Probes for INS

Adrenal cortex steroids are essential for body homeostasis, and adrenal insufficiency is a life-threatening condition. Adrenal endocrine activity is maintained through recruitment of subcapsular progenitor cells that follow a unidirectional differentiation path from zona glomerulosa to zona fasciculata (zF). Here, we show that this unidirectionality is ensured by the histone methyltransferase EZH2. Indeed, we demonstrate that EZH2 maintains adrenal steroidogenic cell differentiation by preventing expression of GATA4 and WT1 that cause abnormal dedifferentiation to a progenitor-like state in Ezh2 KO adrenals. EZH2 further ensures normal cortical differentiation by programming cells for optimal response to adrenocorticotrophic hormone (ACTH)/PKA signaling. This is achieved by repression of phosphodiesterases PDE1B, 3A, and 7A and of PRKAR1B. Consequently, EZH2 ablation results in blunted zF differentiation and primary glucocorticoid insufficiency. These data demonstrate an all-encompassing role for EZH2 in programming steroidogenic cells for optimal response to differentiation signals and in maintaining their differentiated state.

Resolving the spatial distribution of RNA and protein in tissues at subcellular resolution is a challenge in the field of spatial biology. We describe spatial molecular imaging, a system that measures RNAs and proteins in intact biological samples at subcellular resolution by performing multiple cycles of nucleic acid hybridization of fluorescent molecular barcodes. We demonstrate that spatial molecular imaging has high sensitivity (one or two copies per cell) and very low error rate (0.0092 false calls per cell) and background (~0.04 counts per cell). The imaging system generates three-dimensional, super-resolution localization of analytes at ~2 million cells per sample. Cell segmentation is morphology based using antibodies, compatible with formalin-fixed, paraffin-embedded samples. We measured multiomic data (980 RNAs and 108 proteins) at subcellular resolution in formalin-fixed, paraffin-embedded tissues (nonsmall cell lung and breast cancer) and identified >18 distinct cell types, ten unique tumor microenvironments and 100 pairwise ligand-receptor interactions. Data on >800,000 single cells and ~260 million transcripts can be accessed at http://nanostring.com/CosMx-dataset .

Patients with peripheral nerve injury, viral infection or metabolic disorder often suffer neuropathic pain due to inadequate pharmacological options for relief. Developing novel therapies has been challenged by incomplete mechanistic understanding of the cellular microenvironment in sensory nerve that trigger the emergence and persistence of pain. In this study, we report a high resolution transcriptomics map of the cellular heterogeneity of naïve and injured rat sensory nerve covering more than 110,000 individual cells. Annotation reveals distinguishing molecular features of multiple major cell types totaling 45 different subtypes in naïve nerve and an additional 23 subtypes emerging after injury. Ligand-receptor analysis revealed a myriad of potential targets for pharmacological intervention. This work forms a comprehensive resource and unprecedented window into the cellular milieu underlying neuropathic pain and demonstrates that nerve injury is a dynamic process orchestrated by multiple cell types in both the endoneurial and epineurial nerve compartments.

Proper Hedgehog (HH) signaling is essential for embryonic development, while aberrant HH signaling drives pediatric and adult cancers. HH signaling is frequently dysregulated in pancreatic cancer, yet its role remains controversial, with both tumor-promoting and tumor-restraining functions reported. Notably, the GLI family of HH transcription factors (GLI1, GLI2, GLI3), remain largely unexplored in pancreatic cancer. We therefore investigated the individual and combined contributions of GLI1-3 to pancreatic cancer progression. At pre-cancerous stages, fibroblast-specific Gli2/Gli3 deletion decreases immunosuppressive macrophage infiltration and promotes T cell infiltration. Strikingly, combined loss of Gli1/Gli2/Gli3 promotes macrophage infiltration, indicating that subtle changes in Gli expression differentially regulate immune infiltration. In invasive tumors, Gli2/Gli3 KO fibroblasts exclude immunosuppressive myeloid cells and suppress tumor growth by recruiting natural killer cells. Finally, we demonstrate that fibroblasts directly regulate macrophage and T cell migration through the expression of Gli-dependent cytokines. Thus, the coordinated activity of GLI1-3 directs the fibroinflammatory response throughout pancreatic cancer progression.

The lateral habenula (LHb) is an epithalamic brain structure critical for processing and adapting to negative action outcomes. However, despite the importance of LHb to behavior and the clear anatomical and molecular diversity of LHb neurons, the neuron types of the habenula remain unknown. Here, we use high-throughput single-cell transcriptional profiling, monosynaptic retrograde tracing, and multiplexed FISH to characterize the cells of the mouse habenula. We find five subtypes of neurons in the medial habenula (MHb) that are organized into anatomical subregions. In the LHb, we describe four neuronal subtypes and show that they differentially target dopaminergic and GABAergic cells in the ventral tegmental area (VTA). These data provide a valuable resource for future study of habenular function and dysfunction and demonstrate neuronal subtype specificity in the LHb-VTA circuit

In adults, hepatocytes are mainly replenished from the existing progenitor pools of hepatocytes and cholangiocytes during chronic liver injury. However, it is unclear whether other cell types in addition to classical hepatocytes and cholangiocytes contribute to hepatocyte regeneration after chronic liver injuries. Here, we identified a new biphenotypic cell population that contributes to hepatocyte regeneration during chronic liver injuries. We found that a cell population expressed Gli1 and EpCAM (EpCAM+Gli1+), which was further characterized with both epithelial and mesenchymal identities by single-cell RNA sequencing. Genetic lineage tracing using dual recombinases revealed that Gli1+ nonhepatocyte cell population could generate hepatocytes after chronic liver injury. EpCAM+Gli1+ cells exhibited a greater capacity for organoid formation with functional hepatocytes in vitro and liver regeneration upon transplantation in vivo. Collectively, these findings demonstrate that EpCAM+Gli1+ cells can serve as a new source of liver progenitor cells and contribute to liver repair and regeneration.

COVID-19-induced ‘acute respiratory distress syndrome’ (ARDS) is associated with prolonged respiratory failure and high mortality, but the mechanistic basis of lung injury remains incompletely understood. Here, we analyzed pulmonary immune responses and lung pathology in two cohorts of patients with COVID-19 ARDS using functional single cell genomics, immunohistology and electron microscopy. We describe an accumulation of CD163-expressing monocyte-derived macrophages that acquired a profibrotic transcriptional phenotype during COVID-19 ARDS. Gene set enrichment and computational data integration revealed a significant similarity between COVID-19-associated macrophages and profibrotic macrophage populations identified in idiopathic pulmonary fibrosis. COVID-19 ARDS was associated with clinical, radiographic, histopathological, and ultrastructural hallmarks of pulmonary fibrosis. Exposure of human monocytes to SARS-CoV-2, but not Influenza A virus or viral RNA analogs, was sufficient to induce a similar profibrotic phenotype in vitro. In conclusion, we demonstrate that SARS-CoV-2 triggers profibrotic macrophage responses and pronounced fibroproliferative ARDS.

Chromatin remodelers often show broad expression patterns in multiple cell types yet can elicit cell-specific effects in development and diseases. Arid1a binds DNA and regulates gene expression during tissue development and homeostasis. However, it is unclear how Arid1a achieves its functional specificity in regulating progenitor cells. Using the tooth root as a model, we show that loss of Arid1a impairs the differentiation-associated cell cycle arrest of tooth root progenitors through Hedgehog (Hh) signaling regulation, leading to shortened roots. Our data suggest that Plagl1, as a co-factor, endows Arid1a with its cell-type/spatial functional specificity. Furthermore, we show that loss of Arid1a leads to increased expression of Arid1b, which is also indispensable for odontoblast differentiation but is not involved in regulation of Hh signaling. This study expands our knowledge of the intricate interactions among chromatin remodelers, transcription factors, and signaling molecules during progenitor cell fate determination and lineage commitment.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with short overall survival; the standard of care (SoC) is chemotherapy. Immunotherapies in development aim to remodel the stroma by depleting immunosuppressive cell types or using T-cell redirection to kill tumor cells. To date, none of these methods have improved overall survival beyond SoC. Next generation immunotherapies that employ histopathology and molecular subtyping1 for target and patient selection may succeed. Here we leverage a spatial transcriptomics platform (Nanostring Digital Spatial Profiling, DSP) to reveal molecular signaling in tumoral and stromal cells in 57 PDAC patients using tumor microarrays (TMAs). This approach is rapid and clinically relevant as molecular and histology data can be easily bridged.MethodsTMAs generated from surgical resection tissue were commercially sourced. DSP was performed using the CTA RNA panel (1,800 target genes) using PanCK fluorescence for tumor/stroma segmentation. In parallel, slides were chromogenically stained for T-cells (CD3) and macrophages (CD68/CD163). Differential gene expression, gene signature and gene co-expression network analysis was performed using linear models in R.2 3ResultsDifferential gene expression analysis and correlation to IHC confirmed the DSP platform successfully profiled tumor and stromal compartments (figure 1). Immune cell signatures4 and pathway analysis revealed a heterogenous stromal environment. Using a fibroblast gene signature derived from single-cell RNAseq5 we found fibroblast density was positively correlated to PDGFR signaling and MHC-II expression but negatively correlated to B, CD4+ T and neutrophil cell levels (figure 2a). This finding supports the idea that atypical antigen presentation in cancer associated fibroblasts (CAFs) may be exploitable for immunotherapies.6 We constructed a co-expression network from in-situ stromal gene expression and used it to identify receptors coordinately expressed with the immunosuppressive macrophage marker CSF1R as a bispecific antibody partner (figure 2b).7 Classical macrophage markers were identified but also receptors with lesser-known functions in macrophages (TIM3/HAVCR2, FPR3, MS4A6A, LILRB4). Surveying target pairs in this method allows rapid, patient-specific confirmation in serial TMA sections with singleplex IHC or RNAscope.Abstact 928 Figure 1Segmentation strategy and validation of DSP (A) PanCK, CD68 and CD3 staining from two representative tumor cores; (B, C) correlation of gene transcripts in stroma to cell counts from chromogenic staining; (D) heatmap of selected genes differentially expressed in tumor and stroma (n=57 patients).Abstract 928 Figure 2Exploration of the stromal compartment in PDAC TMAs. (A) Heatmap of selected cell type and gene signatures from gene expression in the stroma, color represents single sample enrichment score using GSVA method; (B) a gene co-expression subnetwork in the stroma centered on CSF1R, edge thickness represents strength of correlation, green nodes have evidence for cell surface expression based on proteomic profiling.7ConclusionsIn this study we were able to recapitulate known PDAC biology using very small samples of primary tumors. The combination of TMAs and DSP enables a rapid validation of targets and hypothesis generation for bispecific parings. Further analysis of untreated (n=14) and post-adjuvant chemotherapy (n=26) patients using RNA DSP, IHC and bulk RNAseq is under way. Results from this cohort will enable an integrated histopathology and molecular approach to developing next-generation immunotherapies.ReferencesCollisson EA, Bailey P, Chang DK, Biankin AV. Molecular subtypes of pancreatic cancer. Nat Rev Gastroenterol Hepatol 2019 April;16(4):207-220.Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK (2015). “limma powers differential expression analyses for RNA-sequencing and microarray studies.” Nucleic Acids Research 43(7):e47.Hänzelmann S, Castelo R, Guinney J (2013). “GSVA: gene set variation analysis for microarray and RNA-Seq data.” BMC Bioinformatics 14,7.Charoentong P, Finotello F, Angelova M, Mayer C, Efremova M, Rieder D, Hackl H, Trajanoski Z. Pan-cancer immunogenomic analyses reveal genotype-immunophenotype relationships and predictors of response to checkpoint blockade. Cell Rep 2017 January 3;18(1):248-262.Tirosh I, Izar B, Prakadan SM, Wadsworth MH 2nd, Treacy D, Trombetta JJ, Rotem A, Rodman C, Lian C, Murphy G, Fallahi-Sichani M, Dutton-Regester K, Lin JR, Cohen O, Shah P, Lu D, Genshaft AS, Hughes TK, Ziegler CG, Kazer SW, Gaillard A, Kolb KE, Villani AC, Johannessen CM, Andreev AY, Van Allen EM, Bertagnolli M, Sorger PK, Sullivan RJ, Flaherty KT, Frederick DT, Jané-Valbuena J, Yoon CH, Rozenblatt-Rosen O, Shalek AK, Regev A, Garraway LA. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science 2016 April 8;352(6282):189-96.Elyada E, Bolisetty M, Laise P, Flynn WF, Courtois ET, Burkhart RA, Teinor JA, Belleau P, Biffi G, Lucito MS, Sivajothi S, Armstrong TD, Engle DD, Yu KH, Hao Y, Wolfgang CL, Park Y, Preall J, Jaffee EM, Califano A, Robson P, Tuveson DA. Cross-species single-cell analysis of pancreatic ductal adenocarcinoma reveals antigen-presenting cancer-associated fibroblasts. Cancer Discov 2019 August;9(8):1102-1123. Bausch-Fluck D, Hofmann A, Bock T, Frei AP, Cerciello F, Jacobs A, Moest H, Omasits U, Gundry RL, Yoon C, Schiess R, Schmidt A, Mirkowska P, Härtlová A, Van Eyk JE, Bourquin JP, Aebersold R, Boheler KR, Zandstra P, Wollscheid B. A mass spectrometric-derived cell surface protein atlas. PLoS One 2015 April 20;10(3):e0121314.Ethics ApprovalSpecimens were harvested from unused tissue after a surgical tumor resection procedure. A discrete legal consent form from both hospital and individuals was obtained by the commercial tissue vendor BioMax US for all samples analyzed in this abstract. All human tissues are collected under HIPPA approved protocols.ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

The field of osteoarthritis (OA) biology is rapidly evolving and brilliant progress has been made this year as well. Landmark studies of OA biology published in 2021 and early 2022 were selected through PubMed search by personal opinion. These papers were classified by their molecular mechanisms, and it was largely divided into the intracellular signaling mechanisms and the inter-compartment interaction in chondrocyte homeostasis and OA progression. The intracellular signaling mechanisms involving OA progression included (1) Piezo1/transient receptor potential channels of the vanilloid subtype (TRPV) 4-mediated calcium signaling, (2) mechanical load-F-box and WD repeat domain containing 7 (FBXW7) in chondrocyte senescence, (3) mechanical loading-primary cilia-hedgehog signaling, (4) low grade inflammation by toll-like receptor (TLR)-CD14-lipopolysaccharide-binding protein (LBP) complex and inhibitor of NF-κB kinase (IKK) β-nuclear factor kappa B (NF-κB) signaling, (5) selenium pathway and reactive oxygen species (ROS) production, (6) G protein-coupled receptor (GPCR) and cyclic adenosine monophosphate (cAMP) signaling, (7) peroxisome proliferator-activated receptor α (PPARα)-acyl-CoA thioesterase 12 (ACOT12)-mediated de novo lipogenesis and (8) hypoxia-disruptor of telomeric silencing 1-like (DOT1L)-H3-lysine 79 (H3K79) methylation pathway. The studies on inter-compartment or intercellular interaction in OA progression included the following subjects; (1) the anabolic role of lubricin, glycoprotein from superficial zone cells, (2) osteoclast-chondrocyte interaction via exosomal miRNA and sphingosine 1-phosphate (S1P), (3) senescent fibroblast-like synoviocyte and chondrocyte interaction, (4) synovial macrophage and chondrocyte interaction through Flightless I, (5) αV integrin-mediated transforming growth factor beta (TGFβ) activation by mechanical loading, and (6) osteocytic TGFβ in subchondral bone thickening. Despite the disastrous Covid-19 pandemic, many outstanding studies have expanded the boundary of OA biology. They provide both critical insight into the pathophysiology as well as clues for the treatment of OA.