Search

Recent single-cell studies of cancer in both mice and humans have identified the emergence of a myofibroblast population specifically marked by the highly restricted leucine-rich-repeat-containing protein 15 (LRRC15)1–3. However, the molecular signals that underlie the development of LRRC15+ cancer-associated fibroblasts (CAFs) and their direct impact on anti-tumour immunity are uncharacterized. Here in mouse models of pancreatic cancer, we provide in vivo genetic evidence that TGFβ receptor type 2 signalling in healthy dermatopontin+ universal fibroblasts is essential for the development of cancer-associated LRRC15+ myofibroblasts. This axis also predominantly drives fibroblast lineage diversity in human cancers. Using newly developed Lrrc15–diphtheria toxin receptor knock-in mice to selectively deplete LRRC15+ CAFs, we show that depletion of this population markedly reduces the total tumour fibroblast content. Moreover, the CAF composition is recalibrated towards universal fibroblasts. This relieves direct suppression of tumour-infiltrating CD8+ T cells to enhance their effector function and augments tumour regression in response to anti-PDL1 immune checkpoint blockade. Collectively, these findings demonstrate that TGFβ-dependent LRRC15+ CAFs dictate the tumour-fibroblast setpoint to promote tumour growth. These cells also directly suppress CD8+ T cell function and limit responsiveness to checkpoint blockade. Development of treatments that restore the homeostatic fibroblast setpoint by reducing the population of pro-disease LRRC15+ myofibroblasts may improve patient survival and response to immunotherapy.

Fibroblasts are non-haematopoietic structural cells that define the architecture of organs, support the homeostasis of tissue-resident cells and have key roles in fibrosis, cancer, autoimmunity and wound healing1. Recent studies have described fibroblast heterogeneity within individual tissues1. However, the field lacks a characterization of fibroblasts at single-cell resolution across tissues in healthy and diseased organs. Here we constructed fibroblast atlases by integrating single-cell transcriptomic data from about 230,000 fibroblasts across 17 tissues, 50 datasets, 11 disease states and 2 species. Mouse fibroblast atlases and a DptIRESCreERT2 knock-in mouse identified two universal fibroblast transcriptional subtypes across tissues. Our analysis suggests that these cells can serve as a reservoir that can yield specialized fibroblasts across a broad range of steady-state tissues and activated fibroblasts in disease. Comparison to an atlas of human fibroblasts from perturbed states showed that fibroblast transcriptional states are conserved between mice and humans, including universal fibroblasts and activated phenotypes associated with pathogenicity in human cancer, fibrosis, arthritis and inflammation. In summary, a cross-species and pan-tissue approach to transcriptomics at single-cell resolution has identified key organizing principles of the fibroblast lineage in health and disease. Single-cell and genetic tools are used to characterize the diversity of fibroblasts across healthy and perturbed tissues in mice and humans.

Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell-zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreERT2 knock-in mice enabled localization, multi-omics characterization and genetic depletion of Grem1+ FRCs. Grem1+ FRCs primarily localize at T-B cell junctions of SLOs, neighboring pre-dendritic cells and conventional dendritic cells (cDCs). As such, their depletion resulted in preferential loss and decreased homeostatic proliferation and survival of resident cDCs and compromised T cell immunity. Trajectory analysis of human LN scRNA-seq data revealed expression similarities to murine FRCs, with GREM1+ cells marking the endpoint of both trajectories. These findings illuminate a new Grem1+ fibroblastic niche in LNs that functions to maintain the homeostasis of lymphoid tissue-resident cDCs.

Recent single-cell studies of cancer in both mice and humans have identified the emergence of a myofibroblast population specifically marked by the highly restricted leucine-rich-repeat-containing protein 15 (LRRC15)

Immunotherapies have shown unprecedented clinical benefits in several malignancies1–3. However, clinical responses remain variable and unpredictable, indicating the need to develop predictive platforms that can improve patient stratification4. Phenotyping of tumors into hot, altered, or cold5 based on assessment of only T-cell infiltration in static tumor biopsies provides suboptimal prediction of immunotherapy response6,7. In vivo dynamic mechanisms within the tumor microenvironment such as tumor angiogenesis and leukocyte trafficking5,8,9 also play a central role in modulating anti-tumor immunity and therefore immunotherapy response. Here, we report novel tumor immune microenvironment (TiME) phenotyping in vivo in patients with non-invasive spatially-resolved cellular-level imaging based on endogenous contrast. Investigating skin cancers as a model, with reflectance confocal microscopy (RCM) imaging10, we determined four major phenotypes with variable prevalence of vasculature (Vasc) and inflammation (Inf) features: VaschiInfhi, VaschiInflo, VascloInfhi and Vascmed/hiInflo. The VaschiInfhi phenotype correlates with high immune activation, exhaustion, and vascular signatures while VaschiInflo with endothelial anergy and immune exclusion. Automated quantification of TiME features demonstrates moderate-high accuracy and correlation with corresponding gene expression. Prospectively analyzed response to topical immunotherapy show highest response in VascloInfhi, and reveals the added value of vascular features in predicting treatment response. Our novel in vivo cellular-level imaging and phenotyping approach can potentially advance our fundamental understanding of TiME, develop robust predictors for immunotherapy outcomes and identify novel targetable pathways in future.

In vivo phenotyping of tumor-immune microenvironment (TiME) for predicting response to immunotherapy in patients using non-invasive reflectance confocal microscopy (RCM) demonstrates high molecular and treatment response correlation.

The FoxA transcription factors are critical for liver development through their pioneering activity, which initiates a highly complex regulatory network thought to become progressively resistant to the loss of any individual hepatic transcription factor via mutual redundancy. To investigate the dispensability of FoxA factors for maintaining this regulatory network, we ablated all FoxA genes in the adult mouse liver. Remarkably, loss of FoxA caused rapid hepatocyte dedifferentiation manifested by a massive reduction in the expression of key liver genes. Interestingly, expression of these genes was reduced back to the low levels of the fetal prehepatic endoderm stage, leading to necrosis and lethality within days. Mechanistically, we found FoxA proteins to be required for maintaining enhancer activity, chromatin accessibility, nucleosome positioning and binding by HNF4α. Thus, the FoxA factors act continuously, guarding hepatic enhancer activity throughout life.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 activation (CRISPRa) systems have enabled genetic screens in cultured cell lines to discover and characterize drivers and inhibitors of cancer cell growth. We adapted this system for use in vivo to assess whether modulating endogenous gene expression levels can result in functional outcomes in the native environment of the liver. We engineered the catalytically dead CRISPR-associated 9 (dCas9)-positive mouse, cyclization recombination-inducible (Cre) CRISPRa system for cell type-specific gene activation in vivo. We tested the capacity for genetic screening in live animals by applying CRISPRa in a clinically relevant model of liver injury and repopulation. We targeted promoters of interest in regenerating hepatocytes using multiple single guide RNAs (gRNAs), and employed high-throughput sequencing to assess enrichment of gRNA sequences during liver repopulation and to link specific gRNAs to the initiation of carcinogenesis. All components of the CRISPRa system were expressed in a cell type-specific manner and activated endogenous gene expression in vivo. Multiple gRNA cassettes targeting a proto-oncogene were significantly enriched following liver repopulation, indicative of enhanced division of cells expressing the proto-oncogene. Furthermore, hepatocellular carcinomas developed containing gRNAs that activated this oncogene, indicative of cancer initiation events. Also, we employed our system for combinatorial cancer genetics in vivo as we found that while clonal hepatocellular carcinomas were dependent on the presence of the oncogene-inducing gRNAs, they were depleted for multiple gRNAs activating tumor suppressors.ConclusionThe in vivo CRISPRa platform developed here allows for parallel and combinatorial genetic screens in live animals; this approach enables screening for drivers and suppressors of cell replication and tumor initiation. (Hepatology 2017).

The liver’s extraordinary ability to regenerate has been known since the myth of Prometheus, but the mechanisms involved are still being discovered. Various small animal models have been used in this quest. Two of the most popular include partial hepatectomy (PHx), in which two-thirds of the liver mass is surgically removed to evoke a massive, immediate stimulus for regeneration, and prolonged exposure to toxins that kill liver cells more gradually, provoking chronic regenerative activity. In either case, multiple types of cells must interact effectively to repopulate the organ with functional mature hepatocytes and thus assure ultimate restoration of healthy liver structure and function. This complexity has confounded efforts to distinguish specific changes that occur in cells that repopulate the hepatocyte compartment from changes in other cell populations, including subpopulations of hepatocytes or hepatocyte precursors that do not become regenerative. In the current issue of the JCI, Wang et al. used translating ribosome affinity purification followed by high-throughput RNA sequencing (TRAP-seq) to isolate mRNAs from repopulating hepatocytes in order to profile gene expression specifically in the hepatocytes that regenerate the liver following toxic injury imposed by inherent byproducts of tyrosine metabolism. This innovative methodology can potentially be used to design therapeutic strategies for liver regeneration.

Background: Preclinical studies have shown that activation of the glucocorticoid receptor (GR) leads to resistance to antiandrogens, which is reversed by GR inhibition. ORIC-101 is a potent, selective, orally bioavailable small molecule GR antagonist undergoing clinical development in combination with enzalutamide in patients with metastatic prostate cancer (NCT04033328). Methods: Ten patients were enrolled in the dose escalation portion of the Phase 1b study during which three dose levels of ORIC-101 given once daily were evaluated in combination with 160 mg enzalutamide. Translational markers were measured both pre-treatment and on study, including PD biomarkers, blood cortisol, GR and AR protein levels, and genomic alterations. PD biomarkers FKBP5, GILZ and PER1 were assessed by RT-qPCR in peripheral blood mononuclear cells (PBMCs), collected on multiple days and times in the first two cycles before and after dosing. PD modulation and target coverage were evaluable in 9 out of 10 patients. In tumor biopsies with >50 tumor cells, GR, AR and PTEN protein status were evaluated by immunohistochemistry (IHC). Liquid biopsies were collected before treatment, at the end of Cycle 2, and/or the end of treatment: nuclear GR and ARv7 protein levels were measured on circulating tumor cells (CTCs) by immunofluorescence (Epic Sciences, San Diego, CA), mutations and copy number alterations in cancer genes were captured in plasma with the 500-gene GuardantOMNI® ctDNA assay (Redwood City, CA). Results: PD suppression after one dose of ORIC-101 was observed in PBMCs of all patients. ORIC-101 reached steady-state target engagement within the first cycle in 6 out of 9 evaluable patients across dose levels, including at the RP2D. Nuclear GR protein was detected by IHC in the 6 pre-treatment biopsies, with H-scores ranging from 11 to 298 (median 95, IQR 55-148). Reduction of GR protein was observed in 3 out of 4 matched on study biopsies (pre-treatment H-scores 110-298), indicating potential elimination of GR positive tumor cells, while the fourth patient had low baseline GR levels (H-score 11). Pre-treatment AR protein levels were consistently high (IHC H-scores 230-300). AR alterations observed in ctDNA or CTCs were L702H (n=1 patient), H875Y (n=1), amplification (n=2), and ARv7 (n=2). PTEN protein aberrations were identified in two patients. The amount of tumor shedding in this cohort ranged from 0.24 to 82% (median 2.2%, IQR 0.54-12.1%), comparable to a clinical cohort of ~10,000 prostate tumors profiled with Guardant360® (Guardant Health). Prevalent genomic alterations at time of enrollment included TP53, RB1, BRCA2, ATM and SPOP, with minimal new genomic changes occurring on treatment. Conclusions: Biomarker data from patients enrolled in the Phase 1b study provide evidence of PD modulation, steady-state target coverage, and on-target tumor cell eradication at all dose levels. Tumor characterization in association with clinical outcome provides an opportunity to refine the patient population during the ongoing dose expansion portion of the study. Citation Format: Anneleen Daemen, Shravani Barkund, Ann Johnson, Aleksandr Pankov, Amber W. Wang, Haiying Zhou, Pratik S. Multani, Edna Chow Maneval, Lori S. Friedman, Rupal Patel. Biomarker results supporting selection of RP2D from a phase 1b study of ORIC-101, a glucocorticoid receptor antagonist, in combination with enzalutamide in patients with metastatic prostate cancer progressing on enzalutamide [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P015.

Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell-zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreER

Liver regeneration is impaired in mice with hepatocyte-specific deficiencies in microRNA (miRNA) processing; yet the roles of individual miRNAs or their combinatorial effects in this process are largely unknown. In this study, we sought to identify miRNAs that regulate hepatocyte repopulation following toxic liver injury in a high-throughput manner using the Fah−/− mouse. We constructed plasmid pools encoding over 30,000 tough decoy (TuD) miRNA inhibitors designed to target hepatocyte miRNAs in a pairwise manner. Plasmid libraries were delivered to hepatocytes of Fah−/− mice at the time of liver injury via hydrodynamic tail vein injection and integrated transgene-containing transposons were quantified following repopulation via high-throughput sequencing. Changes in polysome-bound transcripts following miRNA inhibition were determined using translating ribosome affinity purification followed by high-throughput sequencing. Analysis of TuD abundance in hepatocyte genomic DNA and input plasmid pools identified several thousand miRNA inhibitors that were significantly altered following repopulation. We classified a subset of miRNA-binding sites (MBSs) as having strong effect on liver repopulation, thus implicating the targeted hepatocyte miRNAs as regulators of this process. Furthermore, we generated a high-content map of pairwise interactions between 171 MBSs and identified both synergistic and redundant effects. Our study highlights the power of higher-order screens to uncover miRNA functions that would go undetected by individual miRNA perturbations, and provides a new paradigm for the study of epistasis of miRNA activities.

Background & Aims The adult liver is the main detoxification organ and routinely is exposed to environmental insults but retains the ability to restore its mass and function upon tissue damage. However, extensive injury can lead to liver failure, and chronic injury causes fibrosis, cirrhosis, and hepatocellular carcinoma. Currently, the transcriptional regulation of organ repair in the adult liver is incompletely understood. Methods We isolated nuclei from quiescent as well as repopulating hepatocytes in a mouse model of hereditary tyrosinemia, which recapitulates the injury and repopulation seen in toxic liver injury in human beings. We then performed the assay for transposase accessible chromatin with high-throughput sequencing specifically in repopulating hepatocytes to identify differentially accessible chromatin regions and nucleosome positioning. In addition, we used motif analysis to predict differential transcription factor occupancy and validated the in silico results with chromatin immunoprecipitation followed by sequencing for hepatocyte nuclear factor 4α (HNF4α) and CCCTC-binding factor (CTCF). Results Chromatin accessibility in repopulating hepatocytes was increased in the regulatory regions of genes promoting proliferation and decreased in the regulatory regions of genes involved in metabolism. The epigenetic changes at promoters and liver enhancers correspond with the regulation of gene expression, with enhancers of many liver function genes showing a less accessible state during the regenerative process. Moreover, increased CTCF occupancy at promoters and decreased HNF4α binding at enhancers implicate these factors as key drivers of the transcriptomic changes in replicating hepatocytes that enable liver repopulation. Conclusions Our analysis of hepatocyte-specific epigenomic changes during liver repopulation identified CTCF and HNF4α as key regulators of hepatocyte proliferation and regulation of metabolic programs. Thus, liver repopulation in the setting of toxic injury makes use of both general transcription factors (CTCF) for promoter activation, and reduced binding by a hepatocyte-enriched factor (HNF4α) to temporarily limit enhancer activity. All sequencing data in this study were deposited to the Gene Expression Omnibus database and can be downloaded with accession number GSE109466., Graphical abstract

Background & Aims Liver regeneration is impaired in mice with hepatocyte-specific deficiencies in microRNA (miRNA) processing, but it is not clear which miRNAs regulate this process. We developed a high-throughput screen to identify miRNAs that regulate hepatocyte repopulation after toxic liver injury using fumarylacetoacetate hydrolase–deficient mice. Methods We constructed plasmid pools encoding more than 30,000 tough decoy miRNA inhibitors (hairpin nucleic acids designed to specifically inhibit interactions between miRNAs and their targets) to target hepatocyte miRNAs in a pairwise manner. The plasmid libraries were delivered to hepatocytes in fumarylacetoacetate hydrolase–deficient mice at the time of liver injury via hydrodynamic tail-vein injection. Integrated transgene-containing transposons were quantified after liver repopulation via high-throughput sequencing. Changes in polysome-bound transcripts after miRNA inhibition were determined using translating ribosome affinity purification followed by high-throughput sequencing. Results Analyses of tough decoy abundance in hepatocyte genomic DNA and input plasmid pools identified several thousand miRNA inhibitors that were significantly depleted or increased after repopulation. We classified a subset of miRNA binding sites as those that have strong effects on liver repopulation, implicating the targeted hepatocyte miRNAs as regulators of this process. We then generated a high-content map of pairwise interactions between 171 miRNA-binding sites and identified synergistic and redundant effects. Conclusions We developed a screen to identify miRNAs that regulate liver repopulation after injury in live mice.

The loss of insulin-secreting β cells is characteristic among type I and type II diabetes. Stimulating proliferation to expand sources of β cells for transplantation remains a challenge because adult β cells do not proliferate readily. The cell cycle inhibitor p57 has been shown to control cell division in human β cells. Expression of p57 is regulated by the DNA methylation status of the imprinting control region 2 (ICR2), which is commonly hypomethylated in Beckwith-Wiedemann syndrome patients who exhibit massive β cell proliferation. We hypothesized that targeted demethylation of the ICR2 using a transcription activator-like effector protein fused to the catalytic domain of TET1 (ICR2-TET1) would repress p57 expression and promote cell proliferation. We report here that overexpression of ICR2-TET1 in human fibroblasts reduces p57 expression levels and increases proliferation. Furthermore, human islets overexpressing ICR2-TET1 exhibit repression of p57 with concomitant upregulation of Ki-67 while maintaining glucose-sensing functionality. When transplanted into diabetic, immunodeficient mice, the epigenetically edited islets show increased β cell replication compared with control islets. These findings demonstrate that epigenetic editing is a promising tool for inducing β cell proliferation, which may one day alleviate the scarcity of transplantable β cells for the treatment of diabetes.

Development in mammals is accompanied by specific de novo and demethylation events that are thought to stabilize differentiated cell phenotypes. We demonstrate that a large percentage of the tissue-specific methylation pattern is generated postnatally. Demethylation in the liver is observed in thousands of enhancer-like sequences associated with genes that undergo activation during the first few weeks of life. Using a conditional gene ablation strategy we show that the removal of these methyl groups is stable and necessary for assuring proper hepatocyte gene expression and function through its effect on chromatin accessibility. These postnatal changes in methylation come about through exposure to hormone signaling. These results define the molecular rules of 5-methyl-cytosine regulation as an epigenetic mechanism underlying cellular responses to a changing environment., Here the authors show that a large fraction of the tissue-specific methylation pattern is generated postnatally. These changes, which occur in response to hormone signaling, appear to play a major role in the regulation of gene expression and tissue maturation in the liver.

Summary Objective MiR-146a exerts negative control on inflammatory responses by suppressing cytokine-induced expression of interleukin-1 receptor-associated kinase-1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) by impairing NF-κB activity and inhibiting the expression of target genes. Recent study suggests that histone deacetylases (HDACs) are involved in the regulation of microRNA (miRNA) expression. Therefore, we determined whether HDAC inhibitors can increase miR-146a expression, thereby inhibiting interleukin-1β (IL-1β)-induced signaling in osteoarthritis fibroblast-like synoviocytes (OA-FLS). Method MiRNA expression was analyzed using real-time PCR. IL-1β-induced downstream signals and cytokine expression were evaluated using Western blotting and ELISA. Transcription factors regulating promoter activation were identified using chromatin immunoprecipitation assays. Results IL-1β treatment of OA-FLS induced a mild (1.7-fold) increase in miR-146a expression that was unable to appropriately downregulate IRAK1 and TRAF6 expression. HDAC inhibitors, SAHA (vorinostat), and LBH589 (panobinostat) significantly (6.1- and 5.4-fold) elevated miR-146a expression by increasing the binding of the transcription factor NF-κB to the miR-146a promoter, and negatively regulated IL-1β-induced IKK/IκB/p65 phosphorylation signaling and IL-6 secretion. The increase in miR-146a expression induced by the HDAC inhibitors was prevented by transfection of miR-146a inhibitor or HDAC1 (class I HDAC), HDAC4 (class IIa HDAC), and HDAC6 (class IIb HDAC) overexpression, suggesting that they were due to inhibition of HDAC activity. Conclusions Our study demonstrated that HDAC inhibitor treatment in OA-FLS significantly increased miR-146a expression and mediated markedly negative regulation to inhibit IL-1β-induced signaling and cytokine secretion. Our results indicate the potential rationale of anti-inflammatory effects for HDAC inhibitors.

A series of 2-methyl-1H-indol-3-ethylsulfamoylphenylacrylamides based on LBH589–PXD101 core have been synthesized and evaluated for their histone deacetylase (HDAC) inhibitory and anti-inflammatory activity. In vitro, compounds 9–12 show 2.6-fold better HDAC inhibition and 3-fold better IL-6 suppression compared to LBH589·HCl (1·HCl). Furthermore, these compounds did not show apparent cell viability suppression on macrophages while in contrast, treatment with 1·HCl resulted in significant reduction in cell viability as demonstrated by an MTT assay. Repressed expression of iNOS, COX-2 and reduced phosphorylation of p65 revealed the inhibitory effect of these analogues on inflammatory mediator release which is related to inhibited NF-ĸB signals. (N-Hydroxy-3-{3-[2-(2-methyl-1H-indol-3-yl)-ethylsulfamoyl]-phenyl}-acrylamide) (9), exhibited ability superior to that of 1·HCl, was able to reduce carrageenan-induced acute inflammation in an animal model. Compounds 9–12 have potential anti-inflammatory activity and compound 9 can serve as lead compound for further development.