Search

Background and aims: Dickkopf-1 (DKK1) is associated with poor prognosis in intrahepatic cholangiocarcinoma (iCCA), but the mechanisms behind this are unclear. Here, we show that DKK1 plays an immune regulatory role in vivo and inhibition reduces tumour growth.Methods: Various in vivo GEMM mouse models and patient samples were utilized to assess the effects of tumour specific DKK1 overexpression in iCCA. DKK1-driven changes to the tumour immune microenvironment were characterized by immunostaining and gene expression analysis. DKK1 overexpressing and damage induced models of iCCA were used to demonstrate the therapeutic efficacy of DKK1 inhibition in these contexts using the anti-DKK1 therapeutic, DKN-01.Results: DKK1 overexpression in mouse models of iCCA drives an increase in chemokine and cytokine signalling, the recruitment of regulatory macrophages, and promotes the formation of a tolerogenic niche with higher numbers of regulatory T cells. We show a similar association of DKK1 with FOXP3 and regulatory T cells in patient tissue and gene expression data, demonstrating these effects are relevant to human iCCA. Finally, we demonstrate that inhibition of DKK1 with the monoclonal antibody mDKN-01 is effective at reducing tumour burden in two distinct mouse models of the disease.Conclusion: DKK1 promotes tumour immune evasion in iCCA through the recruitment of immune suppressive macrophages. Targeting DKK1 with a neutralizing antibody is effective at reducing tumour growth in vivo. As such, DKK1 targeted and immune modulatory therapies may be an effective strategy in iCCA patients with high DKK1 tumour expression or tolerogenic immune phenotypes.

Detection and depletion of NK cells in mice with metastatic tumors.

Metastasis-associated macrophages (MAM) promote persistent growth of breast cancer cells at the metastatic site and are, thus, an attractive therapeutic target to treat breast cancer metastasis, a leading cause of cancer-related death in women. However, the precise mechanisms behind MAM-mediated metastatic tumor outgrowth have not been fully elucidated. Using mouse models of metastatic breast cancer, we showed that MAMs uniquely expressed hepatocyte growth factor (HGF) in metastatic tumors. We also demonstrated that a selected population of cancer cells with high metastatic potential (cancer cells that can establish metastatic tumors in mice with higher number and incidence than parental cells) had higher expression of HGF receptor, MNNG HOS transforming gene (MET), and were more responsive to HGF released from macrophages compared with the parental cells. Blockade of MET signaling in cancer cells suppressed metastatic tumor expansion, in part, through activation of natural killer cells. Results from this study suggest an approach to prevent life-threatening metastatic tumor formation using blockade of MAM-induced MET signal activation in metastatic cancer cells.

Supplementary Video S1. Tumor cell intravasation occurs at TMEM along with local, transient blood vessel permeability. Time-lapse microscopy of 155 kDa dextran-TMR extravasation (red in left panel, white right panel) and site of tumor cell intravasation (green cells) at TMEM. Macrophages in cyan. Time is indicated in minutes:seconds.

Supplementary Methods, Figure Legends

Supplementary Figure S1. TMEM formation with tumor stage in PyMT. Supplementary Figures S2. Tumor cell intravasation occurs at TMEM along with local, transient blood vessel permeability. Supplementary Figure S3. Tumor cell intravasation occurs at TMEM with local, transient blood vessel permeability in a human patient-derived xenograft model of triple negative breast cancer. Supplementary Figure S4. The sliding window measurement. Supplementary Figure S5. 155 kDa dextran-TMR does not extravasate. Supplementary Figure S6. VEGFAHi TEMs are enriched at TMEM and sites of pericyte depletion. Supplementary Figure S6. Vascular VE-Cadherin is decreased in the presence of Tie2Hi/VEGFAHi macrophages in TMEM in human breast cancer.

Purpose: Adipocytes represent one of the most abundant constituents of the mammary gland. They are essential for mammary tumor growth and survival. Metabolically, one of the more important fat-derived factors (“adipokines”) is adiponectin (APN). Serum concentrations of APN negatively correlate with body mass index and insulin resistance. To explore the association of APN with breast cancer and tumor angiogenesis, we took an in vivo approach aiming to study its role in the mouse mammary tumor virus (MMTV)-polyoma middle T antigen (PyMT) mammary tumor model.Experimental Design: We compared the rates of tumor growth in MMTV-PyMT mice in wild-type and APN-null backgrounds.Results: Histology and micro-positron emission tomography imaging show that the rate of tumor growth is significantly reduced in the absence of APN at early stages. PyMT/APN knockout mice exhibit a reduction in their angiogenic profile resulting in nutrient deprivation of the tumors and tumor-associated cell death. Surprisingly, in more advanced malignant stages of the disease, tumor growth develops more aggressively in mice lacking APN, giving rise to a larger tumor burden, an increase in the mobilization of circulating endothelial progenitor cells, and a gene expression fingerprint indicative of more aggressive tumor cells.Conclusions: These observations highlight a novel important contribution of APN in mammary tumor development and angiogenesis, indicating that APN has potent angio-mimetic properties in tumor vascularization. However, in tumors deprived of APN, this antiangiogenic stress results in an adaptive response that fuels tumor growth through mobilization of circulating endothelial progenitor cells and the development of mechanisms enabling massive cell proliferation despite a chronically hypoxic microenvironment.

Supplementary Data from Proangiogenic Contribution of Adiponectin toward Mammary Tumor Growth In vivo

PDF file - 42K

Supplementary Movie 2 from Direct Visualization of Macrophage-Assisted Tumor Cell Intravasation in Mammary Tumors

Legends to Supplementary Figures & Movies from Direct Visualization of Macrophage-Assisted Tumor Cell Intravasation in Mammary Tumors

PDF file - 1107K

Supplementary Movie 4 from Direct Visualization of Macrophage-Assisted Tumor Cell Intravasation in Mammary Tumors

Supplementary Figure 3 from Direct Visualization of Macrophage-Assisted Tumor Cell Intravasation in Mammary Tumors

PDF file - 1920K, S2: Assessment of Vegfa and Notch target gene expression in flow sorted vascular endothelial cells.

An important step in the process of metastasis from the primary tumor is invasive spread into the surrounding stroma. Using an in vivo invasion assay, we have previously shown that imposed gradients of epidermal growth factor (EGF) or colony-stimulating factor-1 (CSF-1) can induce invasion through an EGF/CSF-1 paracrine loop between cancer cells and macrophages. We now report that invasion induced by other ligands also relies on this EGF/CSF-1 paracrine invasive loop. Using an in vivo invasion assay, we show that MTLn3 breast cancer cells overexpressing ErbB3 exhibit enhanced invasion compared with control MTLn3 cells in response to the ErbB3 ligand HRG-β1. The invasive response of both MTLn3-ErbB3 and transgenic MMTV-Neu tumors to HRG-β1 is inhibited by blocking EGF receptor, CSF-1 receptor, or macrophage function, indicating that invasiveness to HRG-β1 is dependent on the EGF/CSF-1 paracrine loop. Furthermore, we show that CXCL12 also triggers in vivo invasion of transgenic MMTV-PyMT tumors in an EGF/CSF-1–dependent manner. Although the invasion induced by HRG-β1 or CXCL12 is dependent on the EGF/CSF-1 paracrine loop, invasion induced by EGF is not dependent on HRG-β1 or CXCL12 signaling, showing an asymmetrical relationship between different ligand/receptor systems in driving invasion. Our results identify a stromal/tumor interaction that acts as an engine underlying invasion induced by multiple ligands. [Cancer Res 2009;69(7):3221–7]

Supplementary Movie 1 from Direct Visualization of Macrophage-Assisted Tumor Cell Intravasation in Mammary Tumors

Invasion of tumor cells into the surrounding connective tissue and blood vessels is a key step in the metastatic spread of breast tumors. Although the presence of macrophages in primary tumors is associated with increased metastatic potential, the mechanistic basis for this observation is unknown. Using a chemotaxis-based in vivo invasion assay and multiphoton-based intravital imaging, we show that the interaction between macrophages and tumor cells facilitates the migration of carcinoma cells in the primary tumor. Gradients of either epidermal growth factor (EGF) or colony-stimulating factor 1 (CSF-1) stimulate collection into microneedles of tumor cells and macrophages even though tumor cells express only EGF receptor and macrophages express only CSF-1 receptor. Intravital imaging shows that macrophages and tumor cells migrate toward microneedles containing either EGF or CSF-1. Inhibition of either CSF-1– or EGF-stimulated signaling reduces the migration of both cell types. This work provides the first direct evidence for a synergistic interaction between macrophages and tumor cells during cell migration in vivo and indicates a mechanism for how macrophages may contribute to metastasis.

Apoptosis repressor with caspase recruitment domain (ARC) inhibits both death receptor- and mitochondrial/ER-mediated pathways of apoptosis. Although expressed mainly in terminally differentiated cells, ARC is markedly upregulated in a variety of human cancers, where its potential contributions have not yet been defined. In this study, we provide evidence of multiple critical pathophysiologic functions for ARC in breast carcinogenesis. In the polyoma middle T-antigen (PyMT) transgenic mouse model of breast cancer, in which endogenous ARC is strongly upregulated, deletion of the ARC-encoding gene nol3 decreased primary tumor burden without affecting tumor onset or multiplicity. More notably, ARC deficiency also limited tumor cell invasion and the number of circulating cancer cells, markedly reducing the number of lung metastases. Conversely, ectopic overexpression of ARC in a PyMT-derived metastatic breast cancer cell line increased invasion in vitro and lung metastasis in vivo. We confirmed these results in a humanized orthotopic model based on MDA-MB-231-derived LM2 metastatic breast cancer cells, in which RNAi-mediated knockdown of ARC levels was shown to reduce tumor volume, local invasion, and lung metastases. Lastly, we found that endogenous levels of ARC conferred chemoresistance in primary tumors and invading cell populations. Our results establish that ARC promotes breast carcinogenesis by driving primary tumor growth, invasion, and metastasis as well as by promoting chemoresistance in invasive cells. Cancer Res; 71(24); 7705–15. ©2011 AACR.

Supplementary Methods, Figures 1-2 from The EGF/CSF-1 Paracrine Invasion Loop Can Be Triggered by Heregulin β1 and CXCL12

Supplementary Figure 2 from Direct Visualization of Macrophage-Assisted Tumor Cell Intravasation in Mammary Tumors

Antibody Details

Supplementary Movie 3 from Direct Visualization of Macrophage-Assisted Tumor Cell Intravasation in Mammary Tumors

PDF file - 2729K, S1: Flow sorting of human and mouse tumor-associated macrophages.

Although the presence of macrophages in tumors has been correlated with poor prognosis, until now there was no direct observation of how macrophages are involved in hematogenous metastasis. In this study, we use multiphoton microscopy to show, for the first time, that tumor cell intravasation occurs in association with perivascular macrophages in mammary tumors. Furthermore, we show that perivascular macrophages of the mammary tumor are associated with tumor cell intravasation in the absence of local angiogenesis. These results show that the interaction between macrophages and tumor cells lying in close proximity defines a microenvironment that is directly involved in the intravasation of cancer cells in mammary tumors. [Cancer Res 2007;67(6):2649–56]

Supplementary Figure 1 from Direct Visualization of Macrophage-Assisted Tumor Cell Intravasation in Mammary Tumors

Many studies and reports, both governmental and academic, plus legislation, have suggested and some mandated the establishment of threat assessment teams to protect campuses. These teams have proli...

Objective To investigate the effect of the selective progesterone receptor modulator, telapristone acetate (CDB-4124), on endometrial biology and reproductive outcomes. Ovariectomized and hormone-treated CD1 female mice, CD1 female mice with xenotransplants of reconstructed human endometrial tissue, mated wildtype female mice, and cultured human endometrial stromal cells (hESCs) were treated with CDB-4124, followed by the assessment of endometrial cell deoxyribonucleic acid (DNA) proliferation, stromal decidual response, and embryo implantation. Design Experimental study. Setting Academic research laboratory. Patients Healthy volunteer women from the community were recruited for endometrial biopsies. Animals CD1 out-bred mice (Charles River Laboratories) and nude mice, NU/J (Jackson Laboratories, Bar Harbor, ME). Intervention Treatment of mice and hESCs with CDB-4124. Main Outcome Measure The effect of CDB-4124 on endometrial cell morphology and DNA synthesis, decidual response, and mouse embryo implantation. Results CDB-4124 inhibited estradiol-induced epithelial DNA synthesis in the mouse uterus and xenotransplanted human endometrium. This antiproliferative effect was less than that of progesterone (P4) and was observed when CDB-4124 was administered alone or concomitantly with P4. In the uterine epithelium, CDB-4124 acted as a P4 agonist and partial antagonist. In contrast, CDB-4124 acted as a complete P4 antagonist in the uterine stroma, where it blocked P4’s action to induce a decidual response in the pseudopregnant mouse uterus and wildtype mouse uterus after copulation. In mated female mice, CDB-4124 impaired embryo implantation. Similarly, CDB-4124 inhibited the morphological and biochemical transformations of hESCs to decidual cells in vitro. Conclusion CDB-4124 exerts mixed P4 antagonistic/agonistic effects in the human and mouse endometrium, which result in failed embryo implantation because of the absence of stromal decidualization.

Monocytes are progenitors to macrophages and a subclass of dendritic cells (monocyte-derived dendritic cells, MoDCs), but they also act as circulating sensors that respond to environmental changes and disease. Technological advances have defined the production of classical monocytes in the bone marrow through the identification of lineage-determining transcription factors (LDTFs) and have proposed alternative routes of differentiation. Monocytes released into the circulation can be recruited to tissues by specific chemoattractants where they respond to sequential niche-specific signals that determine their differentiation into terminal effector cells. New aspects of monocyte biology in the circulation are being revealed, exemplified by the influence of cancer on the systemic alteration of monocyte subset abundance and transcriptional profiles. These changes can act to enhance the metastatic spread of primary cancers and may offer therapeutic opportunities.

The advent of Single cell RNA sequencing (scRNA-seq) has improved our understanding of cellular heterogeneity by providing in-depth transcriptome information at the single cell level. Several scRNA-seq papers reported sequencing of the tumor CD45 positive fraction, or selected immune cells, giving access to hundreds of sequencing datasets using multiple platforms. Because of the difficulties in performing gene-to-function studies in the immuno-oncology and more precisely in the macrophage field, this “dataset infodemic” did not translate into an abundance of new validated targets. Human macrophages are studied in vitro mainly using cell lines (such as THP1) or monocyte-derived macrophages (MDM) differentiated from human monocytes obtained from blood of healthy donors. However, cell lines are limited by their lack of physiological relevance as in contrast to primary cells they are mutated and fail to respond to, or do not require, important immunological stimulations. MDM cultures are mainly limited by yield, as monocytes from blood do not proliferate in vitro. Moreover, human primary macrophages are not easily genetically modified due to their extreme resistance to transfection and transduction. This phenomenon can be partially explained by their expression of several restriction enzymes such as APOBEC, TRIM and SAMHD1 which hypermutate or degrade the transfected/transduced genetic payload. All these challenges represent a major barrier for target validation and drug screening in the macrophage field. The main goal of the ENIGMAC™ discovery platform is to support gene-to-function studies and to deliver new validated macrophage targets. The platform is designed to develop novel assay systems, unique genome editing methods, and supported by the multi-omics bioinformatics analyses. We use a human Induced Pluripotent Stem Cell (iPSC) line that yields macrophages phenotypically and functionally very similar to MDM. By employing this iPSC system we can produce millions of macrophages per week, which allows multiple high throughput assays to support target validation and drug discovery. Furthermore, we developed several technologies to allow fast and reliable gene editing of macrophages. Our proprietary toolbox allows us to perform gene Knock In (KI), Knock Out (KO) and Knock Down (KD) with high efficiency both at iPSC and macrophage level while maintaining expression/silencing during macrophage differentiation. KD can be performed using a pooled approach that enables screening by flow cytometry-based phenotypes at large scale. In conclusion, the ENIGMAC™ platform represents a unique tool for gene-to-function studies using human macrophages. Notably, it is disease agnostic and can be integrated with a variety of disease-specific conditions and phenotypic readouts. Citation Format: Martha Lopez-Yrigoyen, Thomas W. Monteiro Crozier, Helena Engman, Yuxin Cui, Moritz Haneklaus, Krzysztof B. Wicher, Stephen Myatt, Jeffrey W. Pollard, Luca Cassetta, Carola Ries. ENIGMACTM discovery platform enables gene to function target validation for macrophage therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2751.

Tumorigenesis is not only determined by the intrinsic properties of cancer cells but also by their interactions with components of the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are among the most abundant immune cells in the TME. During initial stages of tumor development, macrophages can either directly promote antitumor responses by killing tumor cells or indirectly recruit and activate other immune cells. As genetic changes occur within the tumor or T helper 2 (TH 2) cells begin to dominate the TME, TAMs begin to exhibit an immunosuppressive protumor phenotype that promotes tumor progression, metastasis, and resistance to therapy. Thus, targeting TAMs has emerged as a strategy for cancer therapy. To date, TAM targeting strategies have focused on macrophage depletion and inhibition of their recruitment into the TME. However, these strategies have shown limited therapeutic efficacy, although trials are still underway with combination therapies. The fact that macrophages have the potential for antitumor activity has moved the TAM targeting field toward the development of TAM-reprogramming strategies to support this antitumor immune response. Here, we discuss the various roles of TAMs in cancer therapy and their immunosuppressive properties, as well as implications for emerging checkpoint inhibitor-based immunotherapies. We review state-of-the-art TAM-targeting strategies, focusing on current ones at the preclinical and clinical trial stages that aim to reprogram TAMs as an oncological therapy.

At the outset of a clinical relationship, boundaries are created in order to define the therapeutic relationship, outline the treatment process, and explain the limits of confidentiality. Although ...

Metastasis-associated macrophages (MAM) promote persistent growth of breast cancer cells at the metastatic site and are, thus, an attractive therapeutic target to treat breast cancer metastasis, a leading cause of cancer-related death in women. However, the precise mechanisms behind MAM-mediated metastatic tumor outgrowth have not been fully elucidated. Using mouse models of metastatic breast cancer, we showed that MAMs uniquely expressed hepatocyte growth factor (HGF) in metastatic tumors. We also demonstrated that a selected population of cancer cells with high metastatic potential (cancer cells that can establish metastatic tumors in mice with higher number and incidence than parental cells) had higher expression of HGF receptor, MNNG HOS transforming gene (MET), and were more responsive to HGF released from macrophages compared with the parental cells. Blockade of MET signaling in cancer cells suppressed metastatic tumor expansion, in part, through activation of natural killer cells. Results from this study suggest an approach to prevent life-threatening metastatic tumor formation using blockade of MAM-induced MET signal activation in metastatic cancer cells.

We report the novel chemical design of fluorescent activatable chemokines as highly specific functional probes for imaging subpopulations of immune cells in live tumours. Activatable chemokines behave as AND‐gates since they emit only after receptor binding and intracellular activation, showing enhanced selectivity over existing agents. We have applied this strategy to produce mCCL2‐MAF as the first probe for in vivo detection of metastasis‐associated macrophages in a preclinical model of lung metastasis. This strategy will accelerate the preparation of new chemokine‐based probes for imaging immune cell function in tumours., Live show: Fluorescent activatable chemokines have been designed as the first chemical probes for in vivo detection of tumour‐associated macrophages in lung metastasis. Activatable chemokines behave as AND gates since they emit only after receptor binding and intracellular activation, thereby showing enhanced selectivity over existing agents.

Metastasis is the systemic manifestation of cancer and the main cause of death from breast cancer. In mouse models of lung metastases, recruitment of classical monocytes from blood to the lung and their differentiation to metastasis-associated macrophages (MAMs) facilitate cancer cell extravasation, survival, and growth. Ablation of MAMs or their monocytic progenitors inhibits metastasis. We hypothesized that factors controlling macrophage polarization modulate tumor cell extravasation in the lung. We evaluated whether signaling by Th1 or Th2 cytokines in macrophages affected trans-endothelial migration of tumor cells in vitro. Interferon γ and LPS inhibited macrophage-dependent tumor cell extravasation while the Th2 cytokine interleukin-4 (IL4) enhanced this process. We demonstrated that IL4 receptor (IL4rα)null mice develop fewer and smaller lung metastasis. Adoptive transfer of wild type monocytes toIL4rαdeficient mice rescued this phenotype. IL4 signaling in macrophages controls the expression of the chemokine receptor CXCR2, necessary for IL4-mediated tumor cell extravasation in vitro. Furthermore, IL4 signaling in macrophages transcriptionally regulates several other genes already causally associated with lung metastasis including CCL2, CSF1, CCR1, HGF and FLT1. The central role for IL4 signaling in MAMs was confirmed by high-resolution intravital imaging of the lung in mice at the time of metastatic seeding, which showed reduced physical interaction between tumor cells andIL4rα-deficient macrophages. This interaction enhances tumor cell survival. These data indicate that IL4 signaling in monocytes and macrophages is key during seeding and growth of breast metastasis in the lung as it regulates pro-tumoral paracrine signaling between cancer cells and macrophages.

Metastasis is the systemic manifestation of cancer and the main cause of death from breast cancer. In mouse models of lung metastases, recruitment of classical monocytes from blood to the lung and their differentiation to metastasis-associated macrophages (MAMs) facilitate cancer cell extravasation, survival and growth. Ablation of MAMs or their monocytic progenitors inhibits metastasis. We hypothesized that factors controlling macrophage polarization modulate tumor cell extravasation in the lung. We evaluated whether signaling by Th1 or Th2 cytokines in macrophages affected transendothelial migration of tumor cells in vitro. Interferon gamma and LPS inhibited macrophage-dependent tumor cell extravasation while the Th2 cytokine interleukin-4 (IL4) enhanced this process. We demonstrated that IL4 receptor (IL4rα)-null mice developed fewer and smaller lung metastasis in E0771-LG mammary cancer models of this disease. Adoptive transfer of wild-type monocytes to IL4rα-deficient mice partially rescued this phenotype. IL4 signaling in macrophages controlled the expression of the chemokine receptor CXCR2, necessary for IL4-mediated tumor cell extravasation in vitro. Furthermore, IL4 signaling in macrophages regulated the transcript abundance of several other genes already causally associated with mammary cancer lung metastasis including Ccl2, Csf1, Ccr1, Hgf and Flt1. The central role of IL4 signaling in MAMs was confirmed by high-resolution intravital imaging of the lung in mice at the time of metastatic seeding, which showed reduced physical interaction between tumor cells and IL4rα-deficient macrophages. This interaction with wild-type MAMs enhanced tumor cell survival and seeding, which was lost in the IL4rα mice. These data indicate that IL4 signaling in monocytes and macrophages is key during seeding and growth of breast metastasis in the lung, as it regulates pro-tumoral paracrine signaling between cancer cells and macrophages.

Tumor cells metastasize to distant organs through a complex series of events that are driven by tumor intrinsic and extrinsic factors. In particular, non-malignant stromal cells, including immune cells, modify tumor metastatic behavior. Of these cells, tumor-associated innate immune cells, particularly macrophages and neutrophils, suppress the cytotoxic activity of innate and adaptive killer cells and interact with tumor cells to promote their growth and malignancy. These findings in mouse cancer models suggest that targeting these sub-populations of immune cells holds therapeutic promise in treating metastatic disease. In this review, we describe the origin and role of the macrophages, neutrophils, and their progenitors in the metastatic cascade and suggest strategies that might enhance cancer therapy.

Liver cirrhosis is a major cause of death worldwide and is characterized by extensive fibrosis. There are currently no effective antifibrotic therapies available. To obtain a better understanding of the cellular and molecular mechanisms involved in disease pathogenesis and enable the discovery of therapeutic targets, here we profile the transcriptomes of more than 100,000 single human cells, yielding molecular definitions for non-parenchymal cell types that are found in healthy and cirrhotic human liver. We identify a scar-associated TREM2+CD9+ subpopulation of macrophages, which expands in liver fibrosis, differentiates from circulating monocytes and is pro-fibrogenic. We also define ACKR1+ and PLVAP+ endothelial cells that expand in cirrhosis, are topographically restricted to the fibrotic niche and enhance the transmigration of leucocytes. Multi-lineage modelling of ligand and receptor interactions between the scar-associated macrophages, endothelial cells and PDGFRα+ collagen-producing mesenchymal cells reveals intra-scar activity of several pro-fibrogenic pathways including TNFRSF12A, PDGFR and NOTCH signalling. Our work dissects unanticipated aspects of the cellular and molecular basis of human organ fibrosis at a single-cell level, and provides a conceptual framework for the discovery of rational therapeutic targets in liver cirrhosis. Single-cell RNA sequencing is used to characterize and compare the functional diversity of cells from liver biopsies of human scarred and normal liver, and identifies markers for scar-associated macrophages and endothelial cells.