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Liver regeneration following resection is a complex process relying on coordinated pathways and cell types in the remnant organ. Myeloid-Derived Suppressor Cells (MDSCs) have a role in liver regeneration-related angiogenesis but other roles they may play in this process remain to be elucidated. In this study, we sought to examine the effect of G-MDSCs on hepatocytes proliferation and immune modulation during liver regeneration. Global gene expression profiling of regenerating hepatocytes in mice with CD11b + Ly6G + MDSCs (G-MDSCs) depletion revealed disrupted transcriptional progression from day one to day two after major liver resection. Key genes and pathways related to hepatocyte proliferation and immune response were differentially expressed upon MDSC depletion. Hepatocytes cellularity increased when co-cultured with G-MDSCs, or treated with amphiregulin, which G-MDSCs upregulate during regeneration. Cytometry by time-of-flight (CyTOF) analysis of the intra-liver immune milieu upon MDSC depletion during regeneration demonstrated increased natural killer cell proportions, alongside changes in other immune cell populations. Taken together, these results provide evidence that MDSCs contribute to early liver regeneration by promoting hepatocyte proliferation and modulating the intra-liver immune response, and illuminate the multifaceted role of MDSCs in liver regeneration., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Activating EGFR (epidermal growth factor receptor) mutations can be inhibited by specific tyrosine kinase inhibitors (TKIs), which have changed the landscape of lung cancer therapy. However, due to secondary mutations and bypass receptors, such as AXL (AXL receptor tyrosine kinase), drug resistance eventually emerges in most patients treated with the first-, second-, or third-generation TKIs (e.g., osimertinib). To inhibit AXL and resistance to osimertinib, we compare two anti-AXL drugs, an antibody (mAb654) and a TKI (bemcentinib). While no pair of osimertinib and an anti-AXL drug is able to prevent relapses, triplets combining osimertinib, cetuximab (an anti-EGFR antibody), and either anti-AXL drug are initially effective. However, longer monitoring uncovers superiority of the mAb654-containing triplet, possibly due to induction of receptor endocytosis, activation of immune mechanisms, or disabling intrinsic mutators. Hence, we constructed a bispecific antibody that engages both AXL and EGFR. When combined with osimertinib, the bispecific antibody consistently inhibits tumor relapses, which warrants clinical trials., Competing Interests: Declaration of interests We, the authors, plan to file a patent application for the bispecific antibody (EGFR/AXL), namely Bis1, which has been developed in this study., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) and dementia in general are age-related diseases with multiple contributing factors, including brain inflammation. Microglia, and specifically those expressing the AD risk gene TREM2, are considered important players in AD, but their exact contribution to pathology remains unclear. In this study, using high-throughput mass cytometry in the 5×FAD mouse model of amyloidosis, we identified senescent microglia that express high levels of TREM2 but also exhibit a distinct signature from TREM2-dependent disease-associated microglia (DAM). This senescent microglial protein signature was found in various mouse models that show cognitive decline, including aging, amyloidosis and tauopathy. TREM2-null mice had fewer microglia with a senescent signature. Treating 5×FAD mice with the senolytic BCL2 family inhibitor ABT-737 reduced senescent microglia, but not the DAM population, and this was accompanied by improved cognition and reduced brain inflammation. Our results suggest a dual and opposite involvement of TREM2 in microglial states, which must be considered when contemplating TREM2 as a therapeutic target in AD., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The TP53 gene is mutated in approximately 30% of all breast cancer cases. Adipocytes and preadipocytes, which constitute a substantial fraction of the stroma of normal mammary tissue and breast tumors, undergo transcriptional, metabolic, and phenotypic reprogramming during breast cancer development and play an important role in tumor progression. We report here that p53 loss in breast cancer cells facilitates the reprogramming of preadipocytes, inducing them to acquire a unique transcriptional and metabolic program that combines impaired adipocytic differentiation with augmented cytokine expression. This, in turn, promotes the establishment of an inflammatory tumor microenvironment, including increased abundance of Ly6C+ and Ly6G+ myeloid cells and elevated expression of the immune checkpoint ligand PD-L1. We also describe a potential gain-of-function effect of common p53 missense mutations on the inflammatory reprogramming of preadipocytes. Altogether, our study implicates p53 deregulation in breast cancer cells as a driver of tumor-supportive adipose tissue reprogramming, expanding the network of non-cell autonomous mechanisms whereby p53 dysfunction may promote cancer. Further elucidation of the interplay between p53 and adipocytes within the tumor microenvironment may suggest effective therapeutic targets for the treatment of breast cancer patients., Competing Interests: Competing interests statement:K.E.d.V. is consultant for Macomics.

Systemic immunity supports lifelong brain function. Obesity posits a chronic burden on systemic immunity. Independently, obesity was shown as a risk factor for Alzheimer's disease (AD). Here we show that high-fat obesogenic diet accelerated recognition-memory impairment in an AD mouse model (5xFAD). In obese 5xFAD mice, hippocampal cells displayed only minor diet-related transcriptional changes, whereas the splenic immune landscape exhibited aging-like CD4 + T-cell deregulation. Following plasma metabolite profiling, we identified free N-acetylneuraminic acid (NANA), the predominant sialic acid, as the metabolite linking recognition-memory impairment to increased splenic immune-suppressive cells in mice. Single-nucleus RNA-sequencing revealed mouse visceral adipose macrophages as a potential source of NANA. In vitro, NANA reduced CD4 + T-cell proliferation, tested in both mouse and human. In vivo, NANA administration to standard diet-fed mice recapitulated high-fat diet effects on CD4 + T cells and accelerated recognition-memory impairment in 5xFAD mice. We suggest that obesity accelerates disease manifestation in a mouse model of AD via systemic immune exhaustion., (© 2023. The Author(s).)

Numerous methods have recently emerged for ordering single cells along developmental trajectories. However, accurate depiction of developmental dynamics can only be achieved after rescaling the trajectory according to the relative time spent at each developmental point. We formulate a model which estimates local cell densities and fluxes, and incorporates cell division and apoptosis rates, to infer the real-time dimension of the developmental trajectory. We validate the model using mathematical simulations and apply it to experimental high dimensional cytometry data obtained from the mouse thymus to construct the true time profile of the thymocyte developmental process. Our method can easily be implemented in any of the existing tools for trajectory inference., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Breast cancer, the most frequent cancer in women, is generally classified into several distinct histological and molecular subtypes. However, single-cell technologies have revealed remarkable cellular and functional heterogeneity across subtypes and even within individual breast tumors. Much of this heterogeneity is attributable to dynamic alterations in the epigenetic landscape of the cancer cells, which promote phenotypic plasticity. Such plasticity, including transition from luminal to basal-like cell identity, can promote disease aggressiveness. We now report that the tumor suppressor LATS1, whose expression is often downregulated in human breast cancer, helps maintain luminal breast cancer cell identity by reducing the chromatin accessibility of genes that are characteristic of a "basal-like" state, preventing their spurious activation. This is achieved via interaction of LATS1 with the NCOR1 nuclear corepressor and recruitment of HDAC1, driving histone H3K27 deacetylation near NCOR1-repressed "basal-like" genes. Consequently, decreased expression of LATS1 elevates the expression of such genes and facilitates slippage towards a more basal-like phenotypic identity. We propose that by enforcing rigorous silencing of repressed genes, the LATS1-NCOR1 axis maintains luminal cell identity and restricts breast cancer progression., (© 2022. The Author(s).)

Pre-leukemic clones carrying DNMT3A mutations have a selective advantage and an inherent chemoresistance, however the basis for this phenotype has not been fully elucidated. Mutations affecting the gene TP53 occur in pre-leukemic hematopoietic stem/progenitor cells (preL-HSPC) and lead to chemoresistance. Many of these mutations cause a conformational change and some of them were shown to enhance self-renewal capacity of preL-HSPC. Intriguingly, a misfolded P53 was described in AML blasts that do not harbor mutations in TP53, emphasizing the dynamic equilibrium between wild-type (WT) and "pseudo-mutant" conformations of P53. By combining single cell analyses and P53 conformation-specific monoclonal antibodies we studied preL-HSPC from primary human DNMT3A-mutated AML samples. We found that while leukemic blasts express mainly the WT conformation, in preL-HSPC the pseudo-mutant conformation is the dominant. HSPC from non-leukemic samples expressed both conformations to a similar extent. In a mouse model we found a small subset of HSPC with a dominant pseudo-mutant P53. This subpopulation was significantly larger among DNMT3AR882H-mutated HSPC, suggesting that while a pre-leukemic mutation can predispose for P53 misfolding, additional factors are involved as well. Treatment with a short peptide that can shift the dynamic equilibrium favoring the WT conformation of P53, specifically eliminated preL-HSPC that had dysfunctional canonical P53 pathway activity as reflected by single cell RNA sequencing. Our observations shed light upon a possible targetable P53 dysfunction in human preL-HSPC carrying DNMT3A mutations. This opens new avenues for leukemia prevention.

Calorie restriction enhances stem cell self-renewal in various tissues, including the mammary gland. We hypothesized that similar to their intestinal counterparts, mammary epithelial stem cells are insulated from sensing changes in energy supply, depending instead on niche signaling. The latter was investigated by subjecting cultures of mammary epithelial stem cells for 8 days to in vivo paracrine calorie-restriction signals collected from a 4-day-conditioned medium of individual mammary cell populations. Conditioned medium from calorie-restricted non-epithelial cells induced latent cell propagation and mammosphere formation-established markers of stem cell self-renewal. Combined RNA-Seq, immunohistochemistry and immunofluorescence analyses of the non-epithelial population identified macrophages and secreted CSF1 as the energy sensor and paracrine signal, respectively. Calorie restriction-induced pStat6 expression in macrophages suggested that skewing to the M2 phenotype contributes to the sensing mechanism. Enhancing CSF1 signaling with recombinant protein and interrupting the interaction with its highly expressed receptor in the epithelial stem cells by neutralizing antibodies were both affected stem cell self-renewal. In conclusion, combined in vivo, in vitro and in silico studies identified macrophages and secreted CSF1 as the energy sensor and paracrine transmitter, respectively, of the calorie restriction-induced effect on mammary stem cell self-renewal.

Cancer cells are highly heterogeneous at the transcriptional level and epigenetic state. Methods to study epigenetic heterogeneity are limited in throughput and information obtained per cell. Here, we adapted cytometry by time-of-flight (CyTOF) to analyze a wide panel of histone modifications in primary tumor-derived lines of diffused intrinsic pontine glioma (DIPG). DIPG is a lethal glioma, driven by a histone H3 lysine 27 mutation (H3-K27M). We identified two epigenetically distinct subpopulations in DIPG, reflecting inherent heterogeneity in expression of the mutant histone. These two subpopulations are robust across tumor lines derived from different patients and show differential proliferation capacity and expression of stem cell and differentiation markers. Moreover, we demonstrate the use of these high-dimensional data to elucidate potential interactions between histone modifications and epigenetic alterations during the cell cycle. Our work establishes new concepts for the analysis of epigenetic heterogeneity in cancer that could be applied to diverse biological systems., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Mammary epithelial stem cells differentiate to create the basal and luminal layers of the gland. Inducing the number of differentiating bovine mammary stem cells may provide compensating populations for the milk-producing cells that die during lactation. Inhibition of mTOR activity by rapamycin signals self-renewal of intestinal stem cells, with similar consequences in the mouse mammary gland and in bovine mammary implants maintained in mice. The implementation of these results in farm animals for better mammary development and production was studied in 3-month-old calves. mTOR activity decreased by ~50% in mammary epithelial cells subjected to 3-week rapamycin administration, with no negative consequences on mammary morphology or β-casein expression. Subsequently, stem cell self-renewal was induced, reflected by a higher propagation rate of cultures from rapamycin-treated glands compared to respective controls and higher expression of selected markers. Followed by 4-day estrogen and progesterone administration, rapamycin significantly induced proliferation rate. Higher numbers of basal and luminal PCNA+ cells were detected in small ducts near the elongating sites as compared to large ducts, in which only luminal cells were affected. Rapamycin administration resulted in induction of individual milk protein genes' expression, which was negatively correlated to their endogenous levels. The inductive effect of rapamycin on luminal cell number was confirmed in organoid cultures, but milk protein expression decreased, probably due to lack of oscillation in rapamycin levels. In conclusion, intramammary rapamycin administration is an effective methodology to reduce mTOR activity in bovine mammary epithelial cells and consequently, induce stem cell self-renewal. The latent positive effect of rapamycin on epithelial cell proliferation and its potential to improve milk protein expression in calves may have beneficial implications for mature cows., Competing Interests: The authors have declared that no competing interests exist.

Consecutive exposures to different pathogens are highly prevalent and often alter the host immune response. However, it remains unknown how a secondary bacterial infection affects an ongoing adaptive immune response elicited against primary invading pathogens. We demonstrated that recruitment of Sca-1 + monocytes into lymphoid organs during Salmonella Typhimurium (STm) infection disrupted pre-existing germinal center (GC) reactions. GC responses induced by influenza, plasmodium, or commensals deteriorated following STm infection. GC disruption was independent of the direct bacterial interactions with B cells and instead was induced through recruitment of CCR2-dependent Sca-1 + monocytes into the lymphoid organs. GC collapse was associated with impaired cellular respiration and was dependent on TNFα and IFNγ, the latter of which was essential for Sca-1 + monocyte differentiation. Monocyte recruitment and GC disruption also occurred during LPS-supplemented vaccination and Listeria monocytogenes infection. Thus, systemic activation of the innate immune response upon severe bacterial infection is induced at the expense of antibody-mediated immunity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Limited outgrowth development of bovine mammary epithelial stem cells transplanted into de-epithelialized mouse fat pads restricts advanced studies on this productive organ's development and renewal. We challenged the mouse-bovine incompatibility by implanting parenchymal adjacent or distant bovine stromal layers (close and far stroma, respectively) into the mouse fat pad to serve as an endogenous niche for transplanted stem cells. The close stroma better supported stem cell take rate and outgrowth development. The diameter of these open duct-like structures represented and occasionally exceeded that of the endogenous ducts and appeared 8.3-fold wider than the capsule-like structures developed in the mouse fat pad after similar cell transplantation. RNA-Seq revealed lower complement activity in this layer, associated with secretion of specific laminins and WNT proteins favoring epithelial outgrowth development. The close stroma appeared genetically more similar to the parenchyma than to the far stroma due to epithelial characteristics, mainly of fibroblasts, including expression of epithelial markers, milk protein genes, and functional mammary claudins. Gene markers and activators of the mesenchymal-to-epithelial transition were highly enriched in the epithelial gene cluster and may contribute to the acquired epithelial properties of this stromal layer., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: For decades, dementia has been characterized by accumulation of waste in the brain and low-grade inflammation. Over the years, emerging studies highlighted the involvement of the immune system in neurodegenerative disease emergence and severity. Numerous studies in animal models of amyloidosis demonstrated the beneficial role of monocyte-derived macrophages in mitigating the disease, though less is known regarding tauopathy. Boosting the immune system in animal models of both amyloidosis and tauopathy, resulted in improved cognitive performance and in a reduction of pathological manifestations. However, a full understanding of the chain of events that is involved, starting from the activation of the immune system, and leading to disease mitigation, remained elusive. Here, we hypothesized that the brain-immune communication pathway that is needed to be activated to combat tauopathy involves monocyte mobilization via the C-C chemokine receptor 2 (CCR2)/CCL2 axis, and additional immune cells, such as CD4 + T cells, including FOXP3 + regulatory CD4 + T cells., Methods: We used DM-hTAU transgenic mice, a mouse model of tauopathy, and applied an approach that boosts the immune system, via blocking the inhibitory Programmed cell death protein-1 (PD-1)/PD-L1 pathway, a manipulation previously shown to alleviate disease symptoms and pathology. An anti-CCR2 monoclonal antibody (αCCR2), was used to block the CCR2 axis in a protocol that partially eliminates monocytes from the circulation at the time of anti-PD-L1 antibody (αPD-L1) injection, and for the critical period of their recruitment into the brain following treatment., Results: Performance of DM-hTAU mice in short-term and working memory tasks, revealed that the beneficial effect of αPD-L1, assessed 1 month after a single injection, was abrogated following blockade of CCR2. This was accompanied by the loss of the beneficial effect on disease pathology, assessed by measurement of cortical aggregated human tau load using Homogeneous Time Resolved Fluorescence-based immunoassay, and by evaluation of hippocampal neuronal survival. Using both multiparametric flow cytometry, and Cytometry by Time Of Flight, we further demonstrated the accumulation of FOXP3 + regulatory CD4 + T cells in the brain, 12 days following the treatment, which was absent subsequent to CCR2 blockade. In addition, measurement of hippocampal levels of the T-cell chemoattractant, C-X-C motif chemokine ligand 12 (Cxcl12), and of inflammatory cytokines, revealed that αPD-L1 treatment reduced their expression, while blocking CCR2 reversed this effect., Conclusions: The CCR2/CCL2 axis is required to modify pathology using PD-L1 blockade in a mouse model of tauopathy. This modification involves, in addition to monocytes, the accumulation of FOXP3 + regulatory CD4 + T cells in the brain, and the T-cell chemoattractant, Cxcl12.

Some antibacterial therapies entail sequential treatments with different antibiotics, but whether this approach is optimal for anti-cancer tyrosine kinase inhibitors (TKIs) remains open. EGFR mutations identify lung cancer patients who can derive benefit from TKIs, but most patients develop resistance to the first-, second-, and third-generation drugs. To explore alternatives to such whack-a-mole strategies, we simulated in patient-derived xenograft models the situation of patients receiving first-line TKIs. Monotherapies comprising approved first-line TKIs were compared to combinations with antibodies specific to EGFR and HER2. We observed uniform and strong superiority of all drug combinations over the respective monotherapies. Prolonged treatments, high TKI dose, and specificity were essential for drug-drug cooperation. Blocking pathways essential for mitosis (e.g., FOXM1), along with downregulation of resistance-conferring receptors (e.g., AXL), might underlie drug cooperation. Thus, upfront treatments using combinations of TKIs and antibodies can prevent emergence of resistance and hence might replace the widely applied sequential treatments utilizing next-generation TKIs., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Gaucher disease (GD) is currently the focus of considerable attention due primarily to the association between the gene that causes GD (GBA) and Parkinson's disease. Mouse models exist for the systemic (type 1) and for the acute neuronopathic forms (type 2) of GD. Here we report the generation of a mouse that phenotypically models chronic neuronopathic type 3 GD. Gba -/- ;Gba tg mice, which contain a Gba transgene regulated by doxycycline, accumulate moderate levels of the offending substrate in GD, glucosylceramide, and live for up to 10 months, i.e. significantly longer than mice which model type 2 GD. Gba -/- ;Gba tg mice display behavioral abnormalities at ∼4 months, which deteriorate with age, along with significant neuropathology including loss of Purkinje neurons. Gene expression is altered in the brain and in isolated microglia, although the changes in gene expression are less extensive than in mice modeling type 2 disease. Finally, bone deformities are consistent with the Gba -/- ;Gba tg mice being a genuine type 3 GD model. Together, the Gba -/- ;Gba tg mice share pathological pathways with acute neuronopathic GD mice but also display differences that might help understand the distinct disease course and progression of type 2 and 3 patients., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Identification of early processes leading to complex tissue pathologies, such as inflammatory bowel diseases, ‎poses a major scientific and clinical challenge that is imperative for improved diagnosis and treatment. Most studies of inflammation onset focus on cellular processes and signaling molecules, while overlooking the environment in which they take place, the continuously remodeled extracellular matrix. In this study, we used colitis models for investigating extracellular-matrix dynamics during disease onset, while treating the matrix as a complete and defined entity. Through the analysis of matrix structure, stiffness and composition, we unexpectedly revealed that even prior to the first clinical symptoms, the colon displays its own unique extracellular-matrix signature and found specific markers of clinical potential, which were also validated in human subjects. We also show that the emergence of this pre-symptomatic matrix is mediated by subclinical infiltration of immune cells bearing remodeling enzymes. Remarkably, whether the inflammation is chronic or acute, its matrix signature converges at pre-symptomatic states. We suggest that the existence of a pre-symptomatic extracellular-matrix is general and relevant to a wide range of diseases., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The mechanical challenge of attaching elastic tendons to stiff bones is solved by the formation of a unique transitional tissue. Here, we show that murine tendon-to-bone attachment cells are bi-fated, activating a mixture of chondrocyte and tenocyte transcriptomes, under regulation of shared regulatory elements and Krüppel-like factors (KLFs) transcription factors. High-throughput bulk and single-cell RNA sequencing of humeral attachment cells revealed expression of hundreds of chondrogenic and tenogenic genes, which was validated by in situ hybridization and single-molecule ISH. ATAC sequencing showed that attachment cells share accessible intergenic chromatin areas with either tenocytes or chondrocytes. Epigenomic analysis revealed enhancer signatures for most of these regions. Transgenic mouse enhancer reporter assays verified the shared activity of some of these enhancers. Finally, integrative chromatin and motif analyses and transcriptomic data implicated KLFs as regulators of attachment cells. Indeed, blocking expression of both Klf2 and Klf4 in developing limb mesenchyme impaired their differentiation., Competing Interests: SK, TO, MO, SM, DL, SK, RB, SB, LF, HK, TS, TC, SI, IA, AV, EZ No competing interests declared, (© 2021, Kult et al.)

Acute liver failure (ALF) is a fulminant complication of multiple etiologies, characterized by rapid hepatic destruction, multi-organ failure and mortality. ALF treatment is mainly limited to supportive care and liver transplantation. Here we utilize the acetaminophen (APAP) and thioacetamide (TAA) ALF models in characterizing 56,527 single-cell transcriptomes to define the mouse ALF cellular atlas. We demonstrate that unique, previously uncharacterized stellate cell, endothelial cell, Kupffer cell, monocyte and neutrophil subsets, and their intricate intercellular crosstalk, drive ALF. We unravel a common MYC-dependent transcriptional program orchestrating stellate, endothelial and Kupffer cell activation during ALF, which is regulated by the gut microbiome through Toll-like receptor (TLR) signaling. Pharmacological inhibition of MYC, upstream TLR signaling checkpoints or microbiome depletion suppress this cell-specific, MYC-dependent program, thereby attenuating ALF. In humans, we demonstrate upregulated hepatic MYC expression in ALF transplant recipients compared to healthy donors. Collectively we demonstrate that detailed cellular/genetic decoding may enable pathway-specific ALF therapeutic intervention.

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., Competing Interests: Declaration of Interests A patent application has been filed related to this work., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The emerging appreciation of plasticity among pancreatic lineages has created interest in harnessing cellular reprogramming for β cell replacement therapy of diabetes. Current reprogramming methodologies are inefficient, largely because of a limited understanding of the underlying mechanisms. Using an in vitro reprogramming system, we reveal the transcriptional repressor RE-1 silencing transcription factor (REST) as a barrier for β cell gene expression in the reprogramming of pancreatic exocrine cells. We observe that REST-bound loci lie adjacent to the binding sites of multiple key β cell transcription factors, including PDX1. Accordingly, a loss of REST function combined with PDX1 expression results in the synergistic activation of endocrine genes. This is accompanied by increased histone acetylation and PDX1 binding at endocrine gene loci. Collectively, our data identify a mechanism for REST activity involving the prevention of PDX1-mediated activation of endocrine genes and uncover REST downregulation and the resulting chromatin alterations as key events in β cell reprogramming., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Crosstalk between neighboring cells underlies many biological processes, including cell signaling, proliferation and differentiation. Current single-cell genomic technologies profile each cell separately after tissue dissociation, losing information on cell-cell interactions. In the present study, we present an approach for sequencing physically interacting cells (PIC-seq), which combines cell sorting of physically interacting cells (PICs) with single-cell RNA-sequencing. Using computational modeling, PIC-seq systematically maps in situ cellular interactions and characterizes their molecular crosstalk. We apply PIC-seq to interrogate diverse interactions including immune-epithelial PICs in neonatal murine lungs. Focusing on interactions between T cells and dendritic cells (DCs) in vitro and in vivo, we map T cell-DC interaction preferences, and discover regulatory T cells as a major T cell subtype interacting with DCs in mouse draining lymph nodes. Analysis of T cell-DC pairs reveals an interaction-specific program between pathogen-presenting migratory DCs and T cells. PIC-seq provides a direct and broadly applicable technology to characterize intercellular interaction-specific pathways at high resolution.

The responses of cells to their surroundings are mediated by the binding of cell surface proteins (CSPs) to extracellular signals. Such processes are regulated via dynamic changes in the structure, composition, and expression levels of CSPs. In this study, we demonstrate the possibility of decorating bacteria with artificial, self-assembled receptors that imitate the dynamic features of CSPs. We show that the local concentration of these receptors on the bacterial membrane and their structure can be reversibly controlled using suitable chemical signals, in a way that resembles changes that occur with CSP expression levels or posttranslational modifications (PTMs), respectively. We also show that these modifications can endow the bacteria with programmable properties, akin to the way CSP responses can induce cellular functions. By programming the bacteria to glow, adhere to surfaces, or interact with proteins or mammalian cells, we demonstrate the potential to tailor such biomimetic systems for specific applications.

The cullin-RING ubiquitin E3 ligase (CRL) family consists of ~250 complexes that catalyze ubiquitylation of proteins to achieve cellular regulation. All CRLs are inhibited by the COP9 signalosome complex (CSN) through both enzymatic (deneddylation) and nonenzymatic (steric) mechanisms. The relative contribution of these two mechanisms is unclear. Here, we decouple the mechanisms using CSNAP, the recently discovered ninth subunit of the CSN. We find that CSNAP reduces the affinity of CSN toward CRL complexes. Removing CSNAP does not affect deneddylation, but leads to global effects on the CRL, causing altered reproductive capacity, suppressed DNA damage response, and delayed cell cycle progression. Thus, although CSNAP is only 2% of the CSN mass, it plays a critical role in the steric regulation of CRLs by the CSN.

Recruited blood monocytes contribute to the establishment, perpetuation, and resolution of tissue inflammation. Specifically, in the inflamed intestine, monocyte ablation was shown to ameliorate colitis scores in preclinical animal models. However, the majority of intestinal macrophages that seed the healthy gut are also monocyte derived. Monocyte ablation aimed to curb inflammation would therefore likely interfere with intestinal homeostasis. In this study, we used a TLR2 trans -membrane peptide that blocks TLR2 dimerization that is critical for TLR2/1 and TLR2/6 heterodimer signaling to blunt inflammation in a murine colitis model. We show that although the TLR2 peptide treatment ameliorated colitis, it allowed recruited monocytes to give rise to macrophages that lack the detrimental proinflammatory gene signature and reduced potentially damaging neutrophil infiltrates. Finally, we demonstrate TLR blocking activity of the peptide on in vitro cultured human monocyte-derived macrophages. Collectively, we provide a significantly improved anti-inflammatory TLR2 peptide and critical insights in its mechanism of action toward future potential use in the clinic., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Extracellular matrix (ECM) plays an important role in tumor development and dissemination, but few points of therapeutic intervention targeting ECM of the tumor microenvironment have been exploited to date. Recent observations suggest that the enzymatic introduction of disulfide bond cross-links into the ECM may be modulated to affect cancer progression. Specifically, the disulfide bond-forming activity of the enzyme Quiescin sulfhydryl oxidase 1 (QSOX1) is required by fibroblasts to assemble ECM components for adhesion and migration of cancer cells. Based on this finding and the increased QSOX1 expression in the stroma of aggressive breast carcinomas, we developed monoclonal antibody inhibitors with the aim of preventing QSOX1 from participating in pro-metastatic ECM remodeling. Here we show that QSOX1 inhibitory antibodies decreased tumor growth and metastasis in murine cancer models and had added benefits when provided together with chemotherapy. Mechanistically, the inhibitors dampened stromal participation in tumor development, as the tumors of treated animals showed fewer myofibroblasts and poorer ECM organization. Thus, our findings demonstrate that specifically targeting excess stromal QSOX1 secreted in response to tumor-cell signaling provides a means to modulate the tumor microenvironment and may complement other therapeutic approaches in cancer., Competing Interests: CONFLICTS OF INTEREST Deborah Fass, Tal Ilani, and Iris Grossman are listed as inventors on a patent for an antibody inhibitor of QSOX1 and uses of the same.

Monocytes are circulating short-lived macrophage precursors that are recruited on demand from the blood to sites of inflammation and challenge. In steady state, classical monocytes give rise to vasculature-resident cells that patrol the luminal side of the endothelium. In addition, classical monocytes feed macrophage compartments of selected organs, including barrier tissues, such as the skin and intestine, as well as the heart. Monocyte differentiation under conditions of inflammation has been studied in considerable detail. In contrast, monocyte differentiation under non-inflammatory conditions remains less well understood. Here we took advantage of a combination of cell ablation and precursor engraftment to investigate the generation of gut macrophages from monocytes. Collectively, we identify factors associated with the gradual adaptation of monocytes to tissue residency. Moreover, comparison of monocyte differentiation into the colon and ileum-resident macrophages revealed the graduated acquisition of gut segment-specific gene expression signatures., Competing Interests: MG, ST, AS, BB, CC, GS, TS, ED, SB, LC, DL, SJ No competing interests declared, (© 2020, Gross-Vered et al.)

Bone protrusions provide stable anchoring sites for ligaments and tendons and define the unique morphology of each long bone. Despite their importance, the mechanism by which superstructures are patterned is unknown. Here, we identify components of the genetic program that control the patterning of Sox9 + / Scx + superstructure progenitors in mouse and show that this program includes both global and regional regulatory modules. Using light-sheet fluorescence microscopy combined with genetic lineage labeling, we mapped the broad contribution of the Sox9 + / Scx + progenitors to the formation of bone superstructures. Then, by combining literature-based evidence, comparative transcriptomic analysis and genetic mouse models, we identified Gli3 as a global regulator of superstructure patterning, whereas Pbx1 , Pbx2 , Hoxa11 and Hoxd11 act as proximal and distal regulators, respectively. Moreover, by demonstrating a dose-dependent pattern regulation in Gli3 and Pbx1 compound mutations, we show that the global and regional regulatory modules work in a coordinated manner. Collectively, our results provide strong evidence for genetic regulation of superstructure patterning, which further supports the notion that long bone development is a modular process.This article has an associated 'The people behind the papers' interview., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The 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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: Because of emergence of resistance to osimertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI), no targeted treatments are available for patients with lung cancer who lose sensitivity due to new mutations or bypass mechanisms. We examined in animals and in vitro an alternative therapeutic approach making use of antibodies. Experimental Design: An osimertinib-sensitive animal model of lung cancer, which rapidly develops drug resistance, has been employed. To overcome compensatory hyperactivation of ERK, which we previously reported, an anti-EGFR antibody (cetuximab) was combined with other antibodies, as well as with a subtherapeutic dose of osimertinib, and cancer cell apoptosis was assayed. Results: Our animal studies identified a combination of three clinically approved drugs, cetuximab, trastuzumab (an anti-HER2 mAb), and osimertinib (low dose), as an effective and long-lasting treatment that is able to prevent onset of resistance to osimertinib. A continuous schedule of concurrent treatment was sufficient for effective tumor inhibition and for prevention of relapses. Studies employing cultured cells and analyses of tumor extracts indicated that the combination of two mAbs and a subtherapeutic TKI dose sorted EGFR and HER2 for degradation; cooperatively enhanced apoptosis; inhibited activation of ERK; and reduced abundance of several bypass proteins, namely MET, AXL, and HER3. Conclusions: Our in vitro assays and animal studies identified an effective combination of clinically approved drugs that might overcome resistance to irreversible TKIs in clinical settings. The results we present attribute the long-lasting effect of the drug combination to simultaneous blockade of several well-characterized mechanisms of drug resistance. Clin Cancer Res; 24(22); 5610-21. ©2018 AACR See related commentary by Fan and Yu, p. 5499 ., (©2018 American Association for Cancer Research.)

Lung development and function arises from the interactions between diverse cell types and lineages. Using single-cell RNA sequencing (RNA-seq), we characterize the cellular composition of the lung during development and identify vast dynamics in cell composition and their molecular characteristics. Analyzing 818 ligand-receptor interaction pairs within and between cell lineages, we identify broadly interacting cells, including AT2, innate lymphocytes (ILCs), and basophils. Using interleukin (IL)-33 receptor knockout mice and in vitro experiments, we show that basophils establish a lung-specific function imprinted by IL-33 and granulocyte-macrophage colony-stimulating factor (GM-CSF), characterized by unique signaling of cytokines and growth factors important for stromal, epithelial, and myeloid cell fates. Antibody-depletion strategies, diphtheria toxin-mediated selective depletion of basophils, and co-culture studies show that lung resident basophils are important regulators of alveolar macrophage development and function. Together, our study demonstrates how whole-tissue signaling interaction map on the single-cell level can broaden our understanding of cellular networks in health and disease., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: During the process of bioethanol production, cellulose is hydrolyzed into its monomeric soluble units. For efficient hydrolysis, a chemical and/or mechanical pretreatment step is required. Such pretreatment is designed to increase enzymatic digestibility of the cellulose chains inter alia by de-crystallization of the cellulose chains and by removing barriers, such as lignin from the plant cell wall. Biological pretreatment, in which lignin is decomposed or modified by white-rot fungi, has also been considered. One disadvantage in biological pretreatment, however, is the consumption of the cellulose by the fungus. Thus, fungal species that attack lignin with only minimal cellulose loss are advantageous. The secretomes of white-rot fungi contain carbohydrate-active enzymes (CAZymes) including lignin-modifying enzymes. Thus, modification of secretome composition can alter the ratio of lignin/cellulose degradation., Results: Pleurotus ostreatus PC9 was genetically modified to either overexpress or eliminate (by gene replacement) the transcriptional regulator CRE1, known to act as a repressor in the process of carbon catabolite repression. The cre1 -overexpressing transformant demonstrated lower secreted cellulolytic activity and slightly increased selectivity (based on the chemical composition of pretreated wheat straw), whereas the knockout transformant demonstrated increased cellulolytic activity and significantly reduced residual cellulose, thereby displaying lower selectivity. Pretreatment of wheat straw using the wild-type PC9 resulted in 2.8-fold higher yields of soluble sugar compared to untreated wheat straw. The overexpression transformant showed similar yields (2.6-fold), but the knockout transformant exhibited lower yields (1.2-fold) of soluble sugar. Based on proteomic secretome analysis, production of numerous CAZymes was affected by modification of the expression level of cre1 ., Conclusions: The gene cre1 functions as a regulator for expression of fungal CAZymes active against plant cell wall lignocelluloses, hence altering the substrate preference of the fungi tested. While the cre1 knockout resulted in a less efficient biological pretreatment, i.e., less saccharification of the treated biomass, the converse manipulation of cre1 (overexpression) failed to improve efficiency. Despite the inverse nature of the two genetic alterations, the expected "mirror image" (i.e., opposite regulatory response) was not observed, indicating that the secretion level of CAZymes, was not exclusively dependent on CRE1 activity.

Epidermal growth factor receptor ( EGFR ) mutations identify patients with lung cancer who derive benefit from kinase inhibitors. However, most patients eventually develop resistance, primarily due to the T790M second-site mutation. Irreversible inhibitors (e.g., osimertinib/AZD9291) inhibit T790M-EGFR, but several mechanisms, including a third-site mutation, C797S, confer renewed resistance. We previously reported that a triple mixture of monoclonal antibodies, 3×mAbs, simultaneously targeting EGFR, HER2, and HER3, inhibits T790M-expressing tumors. We now report that 3×mAbs, including a triplet containing cetuximab and trastuzumab, inhibits C797S-expressing tumors. Unlike osimertinib, which induces apoptosis, 3×mAbs promotes degradation of the three receptors and induces cellular senescence. Consistent with distinct mechanisms, treatments combining 3×mAbs plus sub-inhibitory doses of osimertinib synergistically and persistently eliminated tumors. Thus, oligoclonal antibodies, either alone or in combination with kinase inhibitors, might preempt repeated cycles of treatment and rapid emergence of resistance., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

To provide new tools for in vitro and in vivo prolactin (PRL) research, novel protocols for large-scale preparation of untagged human PRL (hPRL), a hPRL antagonist (del 1-9-G129R hPRL) that acts as a pure antagonist of hPRL in binding to hPRL receptor extracellular domain (hPRLR-ECD), and hPRLR-ECD are demonstrated. The interaction of del 1-9-G129R hPRL with hPRLR-ECD was demonstrated by competitive non-radioactive binding assay using biotinylated hPRL as the ligand and hPRLR-ECD as the receptor, by formation of stable 1:1 complexes with hPRLR-ECD under non-denaturing conditions using size-exclusion chromatography, and by surface plasmon resonance methodology. In all three types of experiments, the interaction of del 1-9-G129R hPRL was equal to that of unmodified hPRL. Del 1-9-G129R hPRL inhibited the hPRL-induced proliferation of Baf/LP cells stably expressing hPRLR. Overall, the biological properties of del 1-9-G129R hPRL prepared by the protocol described herein were similar to those of the antagonist prepared using the protocol reported in the original study; however, the newly described protocol improved yields by >6-fold. To provide long-lasting hPRL as a new reagent needed for in vivo experiments, we prepared its mono-pegylated analogue and found that pegylation lowers its biological activity in a homologous in vitro assay. As its future use will require the development of a PRL antagonist with highly elevated affinity, del 1-9-G129R hPRL was expressed on the surface of yeast cells. It retained its binding capacity for hPRLR-ECD, and this methodology was shown to be suitable for future development of high-affinity hPRL antagonists using a library of randomly mutated open reading frame of del 1-9-G129R hPRL and selecting high-affinity mutants by yeast surface display methodology., (© The Author(s) 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Cellular functions are strongly dependent on surrounding cells and environmental factors. Current technologies are limited in their ability to characterize the spatial location and gene programs of cells in poorly structured and dynamic niches. We developed a method, NICHE-seq, that combines photoactivatable fluorescent reporters, two-photon microscopy, and single-cell RNA sequencing (scRNA-seq) to infer the cellular and molecular composition of niches. We applied NICHE-seq to examine the high-order assembly of immune cell networks. NICHE-seq is highly reproducible in spatial tissue reconstruction, enabling identification of rare niche-specific immune subpopulations and gene programs, including natural killer cells within infected B cell follicles and distinct myeloid states in the spleen and tumor. This study establishes NICHE-seq as a broadly applicable method for elucidating high-order spatial organization of cell types and their molecular pathways., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Cytotoxic T lymphocytes (CTLs) used in immunotherapy are typically cultured under atmospheric O 2 pressure but encounter hypoxic conditions inside tumors. Activating CTLs under hypoxic conditions has been shown to improve their cytotoxicity in vitro, but the mechanism employed and the implications for immunotherapy remain unknown. We activated and cultured OT-I CD8 T cells at either 1% or 20% O 2 . Hypoxic CTLs survived, as well as normoxic ones, in vitro but killed OVA-expressing B16 melanoma cells more efficiently. Hypoxic CTLs contained similar numbers of cytolytic granules and released them as efficiently but packaged more granzyme-B in each granule without producing more perforin. We imaged CTL distribution and motility inside B16-OVA tumors using confocal and intravital 2-photon microscopy and observed no obvious differences. However, mice treated with hypoxic CTLs exhibited better tumor regression and survived longer. Thus, hypoxic CTLs may perform better in tumor immunotherapy because of higher intrinsic cytotoxicity rather than improved migration inside tumors., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Finding intracellular pathways and molecules that can prevent the proliferation of colon cancer cells can provide significant bases for developing treatments for this disease. Ostreolysin (Oly) is a protein found in the mushroom Pleurotus ostreatus, and we have produced a recombinant version of this protein (rOly).We measured the viability of several colon cancer cells treated with rOly. Xenografts and syngeneic colon cancer cells were injected into in vivo mouse models, which were then treated with this recombinant protein.rOly treatment induced a significant reduction in viability of human and mouse colon cancer cells. In contrast, there was no reduction in the viability of normal epithelial cells from the small intestine. In the search for cellular targets of rOly, we showed that it enhances the anti-proliferative activity of drugs targeting cellular tubulin. This was accompanied by a reduction in the weight and volume of tumours in mice injected with rOly as compared to their respective control mice in two in vivo models.Our results advance the functional understanding of rOly as a potential anti-cancer treatment associated with pro-apoptotic activities preferentially targeting colon cancer cells.

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Innate lymphoid cells (ILCs) are critical modulators of mucosal immunity, inflammation, and tissue homeostasis, but their full spectrum of cellular states and regulatory landscapes remains elusive. Here, we combine genome-wide RNA-seq, ChIP-seq, and ATAC-seq to compare the transcriptional and epigenetic identity of small intestinal ILCs, identifying thousands of distinct gene profiles and regulatory elements. Single-cell RNA-seq and flow and mass cytometry analyses reveal compartmentalization of cytokine expression and metabolic activity within the three classical ILC subtypes and highlight transcriptional states beyond the current canonical classification. In addition, using antibiotic intervention and germ-free mice, we characterize the effect of the microbiome on the ILC regulatory landscape and determine the response of ILCs to microbial colonization at the single-cell level. Together, our work characterizes the spectrum of transcriptional identities of small intestinal ILCs and describes how ILCs differentially integrate signals from the microbial microenvironment to generate phenotypic and functional plasticity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Recent single-cell analysis technologies offer an unprecedented opportunity to elucidate developmental pathways. Here we present Wishbone, an algorithm for positioning single cells along bifurcating developmental trajectories with high resolution. Wishbone uses multi-dimensional single-cell data, such as mass cytometry or RNA-Seq data, as input and orders cells according to their developmental progression, and it pinpoints bifurcation points by labeling each cell as pre-bifurcation or as one of two post-bifurcation cell fates. Using 30-channel mass cytometry data, we show that Wishbone accurately recovers the known stages of T-cell development in the mouse thymus, including the bifurcation point. We also apply the algorithm to mouse myeloid differentiation and demonstrate its generalization to additional lineages. A comparison of Wishbone to diffusion maps, SCUBA and Monocle shows that it outperforms these methods both in the accuracy of ordering cells and in the correct identification of branch points.

Despite initial responses to targeted kinase inhibitors, lung cancer patients presenting with primary epidermal growth factor receptor (EGFR) mutations acquire resistance, often due to a second-site mutation (T790M). However, clinical trials found no survival benefits in patients treated with a monoclonal antibody (mAb) to EGFR that should block activation of the mutated receptor and thus bypass resistance to molecules that target the catalytic or ATP-binding site. Using cell lines with the T790M mutation, we discovered that prolonged exposure to mAbs against only the EGFR triggered network rewiring by (i) stimulating the extracellular signal-regulated kinase (ERK) pathway; (ii) inducing the transcription of HER2 (human epidermal growth factor receptor 2) and HER3, which encode other members of the EGFR family, and the gene encoding HGF, which is the ligand for the receptor tyrosine kinase MET; and (iii) stimulating the interaction between MET and HER3, which promoted MET activity. Supplementing the EGFR-specific mAb with those targeting HER2 and HER3 suppressed these compensatory feedback loops in cultured lung cancer cells. The triple mAb combination targeting all three receptors prevented the activation of ERK, accelerated the degradation of the receptors, inhibited the proliferation of tumor cells but not of normal cells, and markedly reduced the growth of tumors in mice xenografted with cells that were resistant to combined treatment with erlotinib and the single function-blocking EGFR mAb. These findings uncovered feedback loops that enable resistance to treatment paradigms that use a single antibody and indicate a new strategy for the treatment of lung cancer patients., (Copyright © 2015, American Association for the Advancement of Science.)