Universal recording of immune cell interactions in vivo.

Immune cells rely on transient physical interactions with other immune and non-immune populations to regulate their function1. To study these 'kiss-and-run' interactions directly in vivo, we previously developed LIPSTIC (labelling immune partnerships by SorTagging intercellular contacts)2, an approach that uses enzymatic transfer of a labelled substrate between the molecular partners CD40L and CD40 to label interacting cells. Reliance on this pathway limited the use of LIPSTIC to measuring interactions between CD4+ T helper cells and antigen-presenting cells, however. Here we report the development of a universal version of LIPSTIC (uLIPSTIC), which can record physical interactions both among immune cells and between immune and non-immune populations irrespective of the receptors and ligands involved. We show that uLIPSTIC can be used, among other things, to monitor the priming of CD8+ T cells by dendritic cells, reveal the steady-state cellular partners of regulatory T cells and identify germinal centre-resident T follicular helper cells on the basis of their ability to interact cognately with germinal centre B cells. By coupling uLIPSTIC with single-cell transcriptomics, we build a catalogue of the immune populations that physically interact with intestinal epithelial cells at the steady state and profile the evolution of the interactome of lymphocytic choriomeningitis virus-specific CD8+ T cells in multiple organs following systemic infection. Thus, uLIPSTIC provides a broadly useful technology for measuring and understanding cell-cell interactions across multiple biological systems.

Vitamin A resolves lineage plasticity to orchestrate stem cell lineage choices.

Lineage plasticity-a state of dual fate expression-is required to release stem cells from their niche constraints and redirect them to tissue compartments where they are most needed. In this work, we found that without resolving lineage plasticity, skin stem cells cannot effectively generate each lineage in vitro nor regrow hair and repair wounded epidermis in vivo. A small-molecule screen unearthed retinoic acid as a critical regulator. Combining high-throughput approaches, cell culture, and in vivo mouse genetics, we dissected its roles in tissue regeneration. We found that retinoic acid is made locally in hair follicle stem cell niches, where its levels determine identity and usage. Our findings have therapeutic implications for hair growth as well as chronic wounds and cancers, where lineage plasticity is unresolved.

Beyond genetics: driving cancer with the tumour microenvironment behind the wheel.

Cancer has long been viewed as a genetic disease of cumulative mutations. This notion is fuelled by studies showing that ageing tissues are often riddled with clones of complex oncogenic backgrounds coexisting in seeming harmony with their normal tissue counterparts. Equally puzzling, however, is how cancer cells harbouring high mutational burden contribute to normal, tumour-free mice when allowed to develop within the confines of healthy embryos. Conversely, recent evidence suggests that adult tissue cells expressing only one or a few oncogenes can, in some contexts, generate tumours exhibiting many of the features of a malignant, invasive cancer. These disparate observations are difficult to reconcile without invoking environmental cues triggering epigenetic changes that can either dampen or drive malignant transformation. In this Review, we focus on how certain oncogenes can launch a two-way dialogue of miscommunication between a stem cell and its environment that can rewire downstream events non-genetically and skew the morphogenetic course of the tissue. We review the cells and molecules of and the physical forces acting in the resulting tumour microenvironments that can profoundly affect the behaviours of transformed cells. Finally, we discuss possible explanations for the remarkable diversity in the relative importance of mutational burden versus tumour microenvironment and its clinical relevance.

Understanding the microenvironment and how this controls cell fate.

The microenvironment influences cell fate. In this collection of voices, researchers from the fields of cancer and regeneration highlight approaches to establish the importance of the microenvironment and discuss future directions to understand the complex interaction between cells and their surrounding environment and how this impacts on disease and regeneration.

The pioneer factor SOX9 competes for epigenetic factors to switch stem cell fates.

During development, progenitors simultaneously activate one lineage while silencing another, a feature highly regulated in adult stem cells but derailed in cancers. Equipped to bind cognate motifs in closed chromatin, pioneer factors operate at these crossroads, but how they perform fate switching remains elusive. Here we tackle this question with SOX9, a master regulator that diverts embryonic epidermal stem cells (EpdSCs) into becoming hair follicle stem cells. By engineering mice to re-activate SOX9 in adult EpdSCs, we trigger fate switching. Combining epigenetic, proteomic and functional analyses, we interrogate the ensuing chromatin and transcriptional dynamics, slowed temporally by the mature EpdSC niche microenvironment. We show that as SOX9 binds and opens key hair follicle enhancers de novo in EpdSCs, it simultaneously recruits co-factors away from epidermal enhancers, which are silenced. Unhinged from its normal regulation, sustained SOX9 subsequently activates oncogenic transcriptional regulators that chart the path to cancers typified by constitutive SOX9 expression.

Designer bugs as cancer drugs?

Skin microbiota can be engineered to induce antitumor T cells.

Ras drives malignancy through stem cell crosstalk with the microenvironment.

Squamous cell carcinomas are triggered by marked elevation of RAS-MAPK signalling and progression from benign papilloma to invasive malignancy1-4. At tumour-stromal interfaces, a subset of tumour-initiating progenitors, the cancer stem cells, obtain increased resistance to chemotherapy and immunotherapy along this pathway5,6. The distribution and changes in cancer stem cells during progression from a benign state to invasive squamous cell carcinoma remain unclear. Here we show in mice that, after oncogenic RAS activation, cancer stem cells rewire their gene expression program and trigger self-propelling, aberrant signalling crosstalk with their tissue microenvironment that drives their malignant progression. The non-genetic, dynamic cascade of intercellular exchanges involves downstream pathways that are often mutated in advanced metastatic squamous cell carcinomas with high mutational burden7. Coupling our clonal skin HRASG12V mouse model with single-cell transcriptomics, chromatin landscaping, lentiviral reporters and lineage tracing, we show that aberrant crosstalk between cancer stem cells and their microenvironment triggers angiogenesis and TGFß signalling, creating conditions that are conducive for hijacking leptin and leptin receptor signalling, which in turn launches downstream phosphoinositide 3-kinase (PI3K)-AKT-mTOR signalling during the benign-to-malignant transition. By functionally examining each step in this pathway, we reveal how dynamic temporal crosstalk with the microenvironment orchestrated by the stem cells profoundly fuels this path to malignancy. These insights suggest broad implications for cancer therapeutics.

Inflammatory memory and tissue adaptation in sickness and in health.

Our body has a remarkable ability to remember its past encounters with allergens, pathogens, wounds and irritants, and to react more quickly to the next experience. This accentuated sensitivity also helps us to cope with new threats. Despite maintaining a state of readiness and broadened resistance to subsequent pathogens, memories can also be maladaptive, leading to chronic inflammatory disorders and cancers. With the ever-increasing emergence of new pathogens, allergens and pollutants in our world, the urgency to unravel the molecular underpinnings of these phenomena has risen to new heights. Here we reflect on how the field of inflammatory memory has evolved, since 2007, when researchers realized that non-specific memory is contained in the nucleus and propagated at the epigenetic level. We review the flurry of recent discoveries revealing that memory is not just a privilege of the immune system but also extends to epithelia of the skin, lung, intestine and pancreas, and to neurons. Although still unfolding, epigenetic memories of inflammation have now been linked to possible brain disorders such as Alzheimer disease, and to an elevated risk of cancer. In this Review, we consider the consequences-good and bad-of these epigenetic memories and their implications for human health and disease.

The integrated stress response remodels the microtubule-organizing center to clear unfolded proteins following proteotoxic stress.

Cells encountering stressful situations activate the integrated stress response (ISR) pathway to limit protein synthesis and redirect translation to better cope. The ISR has also been implicated in cancers, but redundancies in the stress-sensing kinases that trigger the ISR have posed hurdles to dissecting physiological relevance. To overcome this challenge, we targeted the regulatory node of these kinases, namely, the S51 phosphorylation site of eukaryotic translation initiation factor eIF2α and genetically replaced eIF2α with eIF2α-S51A in mouse squamous cell carcinoma (SCC) stem cells of skin. While inconsequential under normal growth conditions, the vulnerability of this ISR-null state was unveiled when SCC stem cells experienced proteotoxic stress. Seeking mechanistic insights into the protective roles of the ISR, we combined ribosome profiling and functional approaches to identify and probe the functional importance of translational differences between ISR-competent and ISR-null SCC stem cells when exposed to proteotoxic stress. In doing so, we learned that the ISR redirects translation to centrosomal proteins that orchestrate the microtubule dynamics needed to efficiently concentrate unfolded proteins at the microtubule-organizing center so that they can be cleared by the perinuclear degradation machinery. Thus, rather than merely maintaining survival during proteotoxic stress, the ISR also functions in promoting cellular recovery once the stress has subsided. Remarkably, this molecular program is unique to transformed skin stem cells, hence exposing a vulnerability in cancer that could be exploited therapeutically.

Desmoplakin Maintains Transcellular Keratin Scaffolding and Protects From Intestinal Injury.

BACKGROUND & AIMS: Desmosomes are intercellular junctions connecting keratin intermediate filaments of neighboring cells. The cadherins desmoglein 2 (Dsg2) and desmocollin 2 mediate cell-cell adhesion, whereas desmoplakin (Dsp) provides the attachment of desmosomes to keratins. Although the importance of the desmosome-keratin network is well established in mechanically challenged tissues, we aimed to assess the currently understudied function of desmosomal proteins in intestinal epithelia. METHODS: We analyzed the intestine-specific villin-Cre DSP (DSPΔIEC) and the combined intestine-specific DSG2/DSPΔIEC (ΔDsg2/Dsp) knockout mice. Cross-breeding with keratin 8-yellow fluorescent protein knock-in mice and generation of organoids was performed to visualize the keratin network. A Dsp-deficient colorectal carcinoma HT29-derived cell line was generated and the role of Dsp in adhesion and mechanical stress was studied in dispase assays, after exposure to uniaxial cell stretching and during scratch assay. RESULTS: The intestine of DSPΔIEC mice was histopathologically inconspicuous. Intestinal epithelial cells, however, showed an accelerated migration along the crypt and an enhanced shedding into the lumen. Increased intestinal permeability and altered levels of desmosomal proteins were detected. An inconspicuous phenotype also was seen in ΔDsg2/Dsp mice. After dextran sodium sulfate treatment, DSPΔIEC mice developed more pronounced colitis. A retracted keratin network was seen in the intestinal epithelium of DSPΔIEC/keratin 8-yellow fluorescent protein mice and organoids derived from these mice presented a collapsed keratin network. The level, phosphorylation status, and solubility of keratins were not affected. Dsp-deficient HT29 cells had an impaired cell adhesion and suffered from increased cellular damage after stretch. CONCLUSIONS: Our results show that Dsp is required for proper keratin network architecture in intestinal epithelia, mechanical resilience, and adhesion, thereby protecting from injury.

Highly efficient manipulation of nervous system gene expression with NEPTUNE.

Genetic loss and gain of function in mice have typically been studied by using knockout or knockin mice that take months to years to generate. To address this problem for the nervous system, we developed NEPTUNE (NEural Plate Targeting by in Utero NanoinjEction) to rapidly and flexibly transduce the neural plate with virus prior to neurulation, and thus manipulate the future nervous system. Stable integration in >95% of cells in the brain enabled long-term overexpression, and conditional expression was achieved by using cell-type-specific MiniPromoters. Knockdown of Olig2 by using NEPTUNE recapitulated the phenotype of Olig2 -/- embryos. We used NEPTUNE to investigate Sptbn2, mutations in which cause spinocerebellar ataxia type 5. Sptbn2 knockdown induced dose-dependent defects in the neural tube, embryonic turning, and abdominal wall closure, previously unreported functions for Sptbn2. NEPTUNE thus offers a rapid and cost-effective technique to test gene function in the nervous system and can reveal phenotypes incompatible with life.

Environmental control of lineage plasticity and stem cell memory.

Tissue-resident stem cells (SCs) are critical players in the maintenance of tissue homeostasis. SCs reside in complex and uniquely anatomically organized microenvironments (SC niches), that carefully control SC lineage outputs depending on localized tissue needs. Upon environmental perturbations and tissue stressors, SCs respond and restore the tissue to homeostasis, as well as protect it from secondary assaults. Critical to this function are two key processes, SC lineage plasticity and SC memory. In this review, we delineate the multifactorial determinants and key principles underlining these two remarkable SC behaviors. Understanding lineage plasticity and SC memory will be critical not only to design new regenerative therapies but also to determine how these processes are altered in a multitude of pathologies such as cancer and chronic tissue damage.

Author Correction: Extracellular serine controls epidermal stem cell fate and tumour initiation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: Mechanics of a multilayer epithelium instruct tumour architecture and function.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A Metabolic Bottleneck for Stem Cell Transformation.

Although oncogenic mutations predispose tissue stem cells to tumor initiation, the rate-limiting processes for stem cell immortalization remain unknown. In this issue of Cell, Bonnay et al. identify enhanced electron transport chain activity as a critical determinant of this process, establishing metabolic reprogramming as limiting for tumor initiation.

Mechanics of a multilayer epithelium instruct tumour architecture and function.

Loss of normal tissue architecture is a hallmark of oncogenic transformation1. In developing organisms, tissues architectures are sculpted by mechanical forces during morphogenesis2. However, the origins and consequences of tissue architecture during tumorigenesis remain elusive. In skin, premalignant basal cell carcinomas form 'buds', while invasive squamous cell carcinomas initiate as 'folds'. Here, using computational modelling, genetic manipulations and biophysical measurements, we identify the biophysical underpinnings and biological consequences of these tumour architectures. Cell proliferation and actomyosin contractility dominate tissue architectures in monolayer, but not multilayer, epithelia. In stratified epidermis, meanwhile, softening and enhanced remodelling of the basement membrane promote tumour budding, while stiffening of the basement membrane promotes folding. Additional key forces stem from the stratification and differentiation of progenitor cells. Tumour-specific suprabasal stiffness gradients are generated as oncogenic lesions progress towards malignancy, which we computationally predict will alter extensile tensions on the tumour basement membrane. The pathophysiologic ramifications of this prediction are profound. Genetically decreasing the stiffness of basement membranes increases membrane tensions in silico and potentiates the progression of invasive squamous cell carcinomas in vivo. Our findings suggest that mechanical forces-exerted from above and below progenitors of multilayered epithelia-function to shape premalignant tumour architectures and influence tumour progression.

m6A RNA methylation impacts fate choices during skin morphogenesis.

N6-methyladenosine is the most prominent RNA modification in mammals. Here, we study mouse skin embryogenesis to tackle m6A's functions and physiological importance. We first landscape the m6A modifications on skin epithelial progenitor mRNAs. Contrasting with in vivo ribosomal profiling, we unearth a correlation between m6A modification in coding sequences and enhanced translation, particularly of key morphogenetic signaling pathways. Tapping physiological relevance, we show that m6A loss profoundly alters these cues and perturbs cellular fate choices and tissue architecture in all skin lineages. By single-cell transcriptomics and bioinformatics, both signaling and canonical translation pathways show significant downregulation after m6A loss. Interestingly, however, many highly m6A-modified mRNAs are markedly upregulated upon m6A loss, and they encode RNA-methylation, RNA-processing and RNA-metabolism factors. Together, our findings suggest that m6A functions to enhance translation of key morphogenetic regulators, while also destabilizing sentinel mRNAs that are primed to activate rescue pathways when m6A levels drop.

Extracellular serine controls epidermal stem cell fate and tumour initiation.

Tissue stem cells are the cell of origin for many malignancies. Metabolites regulate the balance between self-renewal and differentiation, but whether endogenous metabolic pathways or nutrient availability predispose stem cells towards transformation remains unknown. Here, we address this question in epidermal stem cells (EpdSCs), which are a cell of origin for squamous cell carcinoma. We find that oncogenic EpdSCs are serine auxotrophs whose growth and self-renewal require abundant exogenous serine. When extracellular serine is limited, EpdSCs activate de novo serine synthesis, which in turn stimulates α-ketoglutarate-dependent dioxygenases that remove the repressive histone modification H3K27me3 and activate differentiation programmes. Accordingly, serine starvation or enforced α-ketoglutarate production antagonizes squamous cell carcinoma growth. Conversely, blocking serine synthesis or repressing α-ketoglutarate-driven demethylation facilitates malignant progression. Together, these findings reveal that extracellular serine is a critical determinant of EpdSC fate and provide insight into how nutrient availability is integrated with stem cell fate decisions during tumour initiation.

The aging skin microenvironment dictates stem cell behavior.

Aging manifests with architectural alteration and functional decline of multiple organs throughout an organism. In mammals, aged skin is accompanied by a marked reduction in hair cycling and appearance of bald patches, leading researchers to propose that hair follicle stem cells (HFSCs) are either lost, differentiate, or change to an epidermal fate during aging. Here, we employed single-cell RNA-sequencing to interrogate aging-related changes in the HFSCs. Surprisingly, although numbers declined, aging HFSCs were present, maintained their identity, and showed no overt signs of shifting to an epidermal fate. However, they did exhibit prevalent transcriptional changes particularly in extracellular matrix genes, and this was accompanied by profound structural perturbations in the aging SC niche. Moreover, marked age-related changes occurred in many nonepithelial cell types, including resident immune cells, sensory neurons, and arrector pili muscles. Each of these SC niche components has been shown to influence HF regeneration. When we performed skin injuries that are known to mobilize young HFSCs to exit their niche and regenerate HFs, we discovered that aged skin is defective at doing so. Interestingly, however, in transplantation assays in vivo, aged HFSCs regenerated HFs when supported with young dermis, while young HFSCs failed to regenerate HFs when combined with aged dermis. Together, our findings highlight the importance of SC:niche interactions and favor a model where youthfulness of the niche microenvironment plays a dominant role in dictating the properties of its SCs and tissue health and fitness.

Epithelial cells: liaisons of immunity.

The surface and lining tissues of our body are exposed to the external environment, and as such these epithelial tissues must form structural barriers able to defend against microbes, environmental toxins, and mechanical stress. Their cells are equipped to detect a diverse array of surface perturbations, and then launch signaling relays to the immune system. The aim of these liaisons is to coordinate the requisite immune cell response needed to preserve and/or restore barrier integrity and defend the host. It has been recently appreciated that epithelial cells learn from these experiences. Following inflammatory exposure, long-lived stem cells within the tissue retain an epigenetic memory that endows them with heightened responsiveness to subsequent encounters with stress. Here, we review the recent literature on how epithelial cells sense signals from microbes, allergens, and injury at the tissue surface, and transmit this information to immune cells, while embedding a memory of the experience within their chromatin.