Wnt Signaling Inhibition Prevents Postnatal Inflammation and Disease Progression in Mouse Congenital Myxomatous Valve Disease.

BACKGROUND: Myxomatous valve disease (MVD) is the most common cause of mitral regurgitation, leading to impaired cardiac function and heart failure. MVD in a mouse model of Marfan syndrome includes valve leaflet thickening and progressive valve degeneration. However, the underlying mechanisms by which the disease progresses remain undefined. METHODS: Mice with Fibrillin 1 gene variant Fbn1C1039G/+ recapitulate histopathologic features of Marfan syndrome, and Wnt (Wingless-related integration site) signaling activity was detected in TCF/Lef-lacZ (T-cell factor/lymphoid enhancer factor-ß-galactosidase) reporter mice. Single-cell RNA sequencing was performed from mitral valves of wild-type and Fbn1C1039G/+ mice at 1 month of age. Inhibition of Wnt signaling was achieved by conditional induction of the secreted Wnt inhibitor Dkk1 (Dickkopf-1) expression in periostin-expressing valve interstitial cells of Periostin-Cre; tetO-Dkk1; R26rtTA; TCF/Lef-lacZ; Fbn1C1039G/+ mice. Dietary doxycycline was administered for 1 month beginning with MVD initiation (1-month-old) or MVD progression (2-month-old). Histological evaluation and immunofluorescence for ECM (extracellular matrix) and immune cells were performed. RESULTS: Wnt signaling is activated early in mitral valve disease progression, before immune cell infiltration in Fbn1C1039G/+ mice. Single-cell transcriptomics revealed similar mitral valve cell heterogeneity between wild-type and Fbn1C1039G/+ mice at 1 month of age. Wnt pathway genes were predominantly expressed in valve interstitial cells and valve endothelial cells of Fbn1C1039G/+ mice. Inhibition of Wnt signaling in Fbn1C1039G/+ mice at 1 month of age prevented the initiation of MVD as indicated by improved ECM remodeling and reduced valve leaflet thickness with decreased infiltrating macrophages. However, later, Wnt inhibition starting at 2 months did not prevent the progression of MVD. CONCLUSIONS: Wnt signaling is involved in the initiation of mitral valve abnormalities and inflammation but is not responsible for later-stage valve disease progression once it has been initiated. Thus, Wnt signaling contributes to MVD progression in a time-dependent manner and provides a promising therapeutic target for the early treatment of congenital MVD in Marfan syndrome.

Single Cell Multiomics Identifies Cells and Genetic Networks Underlying Alveolar Capillary Dysplasia.

Rationale: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal developmental disorder of lung morphogenesis caused by insufficiency of FOXF1 (forkhead box F1) transcription factor function. The cellular and transcriptional mechanisms by which FOXF1 deficiency disrupts human lung formation are unknown. Objectives: To identify cell types, gene networks, and cell-cell interactions underlying the pathogenesis of ACDMPV. Methods: We used single-nucleus RNA and assay for transposase-accessible chromatin sequencing, immunofluorescence confocal microscopy, and RNA in situ hybridization to identify cell types and molecular networks influenced by FOXF1 in ACDMPV lungs. Measurements and Main Results: Pathogenic single-nucleotide variants and copy-number variant deletions involving the FOXF1 gene locus in all subjects with ACDMPV (n = 6) were accompanied by marked changes in lung structure, including deficient alveolar development and a paucity of pulmonary microvasculature. Single-nucleus RNA and assay for transposase-accessible chromatin sequencing identified alterations in cell number and gene expression in endothelial cells (ECs), pericytes, fibroblasts, and epithelial cells in ACDMPV lungs. Distinct cell-autonomous roles for FOXF1 in capillary ECs and pericytes were identified. Pathogenic variants involving the FOXF1 gene locus disrupt gene expression in EC progenitors, inhibiting the differentiation or survival of capillary 2 ECs and cell-cell interactions necessary for both pulmonary vasculogenesis and alveolar type 1 cell differentiation. Loss of the pulmonary microvasculature was associated with increased VEGFA (vascular endothelial growth factor A) signaling and marked expansion of systemic bronchial ECs expressing COL15A1 (collagen type XV α 1 chain). Conclusions: Distinct FOXF1 gene regulatory networks were identified in subsets of pulmonary endothelial and fibroblast progenitors, providing both cellular and molecular targets for the development of therapies for ACDMPV and other diffuse lung diseases of infancy.

Lymphangioleiomyomatosis (LAM) Cell Atlas.

Lymphangioleiomyomatosis (LAM) is a rare lung disease of women, causing cystic remodelling of the lung and progressive respiratory failure. The cellular composition, microenvironment and cellular interactions within the LAM lesion remain unclear. To facilitate data sharing and collaborative LAM research, we performed an integrative analysis of single-cell data compiled from lung, uterus and kidney of patients with LAM from three research centres and developed an LAM Cell Atlas (LCA) Web-Portal. The LCA offers a variety of interactive options for investigators to search, visualise and reanalyse comprehensive single-cell multiomics data sets to reveal dysregulated genetic programmes at transcriptomic, epigenomic and cell-cell connectome levels.

LungMAP Portal Ecosystem: Systems-Level Exploration of the Lung.

An improved understanding of the human lung necessitates advanced systems models informed by an ever-increasing repertoire of molecular omics, cellular, imaging, and pathological datasets. To centralize and standardize information across broad lung research efforts we expanded the LungMAP.net website into a new gateway portal. This portal connects a broad spectrum of research networks, bulk and single-cell multi-omics data and a diverse collection of image data that span mammalian lung development, and disease. The data are standardized across species and technologies using harmonized data and metadata models that leverage recent advances including those from the Human Cell Atlas, diverse ontologies, and the LungMAP CellCards initiative. To cultivate future discoveries, we have aggregated a diverse collection of single-cell atlases for multiple species (human, rhesus, mouse), to enable consistent queries across technologies, cohorts, age, disease, and drug treatment. These atlases are provided as independent and integrated queryable datasets, with an emphasis on dynamic visualization, figure generation, re-analysis, cell-type curation, and automated reference-based classification of user-provided single-cell genomics datasets (Azimuth). As this resource grows, we intend to increase the breadth of available interactive interfaces, supported data types, data portals and datasets from LungMAP and external research efforts.

Generation of Pulmonary Endothelial Progenitor Cells for Cell-based Therapy Using Interspecies Mouse-Rat Chimeras.

Rationale: Although pulmonary endothelial progenitor cells (EPCs) hold promise for cell-based therapies for neonatal pulmonary disorders, whether EPCs can be derived from pluripotent embryonic stem cells (ESCs) or induced pluripotent stem cells remains unknown.Objectives: To investigate the heterogeneity of pulmonary EPCs and derive functional EPCs from pluripotent ESCs.Methods: Single-cell RNA sequencing of neonatal human and mouse lung was used to identify the heterogeneity of pulmonary EPCs. CRISPR/Cas9 gene editing was used to genetically label and purify mouse pulmonary EPCs. Functional properties of the EPCs were assessed after cell transplantation into neonatal mice with S52F Foxf1 mutation, a mouse model of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Interspecies mouse-rat chimeras were produced through blastocyst complementation to generate EPCs from pluripotent ESCs for cell therapy in ACDMPV mice.Measurements and Main Results: We identified a unique population of EPCs, FOXF1+cKIT+ EPCs, as a subset of recently described general capillary cells (gCAPs) expressing SMAD7, ZBTB20, NFIA, and DLL4 but lacking mature arterial, venous, and lymphatic markers. FOXF1+cKIT+ gCAPs are reduced in ACDMPV, and their transcriptomic signature is conserved in mouse and human lungs. After cell transplantation into the neonatal circulation of ACDMPV mice, FOXF1+cKIT+ gCAPs engraft into the pulmonary vasculature, stimulate angiogenesis, improve oxygenation, and prevent alveolar simplification. FOXF1+cKIT+ gCAPs, produced from ESCs in interspecies chimeras, are fully competent to stimulate neonatal lung angiogenesis and alveolarization in ACDMPV mice.Conclusions: Cell-based therapy using donor or ESC/induced pluripotent stem cell-derived FOXF1+cKIT+ endothelial progenitors may be considered for treatment of human ACDMPV.

In Vivo Generation of Lung and Thyroid Tissues from Embryonic Stem Cells Using Blastocyst Complementation.

Rationale: The regeneration and replacement of lung cells or tissues from induced pluripotent stem cell- or embryonic stem cell-derived cells represent future therapies for life-threatening pulmonary disorders but are limited by technical challenges to produce highly differentiated cells able to maintain lung function. Functional lung tissue-containing airways, alveoli, vasculature, and stroma have never been produced via directed differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells. We sought to produce all tissue components of the lung from bronchi to alveoli by embryo complementation.Objectives: To determine whether ESCs are capable of generating lung tissue in Nkx2-1-/- mouse embryos with lung agenesis.Methods: Blastocyst complementation was used to produce chimeras from normal mouse ESCs and Nkx2-1-/- embryos, which lack pulmonary tissues. Nkx2-1-/- chimeras were examined using immunostaining, transmission electronic microscopy, fluorescence-activated cell sorter analysis, and single-cell RNA sequencing.Measurements and Main Results: Although peripheral pulmonary and thyroid tissues are entirely lacking in Nkx2-1 gene-deleted embryos, pulmonary and thyroid structures in Nkx2-1-/- chimeras were restored after ESC complementation. Respiratory epithelial cell lineages in restored lungs of Nkx2-1-/- chimeras were derived almost entirely from ESCs, whereas endothelial, immune, and stromal cells were mosaic. ESC-derived cells from multiple respiratory cell lineages were highly differentiated and indistinguishable from endogenous cells based on morphology, ultrastructure, gene expression signatures, and cell surface proteins used to identify cell types by fluorescence-activated cell sorter.Conclusions: Lung and thyroid tissues were generated in vivo from ESCs by blastocyst complementation. Nkx2-1-/- chimeras can be used as "bioreactors" for in vivo differentiation and functional studies of ESC-derived progenitor cells.

Single-Cell Transcriptomic Analysis Identifies a Unique Pulmonary Lymphangioleiomyomatosis Cell.

Rationale: Lymphangioleiomyomatosis (LAM) is a metastatic neoplasm of reproductive-age women associated with mutations in tuberous sclerosis complex genes. LAM causes cystic remodeling of the lung and progressive respiratory failure. The sources and cellular characteristics of LAM cells underlying disease pathogenesis remain elusive.Objectives: Identification and characterization of LAM cells in human lung and uterus using a single-cell approach.Methods: Single-cell and single-nuclei RNA sequencing on LAM (n = 4) and control (n = 7) lungs, immunofluorescence confocal microscopy, ELISA, and aptamer proteomics were used to identify and validate LAMCORE cells and secreted biomarkers, predict cellular origins, and define molecular and cellular networks in LAM.Measurements and Main Results: A unique cell type termed LAMCORE was identified, which was distinct from, but closely related to, lung mesenchymal cells. LAMCORE cells expressing signature genes included known LAM markers such as PMEL, FIGF, CTSK, and MLANA and novel biomarkers validated by aptamer screening, ELISA, and immunofluorescence microscopy. LAM cells in lung and uterus are morphologically indistinguishable and share similar gene expression profiles and biallelic TSC2 mutations, supporting a potential uterine origin for the LAMCORE cell. Effects of LAM on resident pulmonary cell types indicated recruitment and activation of lymphatic endothelial cells.Conclusions: A unique population of LAMCORE cells was identified in lung and uterus of patients with LAM, sharing close transcriptomic identity. LAM cell selective markers, secreted biomarkers, and the predicted cellular molecular features provide new insights into the signaling and transcriptional programs that may serve as diagnostic markers and therapeutic targets to influence the pathogenesis of LAM.

Glucocorticoid regulates mesenchymal cell differentiation required for perinatal lung morphogenesis and function.

While antenatal glucocorticoids are widely used to enhance lung function in preterm infants, cellular and molecular mechanisms by which glucocorticoid receptor (GR) signaling influences lung maturation remain poorly understood. Deletion of the glucocorticoid receptor gene (Nr3c1) from fetal pulmonary mesenchymal cells phenocopied defects caused by global Nr3c1 deletion, while lung epithelial- or endothelial-specific Nr3c1 deletion did not impair lung function at birth. We integrated genome-wide gene expression profiling, ATAC-seq, and single cell RNA-seq data in mice in which GR was deleted or activated to identify the cellular and molecular mechanisms by which glucocorticoids control prenatal lung maturation. GR enhanced differentiation of a newly defined proliferative mesenchymal progenitor cell (PMP) into matrix fibroblasts (MFBs), in part by directly activating extracellular matrix-associated target genes, including Fn1, Col16a4, and Eln and by modulating VEGF, JAK-STAT, and WNT signaling. Loss of mesenchymal GR signaling blocked fibroblast progenitor differentiation into mature MFBs, which in turn increased proliferation of SOX9+ alveolar epithelial progenitor cells and inhibited differentiation of mature alveolar type II (AT2) and AT1 cells. GR signaling controls genes required for differentiation of a subset of proliferative mesenchymal progenitors into matrix fibroblasts, in turn, regulating signals controlling AT2/AT1 progenitor cell proliferation and differentiation and identifying cells and processes by which glucocorticoid signaling regulates fetal lung maturation.

Postnatal Alveologenesis Depends on FOXF1 Signaling in c-KIT+ Endothelial Progenitor Cells.

Rationale: Disruption of alveologenesis is associated with severe pediatric lung disorders, including bronchopulmonary dysplasia (BPD). Although c-KIT+ endothelial cell (EC) progenitors are abundant in embryonic and neonatal lungs, their role in alveolar septation and the therapeutic potential of these cells remain unknown.Objectives: To determine whether c-KIT+ EC progenitors stimulate alveologenesis in the neonatal lung.Methods: We used single-cell RNA sequencing of neonatal human and mouse lung tissues, immunostaining, and FACS analysis to identify transcriptional and signaling networks shared by human and mouse pulmonary c-KIT+ EC progenitors. A mouse model of perinatal hyperoxia-induced lung injury was used to identify molecular mechanisms that are critical for the survival, proliferation, and engraftment of c-KIT+ EC progenitors in the neonatal lung.Measurements and Main Results: Pulmonary c-KIT+ EC progenitors expressing PECAM-1, CD34, VE-Cadherin, FLK1, and TIE2 lacked mature arterial, venal, and lymphatic cell-surface markers. The transcriptomic signature of c-KIT+ ECs was conserved in mouse and human lungs and enriched in FOXF1-regulated transcriptional targets. Expression of FOXF1 and c-KIT was decreased in the lungs of infants with BPD. In the mouse, neonatal hyperoxia decreased the number of c-KIT+ EC progenitors. Haploinsufficiency or endothelial-specific deletion of Foxf1 in mice increased apoptosis and decreased proliferation of c-KIT+ ECs. Inactivation of either Foxf1 or c-Kit caused alveolar simplification. Adoptive transfer of c-KIT+ ECs into the neonatal circulation increased lung angiogenesis and prevented alveolar simplification in neonatal mice exposed to hyperoxia.Conclusions: Cell therapy involving c-KIT+ EC progenitors can be beneficial for the treatment of BPD.

SLICE: determining cell differentiation and lineage based on single cell entropy.

A complex organ contains a variety of cell types, each with its own distinct lineage and function. Understanding the lineage and differentiation state of each cell is fundamentally important for the ultimate delineation of organ formation and function. We developed SLICE, a novel algorithm that utilizes single-cell RNA-seq (scRNA-seq) to quantitatively measure cellular differentiation states based on single cell entropy and predict cell differentiation lineages via the construction of entropy directed cell trajectories. We validated our approach using three independent data sets with known lineage and developmental time information from both Homo sapiens and Mus musculus. SLICE successfully measured the differentiation states of single cells and reconstructed cell differentiation trajectories that have been previously experimentally validated. We then applied SLICE to scRNA-seq of embryonic mouse lung at E16.5 to identify lung mesenchymal cell lineage relationships that currently remain poorly defined. A two-branched differentiation pathway of five fibroblastic subtypes was predicted using SLICE. The present study demonstrated the general applicability and high predictive accuracy of SLICE in determining cellular differentiation states and reconstructing cell differentiation lineages in scRNA-seq analysis.

Overexpression of Tbx20 in Adult Cardiomyocytes Promotes Proliferation and Improves Cardiac Function After Myocardial Infarction.

BACKGROUND: Adult mammalian cardiomyocytes (CMs) have the potential to proliferate, but this is not sufficient to generate adequate CMs after myocardial infarction (MI). The transcription factor Tbx20 is required for CM proliferation during development and adult CM homeostasis. The ability of Tbx20 overexpression (Tbx20(OE)) to promote adult CM proliferation and to improve cardiac function after MI was examined. METHODS AND RESULTS: Tbx20(OE) was induced specifically in adult mouse differentiated CMs. Increased CM proliferation and fetal-like characteristics were found in Tbx20(OE) hearts compared with controls without causing pathology 4 weeks after Tbx20(OE) at baseline. Moreover, Tbx20(OE) in adult CM after MI significantly improved survival, cardiac function, and infarct size 4 weeks after MI. Improved cardiac repair, as indicated by increased CM proliferation and capillary density, was observed in the MI border zone of Tbx20(OE) hearts compared with controls. Expression of proliferation activator (cyclin D1, E1, and IGF1) and fetal contractile protein (ssTNI, ßMHC) mRNA was increased whereas negative cell-cycle regulators (p21, Meis1) were decreased in Tbx20(OE) hearts compared with controls under both baseline and MI conditions. Tbx20(OE) in adult hearts activates multiple proproliferation pathways, including Akt, YAP and BMP. Interestingly, p21, Meis1, and a novel cell-cycle inhibitory gene, Btg2, are directly bound and repressed by Tbx20 with induction of proliferation in neonatal CM. CONCLUSIONS: Tbx20(OE), specifically in adult CM, activates multiple cardiac proliferative pathways, directly represses cell-cycle inhibitory genes p21, Meis1, and Btg2, promotes adult CM proliferation; and preserves cardiac performance after MI.

Lung Gene Expression Analysis (LGEA): an integrative web portal for comprehensive gene expression data analysis in lung development.

'LungGENS', our previously developed web tool for mapping single-cell gene expression in the developing lung, has been well received by the pulmonary research community. With continued support from the 'LungMAP' consortium, we extended the scope of the LungGENS database to accommodate transcriptomics data from pulmonary tissues and cells from human and mouse at different stages of lung development. Lung Gene Expression Analysis (LGEA) web portal is an extended version of LungGENS useful for the analysis, display and interpretation of gene expression patterns obtained from single cells, sorted cell populations and whole lung tissues. The LGEA web portal is freely available at http://research.cchmc.org/pbge/lunggens/mainportal.html.

SINCERA: A Pipeline for Single-Cell RNA-Seq Profiling Analysis.

A major challenge in developmental biology is to understand the genetic and cellular processes/programs driving organ formation and differentiation of the diverse cell types that comprise the embryo. While recent studies using single cell transcriptome analysis illustrate the power to measure and understand cellular heterogeneity in complex biological systems, processing large amounts of RNA-seq data from heterogeneous cell populations creates the need for readily accessible tools for the analysis of single-cell RNA-seq (scRNA-seq) profiles. The present study presents a generally applicable analytic pipeline (SINCERA: a computational pipeline for SINgle CEll RNA-seq profiling Analysis) for processing scRNA-seq data from a whole organ or sorted cells. The pipeline supports the analysis for: 1) the distinction and identification of major cell types; 2) the identification of cell type specific gene signatures; and 3) the determination of driving forces of given cell types. We applied this pipeline to the RNA-seq analysis of single cells isolated from embryonic mouse lung at E16.5. Through the pipeline analysis, we distinguished major cell types of fetal mouse lung, including epithelial, endothelial, smooth muscle, pericyte, and fibroblast-like cell types, and identified cell type specific gene signatures, bioprocesses, and key regulators. SINCERA is implemented in R, licensed under the GNU General Public License v3, and freely available from CCHMC PBGE website, https://research.cchmc.org/pbge/sincera.html.

'LungGENS': a web-based tool for mapping single-cell gene expression in the developing lung.

We developed LungGENS (Lung Gene Expression iN Single-cell), a web-based bioinformatics resource for querying single-cell gene expression databases by entering a gene symbol or a list of genes or selecting a cell type of their interest. Gene query provides quantitative RNA expression of the gene of interest in each lung cell type. Cell type query returns associated selective gene signatures and genes encoding cell surface markers and transcription factors in interactive heatmap and tables. LungGENS will be broadly applicable in respiratory research, providing a cell-specific RNA expression resource at single-cell resolution. LungGENS is freely available for non-commercial use at https://research.cchmc.org/pbge/lunggens/default.html.

Identification of endothelial and mesenchymal FOXF1 enhancers involved in alveolar capillary dysplasia.

Mutations in the FOXF1 gene, a key transcriptional regulator of pulmonary vascular development, cause Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins, a lethal lung disease affecting newborns and infants. Identification of new FOXF1 upstream regulatory elements is critical to explain why frequent non-coding FOXF1 deletions are linked to the disease. Herein, we use multiome single-nuclei RNA and ATAC sequencing of mouse and human patient lungs to identify four conserved endothelial and mesenchymal FOXF1 enhancers. We demonstrate that endothelial FOXF1 enhancers are autoactivated, whereas mesenchymal FOXF1 enhancers are regulated by EBF1 and GLI1. The cell-specificity of FOXF1 enhancers is validated by disrupting these enhancers in mouse embryonic stem cells using CRISPR/Cpf1 genome editing followed by lineage-tracing of mutant embryonic stem cells in mouse embryos using blastocyst complementation. This study resolves an important clinical question why frequent non-coding FOXF1 deletions that interfere with endothelial and mesenchymal enhancers can lead to the disease.

Single-Cell Transcriptomic Profiling Identifies Molecular Phenotypes of Newborn Human Lung Cells.

While animal model studies have extensively defined the mechanisms controlling cell diversity in the developing mammalian lung, there exists a significant knowledge gap with regards to late-stage human lung development. The NHLBI Molecular Atlas of Lung Development Program (LungMAP) seeks to fill this gap by creating a structural, cellular and molecular atlas of the human and mouse lung. Transcriptomic profiling at the single-cell level created a cellular atlas of newborn human lungs. Frozen single-cell isolates obtained from two newborn human lungs from the LungMAP Human Tissue Core Biorepository, were captured, and library preparation was completed on the Chromium 10X system. Data was analyzed in Seurat, and cellular annotation was performed using the ToppGene functional analysis tool. Transcriptional interrogation of 5500 newborn human lung cells identified distinct clusters representing multiple populations of epithelial, endothelial, fibroblasts, pericytes, smooth muscle, immune cells and their gene signatures. Computational integration of data from newborn human cells and with 32,000 cells from postnatal days 1 through 10 mouse lungs generated by the LungMAP Cincinnati Research Center facilitated the identification of distinct cellular lineages among all the major cell types. Integration of the newborn human and mouse cellular transcriptomes also demonstrated cell type-specific differences in maturation states of newborn human lung cells. Specifically, newborn human lung matrix fibroblasts could be separated into those representative of younger cells (n = 393), or older cells (n = 158). Cells with each molecular profile were spatially resolved within newborn human lung tissue. This is the first comprehensive molecular map of the cellular landscape of neonatal human lung, including biomarkers for cells at distinct states of maturity.

Deciphering Endothelial and Mesenchymal Organ Specification in Vascularized Lung and Intestinal Organoids.

To investigate the co-development of vasculature, mesenchyme, and epithelium crucial for organogenesis and the acquisition of organ-specific characteristics, we constructed a human pluripotent stem cell-derived organoid system comprising lung or intestinal epithelium surrounded by organotypic mesenchyme and vasculature. We demonstrated the pivotal role of co-differentiating mesoderm and endoderm via precise BMP regulation in generating multilineage organoids and gut tube patterning. Single-cell RNA-seq analysis revealed organ specificity in endothelium and mesenchyme, and uncovered key ligands driving endothelial specification in the lung (e.g., WNT2B and Semaphorins) or intestine (e.g., GDF15). Upon transplantation under the kidney capsule in mice, these organoids further matured and developed perfusable human-specific sub-epithelial capillaries. Additionally, our model recapitulated the abnormal endothelial-epithelial crosstalk in patients with FOXF1 deletion or mutations. Multilineage organoids provide a unique platform to study developmental cues guiding endothelial and mesenchymal cell fate determination, and investigate intricate cell-cell communications in human organogenesis and disease. Highlights: BMP signaling fine-tunes the co-differentiation of mesoderm and endoderm.The cellular composition in multilineage organoids resembles that of human fetal organs.Mesenchyme and endothelium co-developed within the organoids adopt organ-specific characteristics.Multilineage organoids recapitulate abnormal endothelial-epithelial crosstalk in FOXF1-associated disorders.

FOXA2 Cooperates with Mutant KRAS to Drive Invasive Mucinous Adenocarcinoma of the Lung.

The endoderm-lineage transcription factor FOXA2 has been shown to inhibit lung tumorigenesis in in vitro and xenograft studies using lung cancer cell lines. However, FOXA2 expression in primary lung tumors does not correlate with an improved patient survival rate, and the functional role of FOXA2 in primary lung tumors remains elusive. To understand the role of FOXA2 in primary lung tumors in vivo, here, we conditionally induced the expression of FOXA2 along with either of the two major lung cancer oncogenes, EGFRL858R or KRASG12D, in the lung epithelium of transgenic mice. Notably, FOXA2 suppressed autochthonous lung tumor development driven by EGFRL858R, whereas FOXA2 promoted tumor growth driven by KRASG12D. Importantly, FOXA2 expression along with KRASG12D produced invasive mucinous adenocarcinoma (IMA) of the lung, a fatal mucus-producing lung cancer comprising approximately 5% of human lung cancer cases. In the mouse model in vivo and human lung cancer cells in vitro, FOXA2 activated a gene regulatory network involved in the key mucous transcription factor SPDEF and upregulated MUC5AC, whose expression is critical for inducing IMA. Coexpression of FOXA2 with mutant KRAS synergistically induced MUC5AC expression compared with that induced by FOXA2 alone. ChIP-seq combined with CRISPR interference indicated that FOXA2 bound directly to the enhancer region of MUC5AC and induced the H3K27ac enhancer mark. Furthermore, FOXA2 was found to be highly expressed in primary tumors of human IMA. Collectively, this study reveals that FOXA2 is not only a biomarker but also a driver for IMA in the presence of a KRAS mutation. SIGNIFICANCE: FOXA2 expression combined with mutant KRAS drives invasive mucinous adenocarcinoma of the lung by synergistically promoting a mucous transcriptional program, suggesting strategies for targeting this lung cancer type that lacks effective therapies.