Fibroblast Subpopulations Research Articles

The Ferroxidase Hephaestin in Lung Cancer: Pathological Significance and Prognostic Value.

Hephaestin (HEPH) belongs to a group of exocytoplasmic ferroxidases which contribute to cellular iron homeostasis by favouring its export. Down-regulation of HEPH expression, possibly by stimulating cell proliferation due to an increase in iron availability, has shown to correlate with poor survival in breast cancer. The lung is particularly sensitive to iron-induced oxidative stress, given the high oxygen tension present, however, HEPH distribution in lung cancer and its influence on prognosis have not been investigated yet. In this study we explored the prognostic value of HEPH and its expression pattern in the most prevalent histotypes of lung cancers, namely lung adenocarcinoma and lung squamous cell carcinoma. In silico analyses, based on UALCAN, Gene Expression Profiling Interactive Analysis (GEPIA) and Kaplan–Meier plotter bioinformatics, revealed a significant correlation between higher levels of HEPH expression and favorable prognosis, in both cancer histotypes. Moreover, TIMER web platform showed a statistically significant association between HEPH expression and cell elements belonging to the tumor microenvironment identified as endothelial cells and a subpopulation of cancer-associated fibroblasts, further confirmed by double immunohistochemical labeling with cell type specific markers. Taken together, these data shed a light on the complex mechanisms of local iron handling lung cancer can exploit to support tumorigenesis.

Dual recombinase mouse model to dissect cell type‐specific role of microRNA‐21 in pancreatic cancer

Background Pancreatic ductal adenocarcinoma (PDAC) is characterized by its highly aggressive cancer cells and extensive stroma reaction. This reactive stroma contains distinct tumor-restraining and tumor-promoting fibroblast subpopulations. We previously studied the effects of global loss of pro-fibrotic non-coding regulatory microRNA-21 (miR-21) in KRas-driven p53-deleted genetically engineered mouse models (GEMMs) of PDAC. The absence of tumor-restraining smooth muscle actin (SMA)–expressing myofibroblasts (myCAFs) and a massive infiltrate of immune cells were the most salient phenotypic features of global miR-21 loss. Stromal miR-21 activity was required for induction of myCAFs in in-vivo isograft transplantation experiments. Objective We hypothesize that cell-intrinsic activity of miR-21 is required for induction and maintenance of myCAFs. To test this hypothesis, we have generated a new dual recombinase mouse model to temporarily and spatially control the dose of miR-21 activity using the tamoxifen-inducible Flp/FRT system in combination with the Cre/LoxP system that activates the KRas-driven GEMMs. Methods We describe the generation of a “flirted” Mir-21 allele flanked by FRT sites using easi-CRISPR technology and the characterization of mouse strains carrying one or two flirted alleles using molecular biology and histological analyses. Results We demonstrate the efficient recombination of flirted Mir-21 allele(s) in compounded strain with constitutive flippase (Rosa26-Flpe) or with tamoxifen-inducible flippase (Rosa26-FlpoER) after a single intraperitoneal injection of 75 mg/kg tamoxifen. We are crossing individual strains to produce a compounded strain with all the genetic elements required for this model. Histological and immunohistochemical characterization of tumors arising from these animals with full or partial loss of miR-21 activity will be presented at the meeting. Conclusions This dual recombinase system will be the first to conditionally delete the Mir-21 gene in myCAFs in well-established PDAC GEMMs. The genetic components of this dual recombinase system could be of general interest to the scientific community to mechanistically dissect the role of miR-21 in other cell types in PDAC by swapping the FlpoER driver strain or in myCAFs in other KRas-driven cancer types by swapping the Cre driver. Gained knowledge of the cell-intrinsic role of miR-21 in myCAFs and other cell types will be crucial to inform best strategies for pharmacological modulation of miR-21 activity in patients for cancer interception and/or treatment.

Single-cell dual-omics reveals the transcriptomic and epigenomic diversity of cardiac non-myocytes.

The precise cellular identity and molecular features of non-myocytes (non-CMs) in a mammalian heart at a single-cell level remain elusive. Depiction of epigenetic landscape with transcriptomic signatures using the latest single-cell multi-omics has the potential to unravel the molecular programs underlying the cellular diversity of cardiac non-myocytes. Here, we characterized the molecular and cellular features of cardiac non-CM populations in the adult murine heart at the single-cell level. Through single-cell dual omics analysis, we mapped the epigenetic landscapes, characterized the transcriptomic profiles and delineated the molecular signatures of cardiac non-CMs in the adult murine heart. Distinct cis-regulatory elements and trans-acting factors for the individual major non-CM cell types (endothelial cells, fibroblast, pericytes, and immune cells) were identified. In particular, unbiased sub-clustering and functional annotation of cardiac fibroblasts (FBs) revealed extensive FB heterogeneity and identified FB sub-types with functional states related to the cellular response to stimuli, cytoskeleton organization, and immune regulation, respectively. We further explored the function of marker genes Hsd11b1 and Gfpt2 that label major FB subpopulations and determined the distribution of Hsd11b1+ and Gfp2+ FBs in murine healthy and diseased hearts. In summary, we characterized the non-CM cellular identity at the transcriptome and epigenome levels using single-cell omics approaches and discovered previously unrecognized cardiac fibroblast subpopulations with unique functional states.

Differential Expression of Insulin-Like Growth Factor 1 and Wnt Family Member 4 Correlates With Functional Heterogeneity of Human Dermal Fibroblasts.

Although human dermis contains distinct fibroblast subpopulations, the functional heterogeneity of fibroblast lines from different donors is under-appreciated. We identified one commercially sourced fibroblast line (c64a) that failed to express α-smooth muscle actin (α-SMA), a marker linked to fibroblast contractility, even when treated with transforming growth factor-β1 (TGF-β1). Gene expression profiling identified insulin-like growth factor 1 (IGF1) as being expressed more highly, and Asporin (ASPN) and Wnt family member 4 (WNT4) expressed at lower levels, in c64a fibroblasts compared to three fibroblast lines that had been generated in-house, independent of TGF-β1 treatment. TGF-β1 increased expression of C-X-C motif chemokine ligand 1 (CXCL1) in c64a cells to a greater extent than in the other lines. The c64a gene expression profile did not correspond to any dermal fibroblast subpopulation identified by single-cell RNAseq of freshly isolated human skin cells. In skin reconstitution assays, c64a fibroblasts did not support epidermal stratification as effectively as other lines tested. In fibroblast lines generated in-house, shRNA-mediated knockdown of IGF1 increased α-SMA expression without affecting epidermal stratification. Conversely, WNT4 knockdown had no consistent effect on α-SMA expression, but increased the ability of fibroblasts to support epidermal stratification. Thus, by comparing the properties of different lines of cultured dermal fibroblasts, we have identified IGF1 and WNT4 as candidate mediators of two distinct dermal functions: myofibroblast formation and epidermal maintenance.

Dermal Fibroblast Heterogeneity and Its Contribution to the Skin Repair and Regeneration.

Significance: Dermal fibroblasts are the major cell type in the skin's dermal layer. These cells originate from distinct locations of the embryo and reside in unique niches in the dermis. Different dermal fibroblasts exhibit distinct roles in skin development, homeostasis, and wound healing. Therefore, these cells are becoming attractive candidates for cell-based therapies in wound healing. Recent Advances: Human skin dermis comprises multiple fibroblast subtypes, including papillary, reticular, and hair follicle-associated fibroblasts, and myofibroblasts after wounding. Recent studies reveal that these cells play distinct roles in wound healing and contribute to diverse healing outcomes, including nonhealing chronic wound or excessive scar formation, such as hypertrophic scars (HTS) and keloids, with papillary fibroblasts having antiscarring and reticular fibroblast scar-forming properties. Critical Issues: The identities and functions of dermal fibroblast subpopulations in many respects remain unknown. In this review, we summarize the current understanding of dermal fibroblast heterogeneity, including their defined cell markers and dermal niches, dynamic changes, and contributions to skin wound healing, with the emphasis on scarless healing, healing with excessive scars (HTS and keloids), chronic wounds, and the potential application of this heterogeneity for developing cell-based therapies that allow wounds to heal faster with less scarring. Future Directions: Heterogeneous dermal fibroblast populations and their functions are poorly characterized. Refining and advancing our understanding of dermal fibroblast heterogeneity and their participation in skin homeostasis and wound healing may create potential therapeutic applications for nonhealing chronic wounds or wounds that heal with excessive scarring.

Single-cell RNA sequencing redefines the mesenchymal cell landscape of mouse endometrium.

The endometrium is a dynamic tissue that exhibits remarkable resilience to repeated episodes of differentiation, breakdown, regeneration, and remodeling. Endometrial physiology relies on a complex interplay between the stromal and epithelial compartments with the former containing a mixture of fibroblasts, vascular, and immune cells. There is evidence for rare populations of putative mesenchymal progenitor cells located in the perivascular niche of human endometrium, but the existence of an equivalent cell population in mouse is unclear. We used the Pdgfrb‐BAC‐eGFP transgenic reporter mouse in combination with bulk and single‐cell RNA sequencing to redefine the endometrial mesenchyme. In contrast to previous reports we show that CD146 is expressed in both PDGFRβ + perivascular cells and CD31 + endothelial cells. Bulk RNAseq revealed cells in the perivascular niche which express the high levels of Pdgfrb as well as genes previously identified in pericytes and/or vascular smooth muscle cells (Acta2, Myh11, Olfr78, Cspg4, Rgs4, Rgs5, Kcnj8, and Abcc9). scRNA‐seq identified five subpopulations of cells including closely related pericytes/vascular smooth muscle cells and three subpopulations of fibroblasts. All three fibroblast populations were PDGFRα+/CD34 + but were distinct in their expression of Ngfr/Spon2/Angptl7 (F1), Cxcl14/Smoc2/Rgs2 (F2), and Clec3b/Col14a1/Mmp3 (F3), with potential functions in the regulation of immune responses, response to wounding, and organization of extracellular matrix, respectively. Immunohistochemistry was used to investigate the spatial distribution of these populations revealing F1/NGFR + cells in most abundance beside epithelial cells. We provide the first definitive analysis of mesenchymal cells in the adult mouse endometrium identifying five subpopulations providing a platform for comparisons between mesenchymal cells in endometrium and other adult tissues which are prone to fibrosis.

Single-Cell Transcriptomic Atlas of Different Human Cardiac Arteries Identifies Cell Types Associated With Vascular Physiology.

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MicroRNA-130a Is Elevated in Thyroid Eye Disease and Increases Lipid Accumulation in Fibroblasts Through the Suppression of AMPK.

PurposeThyroid eye disease (TED) is a condition that causes the tissue behind the eye to become inflamed and can result in excessive fatty tissue accumulation in the orbit. Two subpopulations of fibroblasts reside in the orbit: those that highly express Thy1 (Thy1+) and those with little or no Thy1 (Thy1–). Thy1– orbital fibroblasts (OFs) are more prone to lipid accumulation than Thy1+ OFs. The purpose of this study was to investigate the mechanisms whereby Thy1– OFs more readily accumulate lipid.MethodsWe screened Thy1+ and Thy1– OFs for differences in microRNA (miRNA) expression. The effects of increasing miR-130a levels in OFs was investigated by measuring lipid accumulation and visualizing lipid deposits. To determine if adenosine monophosphate-activated protein kinase (AMPK) is important for lipid accumulation, we performed small interfering RNA (siRNA)-mediated knockdown of AMPKβ1. We measured AMPK expression and activity using immunoblotting for AMPK and AMPK target proteins.ResultsWe determined that miR-130a was upregulated in Thy1– OFs and that miR-130a targets two subunits of AMPK. Increasing miR-130a levels enhanced lipid accumulation and reduced expression of AMPKα and AMPKβ in OFs. Depletion of AMPK also increased lipid accumulation. Activation of AMPK using AICAR attenuated lipid accumulation and increased phosphorylation of acetyl-CoA carboxylase (ACC) in OFs.ConclusionsThese data suggest that when Thy1– OFs accumulate in TED, miR-130a levels increase, leading to a decrease in AMPK activity. Decreased AMPK activity promotes lipid accumulation in TED OFs, leading to excessive fatty tissue accumulation in the orbit.

Transient ACE (Angiotensin-Converting Enzyme) Inhibition Suppresses Future Fibrogenic Capacity and Heterogeneity of Cardiac Fibroblast Subpopulations.

Transient ACE (angiotensin-converting enzyme) inhibition in spontaneously hypertensive rats is known to protect against future injury-induced cardiac inflammation, fibrosis, and dysfunction; however, the mechanisms of protection have not been delineated. Here, we used single-cell RNA sequencing to test the hypothesis that transient ACE inhibitor treatment would induce a persistent shift in cardiac fibroblast subpopulations. Adult male spontaneously hypertensive rats (11 weeks old, hypertensive with cardiac hypertrophy) were treated for 2 weeks with an ACE inhibitor, enalapril (30 mg/kg per day, PO), or water (untreated spontaneously hypertensive rats) followed by a 2-week washout period (n=7/group). Cardiac fibroblasts were isolated from the left ventricle and subjected to single-cell RNA sequencing. Nine clusters of fibroblasts were identified, with 98% of cells in clusters 0 to 6. The transient treatment produced significant changes both within and across clusters. Cluster 1 depicted a highly fibrogenic gene profile, with cluster 6 serving as a gateway to cluster 1. Transient ACE inhibition depleted the gateway and expanded cluster 0, which was the least fibrogenic profile. Moreover, within cluster 1 fibroblasts, ACE inhibition reduced expression of individual fibrosis genes (eg, COL1A1, COL3A1, and FN1; all P<1×10-35). Clusters 2 to 5 reflected proliferative, moderately fibrogenic, translationally active, and less inflammatory subsets of fibroblasts, all of which exhibited attenuated fibrogenic gene expression after transient ACE inhibition. In conclusion, transient ACE inhibition shifts cardiac fibroblast subpopulations and degree of activation resulting in an overall reduced fibrogenic phenotype.

Tumor Cells and Cancer-Associated Fibroblasts: An Updated Metabolic Perspective.

Simple SummaryTumors are a complex ecosystem including not only cancer cells, but also many distinct cell types of the tumor micro-environment. While the Warburg effect assessing high glucose uptake in tumors was recognized a long time ago, metabolic heterogeneity within tumors has only recently been demonstrated. Indeed, several recent studies have highlighted other sources of carbon than glucose, including amino acids, fatty acids and lactate. These newly identified metabolic trajectories modulate key cancer cell features, such as invasion capacities. In addition, cancer metabolic heterogeneity is not restricted to cancer cells. Here, we also describe heterogeneity of Cancer-Associated Fibroblast (CAF) subpopulations and their complex metabolic crosstalk with cancer cells.During the past decades, metabolism and redox imbalance have gained considerable attention in the cancer field. In addition to the well-known Warburg effect occurring in tumor cells, numerous other metabolic deregulations have now been reported. Indeed, metabolic reprograming in cancer is much more heterogeneous than initially thought. In particular, a high diversity of carbon sources used by tumor cells has now been shown to contribute to this metabolic heterogeneity in cancer. Moreover, the molecular mechanisms newly highlighted are multiple and shed light on novel actors. Furthermore, the impact of this metabolic heterogeneity on tumor microenvironment has also been an intense subject of research recently. Here, we will describe the new metabolic pathways newly uncovered in tumor cells. We will also have a particular focus on Cancer-Associated Fibroblasts (CAF), whose identity, function and metabolism have been recently under profound investigation. In that sense, we will discuss about the metabolic crosstalk between tumor cells and CAF.

Functional Dissection of CD26 and Its Pharmacological Inhibition by Sitagliptin During Skin Wound Healing.

BackgroundSkin fibroblasts are primary mediators underlying wound healing and therapeutic targets in scar prevention and treatment. CD26 is a molecular marker to distinguish fibroblast subpopulations and plays an important role in modulating the biological behaviors of dermal fibroblasts and influencing skin wound repair. Therapeutic targeting of specific fibroblast subsets is expected to reduce skin scar formation more efficiently.Material/MethodsSkin burn and excisional wound healing models were surgically established in mice. The expression patterns of CD26 during wound healing were determined by immunohistochemical staining, real-time RT-PCR, and western blot assays. Normal fibroblasts from intact skin (NFs) and fibroblasts in wounds (WFs) were isolated and sorted by fluorescence-activated cell sorting (FACS) into 4 subgroups – CD26+ NFs, CD26− NFs, CD26+ WFs, and CD26− WFs – for comparisons of their capacities of proliferation, migration, and collagen synthesis. Pharmacological inhibition of CD26 by sitagliptin in skin fibroblasts and during wound healing were further assessed both in vitro and in vivo.ResultsIncreased CD26 expression was observed during skin wound healing in both models. The CD26+ fibroblasts isolated from wounds had significantly stronger abilities to proliferate, migrate, and synthesize collagen than other fibroblast subsets. Sitagliptin treatment potently diminished CD26 expression, impaired the proliferation, migration, and collagen synthesis of fibroblasts in vitro, and diminished scar formation in vivo.ConclusionsOur data reveal that CD26 is functionally involved in skin wound healing by regulating cell proliferation, migration, and collagen synthesis in fibroblasts. Pharmacological inhibition of CD26 by sitagliptin might be a viable strategy to reduce skin scar formation.

Three Distinct Stroma Types in Human Pancreatic Cancer Identified by Image Analysis of Fibroblast Subpopulations and Collagen.

Cancer-associated fibroblasts have emerged to be highly heterogenous and can play multifaceted roles in dictating pancreatic ductal adenocarcinoma (PDAC) progression, immunosuppression, and therapeutic response, highlighting the need for a deeper understanding of stromal heterogeneity between patients and even within a single tumor. We hypothesized that image analysis of fibroblast subpopulations and collagen in PDAC tissues might guide stroma-based patient stratification to predict clinical outcomes and tumor characteristics. A novel multiplex IHC-based image analysis system was established to digitally differentiate fibroblast subpopulations. Using whole-tissue slides from 215 treatment-naïve PDACs, we performed concurrent quantification of principal fibroblast subpopulations and collagen and defined three stroma types: collagen-rich stroma, fibroblast activation protein α (FAP)-dominant fibroblast-rich stroma, and α smooth muscle actin (ACTA2)-dominant fibroblast-rich stroma. These stroma types were assessed for the associations with cancer-specific survival by multivariable Cox regression analyses and with clinicopathologic factors, including CD8+ cell density. FAP-dominant fibroblasts and ACTA2-dominant fibroblasts represented the principal distinct fibroblast subpopulations in tumor stroma. Stroma types were associated with patient survival, SMAD4 status, and transcriptome signatures. Compared with FAP-dominant fibroblast-rich stroma, collagen-rich stroma correlated with prolonged survival [HR, 0.57; 95% confidence interval (CI), 0.33-0.99], while ACTA2-dominant fibroblast-rich stroma exhibited poorer prognosis (HR, 1.65; 95% CI, 1.06-2.58). FAP-dominant fibroblast-rich stroma was additionally characterized by restricted CD8+ cell infiltrates and intense neutrophil infiltration. This study identified three distinct stroma types differentially associated with survival, immunity, and molecular features, thereby underscoring the importance of stromal heterogeneity in subtyping pancreatic cancers and supporting the development of antistromal therapies.

Single-Cell Transcriptomic Analysis of Endometriosis Provides Insights Into Fibroblast Fates and Immune Cell Heterogeneity

Background Endometriosis is an oestrogen-dependent disease with an unclear aetiology and pathogenesis affecting 6-10% of the global female population, predominantly those of reproductive age. Herein, we profile the transcriptomes of approximately 55,000 single cells from three groups including ectopic endometrium, eutopic endometrium from women with endometriosis, and eutopic endometrium from healthy women to create a single-cell transcriptome atlas of endometriosis. Results We have identified 9 cell types and performed single-cell analysis of fibroblasts, and determined a potential developmental trajectory associated with endometriosis. We also identified fibroblast subpopulations related to endometriosis development and found that StAR played an important role in this process. Moreover, T cells in endometriosis were less activated or inflammatory with decreased effector CD8 + T cells, while the composition ratio of natural killer cells decreased and the percentage of monocytes/macrophages increased in endometriosis cysts. In addition, the effectiveness of immune cells in endometriosis lesions, eutopic endometrium from women with endometriosis, and eutopic endometrium from healthy women was distinct. Cell-cell interaction analyses highlighted the imbalanced immune environment in endometriosis lesions and immune cells in endometriosis could promote the development of the disease. Conclusion Our study provided a systematic characterisation of endometriosis and insights into the aetiology and pathology of endometriosis.

Human Dermal Fibroblast Subpopulations Are Conserved across Single-Cell RNA Sequencing Studies

On the basis of their differential location within the dermis and of discrete changes in gene and protein expression, two major fibroblast subtypes (papillary and reticular) have traditionally been distinguished. In the last 3 years, a number of research groups have begun to address transcriptomic heterogeneity of human skin cells at the single-cell level by determining mRNA levels of expressed genes through single-cell RNA sequencing technologies. However, the outcome of single-cell RNA sequencing studies is thus far confusing. Very little overlap was found in fibroblast subpopulations, which also varied in number and composition in each dataset. After a careful reappraisal of the transcriptomic data of 13,823 human adult dermal fibroblasts that have been sequenced to date, we show that fibroblasts may robustly be assigned to three major types (axes A‒C), which in turn are composed of 10 major subtypes (clusters), which we denominated A1‒A4, B1 and B2, and C1‒C4. These computationally determined axes and clusters represent the major fibroblast types and subtypes in adult healthy human skin across different datasets, accounting for 92.5% of the sequenced fibroblasts. They thus may provide the basis to improve our understanding of dermal homeostasis and cellular function at the transcriptomic level.

Integrative single-cell transcriptome analysis reveals a subpopulation of fibroblasts associated with favorable prognosis of liver cancer patients

Single-cell transcriptome analysis has provided detailed insights into the ecosystem of liver cancer. However, the changes of the cellular and molecular components of liver tumors in comparison with normal livers have not been described at single-cell level. Here, we performed an integrative single-cell analysis of both normal livers and liver cancers. Principal component analysis was firstly performed to delineate the cell lineages in liver tissues. Differential gene expression within major cell types were then analyzed between tumor and normal samples, thus resolved the cell type-specific molecular alterations in liver cancer development. Moreover, a comparison between liver cancer derived versus normal liver derived cell components revealed that two subpopulations of fibroblasts were exclusively expanded in liver cancer tissues. By further defining subpopulation-specific gene signatures, characterizing their spatial distribution in tumor tissues and investigating their clinical significance, we found that the SPARCL1 positive fibroblasts, representing a group of tumor vessel associated fibroblasts, were related to reduced vascular invasion and prolonged survival of liver cancer patients. Through establishing an in-vitro endothelial-to-mesenchymal transition model, we verified the conversion of the fetal liver sinusoidal endothelial cells into the fibroblast-like cells, demonstrating a possible endothelial cell origination of the SPARCL1 positive fibroblasts. Our study provides new insights into the cell atlas alteration, especially the expanded fibroblasts in liver cancers.

The Balance of Stromal BMP Signaling Mediated by GREM1 and ISLR Drives Colorectal Carcinogenesis

Cancer-associated fibroblasts (CAFs), key constituents of the tumor microenvironment, either promote or restrain tumor growth. Attempts to therapeutically target CAFs have been hampered by our incomplete understanding of these functionally heterogeneous cells. Key growth factors in the intestinal epithelial niche, bone morphogenetic proteins (BMPs), also play a critical role in colorectal cancer (CRC) progression. However, the crucial proteins regulating stromal BMP balance and the potential application of BMP signaling to manage CRC remain largely unexplored. Using human CRC RNA expression data, we identified CAF-specific factors involved in BMP signaling, then verified and characterized their expression in the CRC stroma by in situ hybridization. CRC tumoroids and a mouse model of CRC hepatic metastasis were used to test approaches to modify BMP signaling and treat CRC. We identified Grem1 and Islr as CAF-specific genes involved in BMP signaling. Functionally, GREM1 and ISLR acted to inhibit and promote BMP signaling, respectively. Grem1 and Islr marked distinct fibroblast subpopulations and were differentially regulated by transforming growth factor β and FOXL1, providing an underlying mechanism to explain fibroblast biological dichotomy. In patients with CRC, high GREM1 and ISLR expression levels were associated with poor and favorable survival, respectively. A GREM1-neutralizing antibody or fibroblast Islr overexpression reduced CRC tumoroid growth and promoted Lgr5+ intestinal stem cell differentiation. Finally, adeno-associated virus 8 (AAV8)-mediated delivery of Islr to hepatocytes increased BMP signaling and improved survival in our mouse model of hepatic metastasis. Stromal BMP signaling predicts and modifies CRC progression and survival, and it can be therapeutically targeted by novel AAV-directed gene delivery to the liver.

Decoding myofibroblast origins in human kidney fibrosis.

Kidney fibrosis is the hallmark of chronic kidney disease progression, however, currently no antifibrotic therapies exist. This is largely because the origin, functional heterogeneity and regulation of scar-forming cells during human kidney fibrosis remains poorly understood. Here, using single cell RNA-seq, we profiled the transcriptomes of proximal tubule and non-proximal tubule cells in healthy and fibrotic human kidneys to map the entire human kidney in an unbiased approach. This enabled mapping of all matrix-producing cells at high resolution, revealing distinct subpopulations of pericytes and fibroblasts as the major cellular sources of scar forming myofibroblasts during human kidney fibrosis. We used genetic fate-tracing, time-course single cell RNA-seq and ATAC-seq experiments in mice, and spatial transcriptomics in human kidney fibrosis to functionally interrogate these findings, shedding new light on the origin, heterogeneity and differentiation of human kidney myofibroblasts and their fibroblast and pericyte precursors at unprecedented resolution. Finally, we used this strategy to facilitate target discovery, identifying Nkd2 as a myofibroblast-specific target in human kidney fibrosis.

Abstract PO-039: Spatially discrete signalling niches regulate fibroblast heterogeneity in human lung cancer

Abstract In this study we set out to define the prominent fibroblast subpopulations found in human non-small cell lung cancer. Fibroblasts are functionally heterogeneous cells, capable of promoting and suppressing tumor progression. Across cancer types, the extent and cause of this phenotypic diversity remains unknown. We used single-cell RNA sequencing and multiplexed immunohistochemistry to examine fibroblast heterogeneity in human lung and non-small cell lung cancer (NSCLC) samples. We then performed in silico and in vitro analyses to examine the molecular mechanisms regulating fibroblast phenotypes. Single-cell RNA sequencing identified seven fibroblast subpopulations: including inflammatory fibroblasts and myofibroblasts (representing terminal differentiation states), quiescent fibroblasts, proto-myofibroblasts (x2) and proto-inflammatory fibroblasts (x2). Multiplex immunohistochemistry showed that these fibroblast subpopulations were variably distributed throughout tissues but accumulated at discrete niches associated with differentiation status. Bioinformatics analyses suggested TGF-β1 and IL-1 as key regulators of myofibroblastic and inflammatory differentiation respectively. However, in vitro analyses showed that whilst TGF-β1 stimulation in combination with increased tissue tension could induce myofibroblast marker expression, it failed to fully re-capitulate ex-vivo¬ phenotypes. Similarly, IL-1β treatment only induced upregulation of a subset of inflammatory fibroblast marker genes. In silico modelling of ligand-receptor signaling identified additional pathways and cell interactions likely to be involved in fibroblast activation. This highlighted a potential role for IL-11 and IL-6 (among other ligands) in myofibroblast and inflammatory fibroblast activation respectively. This analysis provides valuable insight into fibroblast subtypes and differentiation mechanisms in NSCLC. Citation Format: Christopher Jon Hanley, Sara Waise, Parker Rachel, Maria-Antoinette Lopez, Julian Taylor, Lucy Kimbley, Jonathon West, Christian Ottensmeier, Mat Rose-Zerilli, Gareth Thomas. Spatially discrete signalling niches regulate fibroblast heterogeneity in human lung cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-039.

Prrx1 Fibroblasts Represent a Pro-fibrotic Lineage in the Mouse Ventral Dermis

SUMMARYFibroblast heterogeneity has been shown within the unwounded mouse dorsal dermis, with fibroblast subpopulations being identified according to anatomical location and embryonic lineage. Using lineage tracing, we demonstrate that paired related homeobox 1 (Prrx1)-expressing fibroblasts are responsible for acute and chronic fibroses in the ventral dermis. Single-cell transcriptomics further corroborated the inherent fibrotic characteristics of Prrx1 fibroblasts during wound repair. In summary, we identify and characterize a fibroblast subpopulation in the mouse ventral dermis with intrinsic scar-forming potential.

Abstract PO-043: Plexiform neurofibroma microenvironment at single-cell resolution

Abstract Plexiform neurofibromas (PN) are benign nerve sheath Schwann cell tumors, which occur in at least 50% of individuals with loss of function mutations in the NF1 tumor suppressor gene, an off signal for RAS-GTPases. Tumors form, at least in part, through activation of the RAS-MAPK pathway in Schwann cells. Though benign, PN can cause significant morbidity and are often inoperable due to integration with vital peripheral nerves. Recent clinical trials have shown that MEK inhibition can shrink most PN, but continued MEK inhibition is required for sustained response. It is recognized that PN contain many cell types in addition to Schwann cells, and that Schwann cells are present in varying proportions. However, biomarkers for many cell types have not yet been identified, and it is not known whether subpopulations of certain cell types exist within neurofibromas. To address these issues, we performed single cell RNA sequencing on PN using a genetically engineered mouse model which closely recapitulates human PN. We have characterized tumor composition, and identified PN Schwann cells, satellite glial cells, endothelial cells, fibroblasts, macrophages, T cells, dendritic cells, and other previously unreported cell types. We identified multiple subpopulations of Schwann cells, macrophages and fibroblasts within benign PN. Cluster by cluster comparison of PN to age-matched healthy nerve and to pretumor nerve tissue from the same mouse model revealed gene regulatory networks that are significantly altered in PN, after loss of NF1 function. Further, inference of cell-cell communication networks shows robust changes in PN that are absent before tumors form, and that are potentially targetable. Comparison to MEK inhibitor treated PN and to human PN sc-RNASeq is ongoing. Taken together, these approaches characterize PN, provide potential biomarkers for cell populations and identify targets for drug treatment. Supported by NIH (NINDS-28840) and the DOD Program on Neurofibromatosis. LK is supported by NIH Ruth L. Kirschstein T32. Citation Format: Leah J. Kershner, Jianqiang Wu, Nathan Salomonis, Nancy Ratner. Plexiform neurofibroma microenvironment at single-cell resolution [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-043.