteins linked to tumor-promoting processes. These data highlighted the importance of testing the human health effects of replacements that are structurally related to chemicals of concern.

Single cell enhancer activity distinguishes GABAergic and cholinergic lineages in embryonic mouse basal ganglia.

Enhancers integrate transcription factor signaling pathways that drive cell fate specification in the developing brain. We paired enhancer labeling and single-cell RNA-sequencing (scRNA-seq) to delineate and distinguish specification of neuronal lineages in mouse medial, lateral, and caudal ganglionic eminences (MGE, LGE, and CGE) at embryonic day (E)11.5. We show that scRNA-seq clustering using transcription factors improves resolution of regional and developmental populations, and that enhancer activities identify specific and overlapping GE-derived neuronal populations. First, we mapped the activities of seven evolutionarily conserved brain enhancers at single-cell resolution in vivo, finding that the selected enhancers had diverse activities in specific progenitor and neuronal populations across the GEs. We then applied enhancer-based labeling, scRNA-seq, and analysis of in situ hybridization data to distinguish transcriptionally distinct and spatially defined subtypes of MGE-derived GABAergic and cholinergic projection neurons and interneurons. Our results map developmental origins and specification paths underlying neurogenesis in the embryonic basal ganglia and showcase the power of scRNA-seq combined with enhancer-based labeling to resolve the complex paths of neuronal specification underlying mouse brain development.

ZipSeq: barcoding for real-time mapping of single cell transcriptomes.

Spatial transcriptomics seeks to integrate single cell transcriptomic data within the three-dimensional space of multicellular biology. Current methods to correlate a cell's position with its transcriptome in living tissues have various limitations. We developed an approach, called 'ZipSeq', that uses patterned illumination and photocaged oligonucleotides to serially print barcodes ('zipcodes') onto live cells in intact tissues, in real time and with an on-the-fly selection of patterns. Using ZipSeq, we mapped gene expression in three settings: in vitro wound healing, live lymph node sections and a live tumor microenvironment. In all cases, we discovered new gene expression patterns associated with histological structures. In the tumor microenvironment, this demonstrated a trajectory of myeloid and T cell differentiation from the periphery inward. A combinatorial variation of ZipSeq efficiently scales in the number of regions defined, providing a pathway for complete mapping of live tissues, subsequent to real-time imaging or perturbation.

Expanding the boundaries of synthetic development.

Embryonic tissue boundaries are critical to not only cement newly patterned structures during development, but also to serve as organizing centers for subsequent rounds of morphogenesis. â€‹Although this latter role is especially difficult to study in vivo, synthetic embryology offers a new vantage point and fresh opportunities. In this review, we cover recent progress towards understanding and controlling in vitro boundaries and how they impact synthetic model systems. A key point this survey highlights is that the outcome of self-organization is strongly dependent on the boundary imposed, and new insight into the complex functions of embryonic boundaries will be necessary to create better self-organizing tissues for basic science, drug development, and regenerative medicine.

MULTI-seq: sample multiplexing for single-cell RNA sequencing using lipid-tagged indices.

Sample multiplexing facilitates scRNA-seq by reducing costs and identifying artifacts such as cell doublets. However, universal and scalable sample barcoding strategies have not been described. We therefore developed MULTI-seq: multiplexing using lipid-tagged indices for single-cell and single-nucleus RNA sequencing. MULTI-seq reagents can barcode any cell type or nucleus from any species with an accessible plasma membrane. The method involves minimal sample processing, thereby preserving cell viability and endogenous gene expression patterns. When cells are classified into sample groups using MULTI-seq barcode abundances, data quality is improved through doublet identification and recovery of cells with low RNA content that would otherwise be discarded by standard quality-control workflows. We use MULTI-seq to track the dynamics of T-cell activation, perform a 96-plex perturbation experiment with primary human mammary epithelial cells and multiplex cryopreserved tumors and metastatic sites isolated from a patient-derived xenograft mouse model of triple-negative breast cancer.

No detectable alloreactive transcriptional responses under standard sample preparation conditions during donor-multiplexed single-cell RNA sequencing of peripheral blood mononuclear cells.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) provides high-dimensional measurements of transcript counts in individual cells. However, high assay costs and artifacts associated with analyzing samples across multiple sequencing runs limit the study of large numbers of samples. Sample multiplexing technologies such as MULTI-seq and antibody hashing using single-cell multiplexing kit (SCMK) reagents (BD Biosciences) use sample-specific sequence tags to enable individual samples to be sequenced in a pooled format, markedly lowering per-sample processing and sequencing costs while minimizing technical artifacts. Critically, however, pooling samples could introduce new artifacts, partially negating the benefits of sample multiplexing. In particular, no study to date has evaluated whether pooling peripheral blood mononuclear cells (PBMCs) from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30 min co-incubation at 4 °C) results in significant changes in gene expression resulting from alloreactivity (i.e., response to non-self). The ability to demonstrate minimal to no alloreactivity is crucial to avoid confounded data analyses, particularly for cross-sectional studies evaluating changes in immunologic gene signatures. RESULTS: Here, we applied the 10x Genomics scRNA-seq platform to MULTI-seq and/or SCMK-labeled PBMCs from a single donor with and without pooling with PBMCs from unrelated donors for 30 min at 4 °C. We did not detect any alloreactivity signal between mixed and unmixed PBMCs across a variety of metrics, including alloreactivity marker gene expression in CD4+ T cells, cell type proportion shifts, and global gene expression profile comparisons using Gene Set Enrichment Analysis and Jensen-Shannon Divergence. These results were additionally mirrored in publicly-available scRNA-seq data generated using a similar experimental design. Moreover, we identified confounding gene expression signatures linked to PBMC preparation method (e.g., Trima apheresis), as well as SCMK sample classification biases against activated CD4+ T cells which were recapitulated in two other SCMK-incorporating scRNA-seq datasets. CONCLUSIONS: We demonstrate that (i) mixing PBMCs from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30 min co-incubation at 4 °C) does not cause an allogeneic response, and (ii) that Trima apheresis and PBMC sample multiplexing using SCMK reagents can introduce undesirable technical artifacts into scRNA-seq data. Collectively, these observations establish important benchmarks for future cross-sectional immunological scRNA-seq experiments.

Quanti.us: a tool for rapid, flexible, crowd-based annotation of images.

We describe Quanti.us , a crowd-based image-annotation platform that provides an accurate alternative to computational algorithms for difficult image-analysis problems. We used Quanti.us for a variety of medium-throughput image-analysis tasks and achieved 10-50× savings in analysis time compared with that required for the same task by a single expert annotator. We show equivalent deep learning performance for Quanti.us-derived and expert-derived annotations, which should allow scalable integration with tailored machine learning algorithms.

Roadmap for the multiscale coupling of biochemical and mechanical signals during development.

The way in which interactions between mechanics and biochemistry lead to the emergence of complex cell and tissue organization is an old question that has recently attracted renewed interest from biologists, physicists, mathematicians and computer scientists. Rapid advances in optical physics, microscopy and computational image analysis have greatly enhanced our ability to observe and quantify spatiotemporal patterns of signalling, force generation, deformation, and flow in living cells and tissues. Powerful new tools for genetic, biophysical and optogenetic manipulation are allowing us to perturb the underlying machinery that generates these patterns in increasingly sophisticated ways. Rapid advances in theory and computing have made it possible to construct predictive models that describe how cell and tissue organization and dynamics emerge from the local coupling of biochemistry and mechanics. Together, these advances have opened up a wealth of new opportunities to explore how mechanochemical patterning shapes organismal development. In this roadmap, we present a series of forward-looking case studies on mechanochemical patterning in development, written by scientists working at the interface between the physical and biological sciences, and covering a wide range of spatial and temporal scales, organisms, and modes of development. Together, these contributions highlight the many ways in which the dynamic coupling of mechanics and biochemistry shapes biological dynamics: from mechanoenzymes that sense force to tune their activity and motor output, to collectives of cells in tissues that flow and redistribute biochemical signals during development.

Paracrine costimulation of IFN-γ signaling by integrins modulates CD8 T cell differentiation.

The cytokine IFN-γ is a critical regulator of immune system development and function. Almost all leukocytes express the receptor for IFN-γ, yet each cell type elicits a different response to this cytokine. Cell type-specific effects of IFN-γ make it difficult to predict the outcomes of the systemic IFN-γ blockade and limit its clinical application, despite many years of research. To better understand the cell-cell interactions and cofactors that specify IFN-γ functions, we focused on the function of IFN-γ on CD8 T cell differentiation. We demonstrated that during bacterial infection, IFN-γ is a dominant paracrine trigger that skews CD8 T cell differentiation toward memory. This skewing is preferentially driven by contact-dependent T cell-T cell (T-T) interactions and the localized IFN-γ secretion among activated CD8 T cells in a unique splenic microenvironment, and is less sensitive to concurrent IFN-γ production by other immune cell populations such as natural killer (NK) cells. Modulation of CD8 T cell differentiation by IFN-γ relies on a nonconventional IFN-γ outcome that occurs specifically within 24 hours following infection. This is driven by IFN-γ costimulation by integrins at T-T synapses, and leads to synergistic phosphorylation of the proximal STAT1 molecule and accelerated IL-2 receptor down-regulation. This study provides evidence of the importance of context-dependent cytokine signaling and gives another example of how cell clusters and the microenvironment drive unique biology.

Dissecting the stem cell niche with organoid models: an engineering-based approach.

For many tissues, single resident stem cells grown in vitro under appropriate three-dimensional conditions can produce outgrowths known as organoids. These tissues recapitulate much of the cell composition and architecture of the in vivo organ from which they derive, including the formation of a stem cell niche. This has facilitated the systematic experimental manipulation and single-cell, high-throughput imaging of stem cells within their respective niches. Furthermore, emerging technologies now make it possible to engineer organoids from purified cellular and extracellular components to directly model and test stem cell-niche interactions. In this Review, we discuss how organoids have been used to identify and characterize stem cell-niche interactions and uncover new niche components, focusing on three adult-derived organoid systems. We also describe new approaches to reconstitute organoids from purified cellular components, and discuss how this technology can help to address fundamental questions about the adult stem cell niche.

Printed droplet microfluidics for on demand dispensing of picoliter droplets and cells.

Although the elementary unit of biology is the cell, high-throughput methods for the microscale manipulation of cells and reagents are limited. The existing options either are slow, lack single-cell specificity, or use fluid volumes out of scale with those of cells. Here we present printed droplet microfluidics, a technology to dispense picoliter droplets and cells with deterministic control. The core technology is a fluorescence-activated droplet sorter coupled to a specialized substrate that together act as a picoliter droplet and single-cell printer, enabling high-throughput generation of intricate arrays of droplets, cells, and microparticles. Printed droplet microfluidics provides a programmable and robust technology to construct arrays of defined cell and reagent combinations and to integrate multiple measurement modalities together in a single assay.

Programmed synthesis of three-dimensional tissues.

Reconstituting tissues from their cellular building blocks facilitates the modeling of morphogenesis, homeostasis and disease in vitro. Here we describe DNA-programmed assembly of cells (DPAC), a method to reconstitute the multicellular organization of organoid-like tissues having programmed size, shape, composition and spatial heterogeneity. DPAC uses dissociated cells that are chemically functionalized with degradable oligonucleotide 'Velcro', allowing rapid, specific and reversible cell adhesion to other surfaces coated with complementary DNA sequences. DNA-patterned substrates function as removable and adhesive templates, and layer-by-layer DNA-programmed assembly builds arrays of tissues into the third dimension above the template. DNase releases completed arrays of organoid-like microtissues from the template concomitant with full embedding in a variety of extracellular matrix (ECM) gels. DPAC positions subpopulations of cells with single-cell spatial resolution and generates cultures several centimeters long. We used DPAC to explore the impact of ECM composition, heterotypic cell-cell interactions and patterns of signaling heterogeneity on collective cell behaviors.

A strategy for tissue self-organization that is robust to cellular heterogeneity and plasticity.

Developing tissues contain motile populations of cells that can self-organize into spatially ordered tissues based on differences in their interfacial surface energies. However, it is unclear how self-organization by this mechanism remains robust when interfacial energies become heterogeneous in either time or space. The ducts and acini of the human mammary gland are prototypical heterogeneous and dynamic tissues comprising two concentrically arranged cell types. To investigate the consequences of cellular heterogeneity and plasticity on cell positioning in the mammary gland, we reconstituted its self-organization from aggregates of primary cells in vitro. We find that self-organization is dominated by the interfacial energy of the tissue-ECM boundary, rather than by differential homo- and heterotypic energies of cell-cell interaction. Surprisingly, interactions with the tissue-ECM boundary are binary, in that only one cell type interacts appreciably with the boundary. Using mathematical modeling and cell-type-specific knockdown of key regulators of cell-cell cohesion, we show that this strategy of self-organization is robust to severe perturbations affecting cell-cell contact formation. We also find that this mechanism of self-organization is conserved in the human prostate. Therefore, a binary interfacial interaction with the tissue boundary provides a flexible and generalizable strategy for forming and maintaining the structure of two-component tissues that exhibit abundant heterogeneity and plasticity. Our model also predicts that mutations affecting binary cell-ECM interactions are catastrophic and could contribute to loss of tissue architecture in diseases such as breast cancer.