Major Cell Types Research Articles

Initial pig developmental stage influences intestinal organoid growth but not cellular composition.

Intestinal organoids are promising tools in the context of animal experiment reduction but a thorough characterization of the impact of the origin of intestinal stem cells (ISC) on organoid phenotype is needed to routinely use this cellular model. Our objective was to evaluate the effect of ISC donor age on the growth, morphology and cellular composition of intestinal organoids derived from pig. Organoids were derived from jejunal and colonic ISC obtained from 1-, 7-, 28-, 36- and 180-day-old pigs and passaged three times. We first confirmed by qPCR that the expression of 18% of the >80 studied genes related to various intestinal functions differed between jejunal and colonic organoids after two passages (p < 0.05). Growth and morphology of organoids depended on intestinal location (greater number and larger organoids derived from colonic than jejunal ISC, p < 0.05) but also pig age. Indeed, when ISC were derived from young piglets, the ratio of organoids to spheroids was greater (p < 0.05), spheroids were larger during the primary culture but smaller after two passages (p < 0.05) and organoids were smaller after one passage (p > 0.05) compared to ISC from older pigs. Finally, no difference in cellular composition, evaluated by immunostaining of markers of the major intestinal cell types (absorptive, enteroendocrine and goblet cells) was observed between organoids originating from 7- or 180-day-old pigs, but differences between intestinal site origins were noticed. In conclusion, while the age of the tissue donor affected organoid growth and morphology, it did not influence the phenotype.

IFN-I promotes T cell-independent immunity and RBC autoantibodies via modulation of B-1 cell subsets in murine SCD.

The pathophysiology of sickle cell disease (SCD) is characterized by hemolytic anemia and vaso-occlusion, although its impact on the adaptive immune responses remains incompletely understood. To comprehensibly profile the humoral immune responses, we immunized SCD mice with T cell-independent (TI) and T cell-dependent (TD) antigens. Our study showed that SCD mice have significantly enhanced type 2 TI (TI-2) immune responses in a manner dependent on the level of type I IFN (IFN-I), while maintaining similar or decreased TD immune responses depending on the route of antigen administration. Consistent with the enhanced TI-2 immune responses in SCD mice, the frequencies of B-1b cells (B-1 cells in humans), a major cell type responding to TI-2 antigens, were significantly increased in both the peritoneal cavity and spleens of SCD mice and in blood of patients with SCD. In support of expanded B-1 cells, elevated levels of anti-red blood cell (RBC) autoantibodies were detected in both SCD mice and patients. Both the levels of TI-2 immune responses and anti-RBC autoantibodies were significantly reduced following IFNAR antibody blockades and in IFNAR-1 deficient SCD mice. Moreover, the alteration of B-1 cell subsets were reversed in IFNAR-1 deficient SCD mice, uncovering a critical role for IFN-I in the enhanced TI-2 immune responses and the increased production of anti-RBC autoantibodies by modulating the innate B-1 cell subsets in SCD. Overall, our study provides experimental evidence that the modulation of B-1 cells and IFN-I can regulate TI immune responses and the levels of anti-RBC autoantibodies in SCD.

The roles of G protein-coupled receptor genes and the tumor microenvironment in esophageal squamous cell carcinoma

Abstract Objectives Esophageal squamous cell carcinoma (ESCA) is a challenging disease characterized by a high mortality rate. Understanding the prognostic relationship between G protein-coupled receptors (GPR) and ESCA is critical for improving patient outcomes, yet this connection remains to be fully explored. Methods In this study, we examined the roles of GPR genes and the tumor microenvironment (TME) in ESCA development and progression. Cox regression and Kaplan–Meier analysis demonstrated the predictive value of these genes. Our analysis of TME cell-cell communication revealed extensive interactions, particularly involving neutrophils. We also assessed the combined predictive value of GPR genes, TME score, and tumor mutation burden (TMB) for patient prognosis in ESCA, ultimately constructing a GPR-TME-TMB classifier for prognosis prediction. Results We identified significant differences in GPR gene expression between normal and tumor tissues, with four genes (GPER1, GPR82, FFAR2, and HCAR3) correlating with patient prognosis. Single-cell RNA sequencing analysis revealed 10 major cell types in the TME, with GPR gene expression highly enriched in neutrophils. Our findings indicate that the GPR-TME-TMB classifier is strongly associated with patient prognoses. Additionally, our results align with previous studies on the roles of GPR genes and the TME in ESCA. Conclusions Our results suggest that GPR-related genes play a role in ESCA progression and are strongly associated with TME in ESCA. We constructed a GPR-TME classifier for ESCA to provide new directions for the treatment and prognosis of ESCA patients.

Innovative strategies to optimise colorectal cancer immunotherapy through molecular mechanism insights

BackgroundColorectal cancer (CRC) is a leading cause of cancer-related deaths globally. The heterogeneity of the tumor microenvironment significantly influences patient prognosis, while the diversity of tumor cells shapes its unique characteristics. A comprehensive analysis of the molecular profile of tumor cells is crucial for identifying novel molecular targets for drug sensitivity analysis and for uncovering the pathophysiological mechanisms underlying CRC.MethodsWe utilized single-cell RNA sequencing technology to analyze 13 tissue samples from 4 CRC patients, identifying key cell types within the tumor microenvironment. Intercellular communication was assessed using CellChat, and a risk score model was developed based on eight prognostic genes to enhance patient stratification for immunotherapeutic approaches. Additionally, in vitro experiments were performed on DLX2, a gene strongly associated with poor prognosis, to validate its potential role as a therapeutic target in CRC progression.ResultsEight major cell types were identified across the tissue samples. Within the tumor cell population, seven distinct subtypes were recognized, with the C0 FXYD5+ tumor cells subtype being significantly linked to cancer progression and poor prognosis. CellChat analysis indicated extensive communication among tumor cells, fibroblasts, and immune cells, underscoring the complexity of the tumor microenvironment. The risk score model demonstrated high accuracy in predicting 1-, 3-, and 5-year survival rates in CRC patients. Enrichment analysis revealed that the C0 FXYD5+ tumor cell subtype exhibited increased energy metabolism, protein synthesis, and oxidative phosphorylation, contributing to its aggressive behavior. In vitro experiments confirmed DLX2 as a critical gene associated with poor prognosis, suggesting its viability as a target for improving drug sensitivity.ConclusionIn summary, this study advances our understanding of CRC progression by identifying critical tumor subtypes, molecular pathways, and prognostic markers that can inform innovative strategies for predicting and enhancing drug sensitivity. These findings hold promise for optimizing immunotherapeutic approaches and developing new targeted therapies, ultimately aiming to improve patient outcomes in CRC.

Generation and long-term culture of human cerebellar organoids from pluripotent stem cells.

The advancement of research on human cerebellar development and diseases has been hindered by the lack of a cell-based system that mirrors the cellular diversity and functional characteristics of the human cerebellum. Here, we describe our protocol for a human pluripotent stem cell-derived human cerebellar organoid (hCerO) model, which successfully replicates the cellular diversity of the fetal cerebellum along with some of its distinct cytoarchitectural features. Our approach involves the patterning of human pluripotent stem cells, resulting in the generation of both cerebellar excitatory and inhibitory progenitor populations-specifically, the rhombic lip and ventricular zone progenitors, respectively. This patterning strategy leads to the reproducible differentiation of the major neurons of the cerebellum such as granule cells and Purkinje cells within just one month of culture. hCerOs serve as platforms for molecular, cellular and functional assays, including single-cell transcriptomics, immunohistochemistry and investigations into calcium dynamics and electrophysiological properties. Remarkably, the cultivation of hCerOs for up to 8 months enables the healthy survival and maturation of Purkinje cells, which exhibit molecular and electrophysiological features akin to their in vivo counterparts. Overall, our protocol generates and allows for the long-term culture of all major cell types within the cerebellum. Consequently, this significant advancement provides the developmental neurobiology field with a robust platform for exploring both cerebellar development and diseases within an all-human system. This protocol can be easily implemented by a technician with cell culture experience and takes 1-2 months to complete with an option for extended maturation over the course of several months.

'Iterative Bleaching Extends Multiplexity' facilitates simultaneous identification of all major retinal cell types.

To understand the multicellular composition of tissues, and how it is altered during development, ageing and/or disease, we must visualise the complete cellular landscape. Currently, this is hindered by our limited ability to combine multiple cellular markers. To overcome this, we adapted a highly multiplexed immunofluorescence (IF) technique called 'Iterative Bleaching Extends Multiplexity' (IBEX) to the zebrafish retina. We optimised fluorescent antibody micro-conjugation to perform sequential rounds of labelling on a single tissue to simultaneously visualise all major retinal cell types with 11 cell-specific antibodies. We further adapted IBEX to be compatible with fluorescent transgenic reporter lines, in situ hybridisation chain reaction (HCR), and whole-mount immunofluorescence (WMIF). We applied IBEX at multiple stages to study the spatial and temporal relationships between glia and neurons during retinal development. Finally, we demonstrate the utility of IBEX across species by testing it on the turquoise killifish (Nothobranchius furzeri) and African clawed frog (Xenopus laevis) to glean large amounts of information from precious tissues. These techniques will revolutionise our ability to visualise multiple cell types in any organism where antibodies are readily available.

CosGeneGate selects multi-functional and credible biomarkers for single-cell analysis.

Selecting representative genes or marker genes to distinguish cell types is an important task in single-cell sequencing analysis. Although many methods have been proposed to select marker genes, the genes selected may have redundancy and/or do not show cell-type-specific expression patterns to distinguish cell types. Here, we present a novel model, named CosGeneGate, to select marker genes for more effective marker selections. CosGeneGate is inspired by combining the advantages of selecting marker genes based on both cell-type classification accuracy and marker gene specific expression patterns. We demonstrate the better performance of the marker genes selected by CosGeneGate for various downstream analyses than the existing methods with both public datasets and newly sequenced datasets. The non-redundant marker genes identified by CosGeneGate for major cell types and tissues in human can be found at the website as follows: https://github.com/VivLon/CosGeneGate/blob/main/marker gene list.xlsx.

The Role of Urinary Extracellular Vesicles in Kidney Cancer: Diagnostic and Therapeutic Potential

Renal cancer ranks as the 14th most common cancer globally, with renal cell carcinoma (RCC) being the primary variant, arising from renal tubular epithelial cells; clear cell RCC constitutes about 80% of cases. Despite their limitations, surgery and targeted therapy remain the mainstays of RCC treatment. Regardless of advancements in RCC research, substantial obstacles continue to exist, such as delayed diagnosis, advanced distant metastasis, and drug resistance. As urine is an easily accessible biofluid, the identification of EVs has paved the way for novel biomarker research. Urinary extracellular vesicles (uEVs) are a novel source of biomarkers with potential applications in cancer detection and management, utilizing a less invasive approach. New data indicate that uEVs are crucial in several areas of RCC, containing tumor development, metastasis, immune evasion, and response to drugs. These vesicles facilitate intercellular communication by transporting a variety of bioactive substances, including RNA, DNA, proteins, and lipids, and are released into the extracellular space by the majority of cell types. uEVs RNAs and proteins are presently being investigated for their possible application as diagnostic biomarkers for different types of kidney cancer. This review summarizes the most recent research examining the potential of uEVs cargo as a biomarker for the diagnosis, prognosis, and treatment of renal cancer.

Abstract B004: Identification of distinct subpopulations of cancer associated fibroblasts in oral squamous cell carcinoma by imaging mass cytometry

Abstract Background: Although cancer associated fibroblast (CAF) heterogeneity is recognized in oral squamous cell carcinoma (OSCC), the phenotype and role of CAF subpopulations in OSCC is still unknown. Objectives: This study aimed to dissect CAF heterogeneity and spatial location by using imaging mass cytometry on formalin fixed paraffin embedded (FFPE) tissues from primary OSCC. Methods: Imaging mass cytometry (Hyperion technology) was used to identify 24 markers (E-cadherin, EGFR, Ki-67, α-SMA, CD140β, FAP, FSP-1/S100A4, CD90/Thy-1, integrin α-11, collagen-1, tenascin-C, caveolin-1, CD4, CD8a, CD16, CD20, CD68, CD163, FoxP3, granzyme B, CD31, podoplanin, CD146, and YAP-1) in HPV-negative OSCC lesions (n=10). Tissue images were segmented employing the Steinbock framework. Unsupervised clustering of single-cell data was carried out using a bioinformatics pipeline developed in R language. Results: Unsupervised clustering identified several major cell types, with E-cadherin+ cells being the most abundant (30%) and CD146+ being the least abundant cell type (0.62%). Within the immune compartment CD4+ cells were most abundant (11.72%) followed by CD8+ cells (10.6%) and CD68+ cells (10.25%). Cells negative for other major lineage markers and positive for stromal markers were considered as CAFs and they clustered in 6 clusters defined as CAF1 to CAF6. CAF1-3 were high in FAP and CD140β while CAF 4-6 were low expressing FAP and CD140β. CAF1 and 6 expressed high αSMA, while CAF 3 and 5 expressed high collagen-1. CAF6 had high expression of integrin α-11. CAF1 and 3 subtypes were found closer to immune cells whereas CAF2 and 6 were closely located to cancer cells. Conclusions: We identified six distinct subpopulations of CAFs in OSCC by using imaging mass cytometry. Their clinical implications can be further revealed by using this method on larger patient cohorts with FFPE tissues available in diagnostic biobanks. Citation Format: Stian Tornaas, Siren Fromreide, Dimitrios Kleftogiannis, Hans Jørgen Aarstad, Olav K. Vintermyr, Lars Akslen, Harsh Dongre, Daniela Elena Costea. Identification of distinct subpopulations of cancer associated fibroblasts in oral squamous cell carcinoma by imaging mass cytometry [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B004.

Abstract A039: Characterization of the heterogeneous nature of the peritumoral zone of glioblastomas and its relevance for the clinical progression of the disease

Abstract Glioblastoma (GBM) is one of the most aggressive forms of cancer, with a recurrence rate of nearly 100% within 12 months of diagnosis. This is attributable to the persistence of residual tumor cells following surgical resection, which can repopulate the tumor even after the administration of aggressive chemoradiotherapy. These cells are embedded in a specific microenvironment in the peritumoral zone (PTZ), which is formed by their interaction with the brain parenchyma. The PTZ can be classified radiologically into two distinct categories: areas of clear tumor infiltration (non-contrast-enhancing (non-CE) tumor) and areas of vascular edema. Recent data suggest that the extent of surgical resection to non-CE regions may confer substantial benefits to patients with GBM. However, while the tumor core niche has been the subject of extensive study, there is a paucity of knowledge regarding the nature of the PTZ. In this study, we prospectively collected a large cohort of radiologically guided biopsies from GBM patients and performed a comprehensive cellular analysis. To this end, we initially integrated publicly available single-cell RNA sequencing (scRNA-seq) datasets from non-pathogenic human brains and gliomas, resulting in an integrated atlas representing the major cell types: immune (myeloid cells and T cells), astrocytic, oligodendroglial, and vascular. By means of a meticulous multi-stage examination of differentially expressed genes in glioma and cell type-specific markers, we were able to identify several genes that could serve to characterize the cellular content of the biopsies obtained from the tumor core and the periphery, comprising non-CE tumor, edema, and what was deemed "normal" tissue. The application of unsupervised clustering enabled the separation of the most pathogenic samples (including CE and non-CE tumor biopsies) based on the enrichment of proliferative markers and the pronounced absence of normal endothelial and oligodendrocyte expression. The tumorigenic samples were clustered into two main groups: one group that was enriched in astrocytic and proliferative markers and another one that was enriched in myeloid cells. Furthermore, our findings indicate the existence of inter- and intra-tumor heterogeneity in the cellular composition of the PTZ in GBM, with observed differences associated with the degree of tumor infiltration, but also patient age and gender. In particular, a distinct pathological pattern was discerned in select edema and normal biopsies from multiple patients, particularly those of an advanced age. Moreover, several markers of neural and immune cells associated with a favorable prognosis were identified, though only when expressed in the periphery. In conclusion, our study offers a comprehensive scRNA-seq resource for investigating the involvement of diverse cell types in the pathobiology of GBM. Additionally, it highlights the heterogeneous nature of the glioma periphery, which could explain the gender and age-related prognostic differences that have been observed among patients with GBM. Citation Format: Olaya de Dios, María de los Ángeles Ramírez-González, Beatriz Herranz, Juan Romero, Ana Ramos, Juan Manuel Sepúlveda-Sánchez, Ricardo Gargini, Aurelio Hernández-Laín, Pedro González, Luis Jiménez-Roldán, Guillermo García-Posadas, Ángel Pérez-Núñez, Pilar Sánchez-Gómez. Characterization of the heterogeneous nature of the peritumoral zone of glioblastomas and its relevance for the clinical progression of the disease [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A039.

Abstract B006: Role of the BAF complex in pancreatic cancer-associated fibroblast function

Abstract Fibroblasts comprise the majority cell type within connective tissue and play an important role in the support and repair of every organ. This homeostatic support occurs via the activation of fibroblasts (e.g. myofibroblasts) that then secrete growth factors, cytokines, and components of the extracellular matrix (ECM) that can further modulate residential cells. It has been demonstrated that cell-derived ECMs (CDMs) alone, generated in vitro, are sufficient to activate or maintain naive fibroblasts depending on the activation status of the fibroblasts that generated the CDM. Using this CDM system, we published that Netrin G1 is a regulator of pancreatic cancer-associated fibroblast (CAF) function that is pathologically expressed, and that its expression is dependent on the ECM. The mechanistic details behind how the ECM regulates fibroblasts’ gene expression to alter its function and activation status, however, is currently unknown. We previously showed that loss of the β5 integrin subunit negatively impacts the ability of pancreatic CAFs to maintain a constitutively activated state and dampens their pro- tumorigenic functions. RNA sequencing of these β5-deficient CAFs showed altered expression of Netrin G1, as well as members of the BAF (BRG/BRM-associated factor) family of chromatin remodelers. The goal of this project is to elucidate the role of the BAF complex in mediating the outside-in signaling from the ECM. To determine the role of the BAF complex on fibroblast activation and function, knockdown (KD) cell lines for the ATP-subunits Brg1 and Brm1 were generated in patient-derived pancreatic CAFs. Akin to β5 KD CAFs, loss of both Brg1 and Brm1 decreased the expression of published TGFβ-dependent markers of pancreatic CAF activation. However, unlike β5 KD CAFs that are unable to generate substantial, aligned CDMs, Brg1 and Brm1 KD CAFs had no effect on matrix deposition or alignment; like Netrin G1 KD CAFs that have decreased pro-tumor functions. Indeed, loss of Brm1 appears to decrease Netrin G1 akin to β5 KD CAFs. Expression levels of Netrin G1 were not restored in β5 nor Brm1 KD CAFs treated with recombinant TGFβ-1 during CDM production, whereas the TGFβ-dependent activation markers were. Collectively, these data begin to suggest that pro-tumorigenic ECM- mediated signaling may alter fibroblast gene expression via the BAF complex in a TGFβ dependent manner, opening the door to epigenetic drug targets as a new therapeutic access point to normalize the tumor-enabling microenvironment in pancreatic cancer patients. Citation Format: Jaye C. Gardiner, Khoa A. Tran, Janusz Franco-Barraza, Edna Cukierman. Role of the BAF complex in pancreatic cancer-associated fibroblast function [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B006.

Mechanisms driving epigenetic and transcriptional responses of microglia in a neurodegenerative lysosomal storage disorder model.

Lysosomal dysfunction is causally linked to neurodegeneration in many lysosomal storage disorders (LSDs) and is associated with various age-related neurodegenerative diseases 1,2 , but there is limited understanding of the mechanisms by which altered lysosomal function leads to changes in gene expression that drive pathogenic cellular phenotypes. To investigate this question, we performed systematic imaging, transcriptomic, and epigenetic studies of major brain cell types in Sgsh null (KO) mice, a preclinical mouse model for Sanfilippo syndrome (Mucopolysaccharidosis Type IIIA, MPS-IIIA) 3,4 . MPS-IIIA is a neurodegenerative LSD caused by homozygous loss-of-function (LoF) mutations in SGSH which results in severe early-onset developmental, behavioral, and neurocognitive impairment 5-15 . Electron microscopy, immunohistochemistry, and single-nucleus RNA-sequencing analysis revealed microglia as the cell type exhibiting the most dramatic phenotypic alterations in Sgsh KO mice. Further temporal analysis of microglia gene expression showed dysregulation of genes associated with lysosomal function and immune signaling pathways beginning early in the course of the disease. Sgsh deficiency similarly resulted in increases in open chromatin and histone acetylation at thousands of putative microglia-specific enhancers associated with upregulated genes but had much less impact on the epigenetic landscapes of neurons or oligodendrocytes. We provide evidence for dominant and context-dependent roles of members of the MITF/TFE family as major drivers of microglia-specific epigenetic and transcriptional changes resulting from lysosomal stress that are dependent on collaborative interactions with PU.1/ETS and C/EBP transcription factors. Lastly, we show that features of the transcriptomic and epigenetic alterations observed in murine Sgsh deficiency are also observed in microglia derived from mouse models of age-related neurodegeneration and in human Alzheimer's disease patients, revealing common and disease-specific transcriptional mechanisms associated with disease-associated microglia phenotypes.

Single nucleus RNA sequencing reveals cellular and molecular responses to vanadium exposure in duck kidneys

Vanadium (V) exposure is known to induce renal toxicity, yet its specific effects on renal cell types and molecular mechanisms remain incompletely understood. We used single nucleus RNA sequencing (snRNA-seq) to characterize the impact of V on duck kidney cells at a cellular resolution. Following a 44-day exposure, immunofluorescence analysis revealed a significant increase in α-SMC expression in the renal interstitium, indicative of fibrotic response. SnRNA-seq identified 12 major cell types organized into 19 clusters within the kidney. Significant changes in cell composition were observed, notably an increase in proximal tubule cells (PT2 subtype), glomerular endothelial cells, principal cells, and alterations in immune cell proportions, while collecting duct intercalated cells (CD-IC) and thick ascending limb showed decreased percentages. Differential gene expression analysis highlighted pathways implicated in V toxicity across different cell types. Changes in drug metabolism-cytochrome P450, butanoate metabolism, and actin cytoskeleton regulation were exhibited by PT cells. Alterations in collecting duct secretion, oxidative phosphorylation, and bicarbonate reclamation pathways were shown in CD-IC cells. Furthermore, immune cells displayed changes in T cell receptor and chemokine signaling pathways, indicative of altered immune responses. Taken together, these findings contribute to a better shedding light on the pathogenic mechanisms of V induced renal injury.

Abstract 4137845: A single-cell lung atlas of human pulmonary arterial hypertension

Introduction: Pulmonary arterial hypertension (PAH) is a disaster disease characterized by obliterative vascular remodeling and persistent increase of vascular resistance, leading to right heart failure and premature death. Understanding the cellular and molecular mechanisms will help develop novel therapeutic approaches for PAH patients. Hypothesis: We hypothesis that single-cell RNA sequencing analysis can help understand the cellular and molecular mechanisms that drive the disease initiation and progression. Methods: 10 healthy donors, 10 idiopathic PAH, 10 drug and toxin induced PAH, 10 systemic and pulmonary shunting induced PAH patients from both male and female, and different ethical groups and races, and ages were obtained from PHBI. Lung tissue specimens were processed for fixed scRNA-seq. The cell proportion change, gene expression, and pathway analysis were evaluated between different disease subclasses, sex, age on different cell types. Results: Our analysis reveals 317,414 cells, 33 clusters and 9 major cell types, including endothelial cells (ECs), alveolar cells, fibroblast, smooth muscle cells (SMCs), pericytes, myeloid cells, lymphocytes, airway epithelial cells, platelet. Cell proportion analysis demonstrated an increase in the proportions of venous ECs, adventitial fibroblasts and myofibroblasts, alveolar macrophages, B cells, plasma cells and a reduction in capillary ECs 2 and pericytes in PAH lungs. KEGG Pathway analysis showed that upregulated pathways related Herpes simplex virus 1 infection, ECM-receptor interaction, Complement and coagulation cascades, Hedgehog signaling, TGF-beta signaling pathway, and downregulated pathways related to IL-17 signaling, TNF signaling, MAPK signaling pathway were enriched in the PAH patients. Female PAH patients exhibit an increase of myofibroblasts and platelets proportion compared to male PAH patients. MsigDB Hallmark Pathway analysis showed that p53 pathway was upregulated, whereas Inflammatory Response, Hypoxia, Epithelial Mesenchymal Transition were downregulated in female PAH patients. PAH patients older than 21 years exhibit an increase in the cell proportion of Platelet, SMCs, Arterial ECs and Venous ECs, and alveolar macrophages and a reduction of B cells and plasma cells. Conclusions: Our integrated analysis of human PAH lung atlas dataset provides a comprehensive understanding of lung cell populations and molecular signature in PAH patients with different sex, age and subclasses

Abstract 4138606: Toll-like Receptor 4 Signaling Establishes Trained Innate Immunity Through Interferon-Mediated Epigenetic Modifications Leading to Cardioprotection in a Stress-induced Cardiomyopathy Model

Introduction: The mechanisms responsible for establishing preconditioning-induced cardioprotection remain unknown. We have shown that a high dose of isoproterenol (ISO) induces cardioprotection against a second ISO dose in mice. The durability of protection and the lack of an innate immune response suggests trained immunity as a novel cardioprotective mechanism. Hypothesis: We hypothesize that cardioprotection is conferred through trained immunity, by interferon signaling downstream of necrotic cardiac material-mediated Toll-like receptor 4 (TLR4) activation. Methods: Wild-type C57BL/6J mice were intraperitoneally injected with TLR agonists or diluent, and challenged with 300 mg/kg ISO 7 days later. Mice were assessed by 2-D echocardiography, serum cardiac troponin levels, flow cytometry immune cell counts, and Multiome (single nuclei RNA+ATAC) sequencing. Results: The TLR4 agonist lipopolysaccharide (LPS) induced cardioprotection against ISO injury, with mice having enhanced survival (P=0.049) and no changes in cardiac troponin levels (P&gt;0.99), cardiac neutrophil influx (P&gt;0.99) or left ventricular motion (P=0.057) relative to baseline values before injury. Treating LPS-injected mice with β-glucan reversed the effects of LPS on immune cells and abolished cardioprotection. Multiome analysis of genes linked to chromatin peaks with increased accessibility in LPS+vehicle (protected) compared to LPS+β-glucan and diluent control (non-protected) hearts revealed the interferon pathway to be up-regulated across all major cell types. Modulation of interferon signaling with monoclonal antibodies against type 1+2 interferon receptors abolished cardioprotection in LPS-treated mice, whereas pre-treatment with recombinant type 1+2 interferons induced cardioprotection. Importantly, interferon-treated hearts shared similar chromatin accessibility features and enriched transcription factor motifs, including interferon-specific motifs, with LPS-protected hearts across cell types, particularly among non-cardiomyocytes. Conclusions: TLR4-induced interferon signaling is sufficient and in part necessary for cardioprotection against ISO injury. Moreover, our findings show that epigenetic modifications downstream of interferon signaling lead to cardioprotection consistent with trained innate immunity.

TMIC-69. HUMAN ORGANOID TUMOR TRANSPLANTATION IDENTIFIES FUNCTIONAL GLIOBLASTOMA MICROENVIRONMENTAL COMMUNICATION MEDIATED BY PTPRZ1

Abstract Glioblastoma, the most aggressive form of primary brain cancer, is driven by both intrinsic cellular properties and extrinsic factors from the tumor microenvironment (TME). Here, we leverage our novel human organoid tumor transplantation (HOTT) system to explore how external cues modulate glioblastoma cell type specification, heterogeneity, and migration. Comparing the single-cell transcriptomes of HOTT and matched primary tumors, we found that HOTT recapitulates not only core features of major patient tumor cell types but also those of peritumor non-neoplastic cell types. This enables us to explore tumor-TME interactions and identify PTPRZ1, a receptor tyrosine phosphatase implicated in brain tumor migration, as a key mediator in intercellular communication. This finding was further confirmed by interrogation of published datasets from patients containing non-neoplastic microenvironmental cells. PTPRZ1 activation, resulting from the reactivation of developmental programs in both the tumor and its TME, highlights the relevance of the organoid system for modeling the GBM microenvironment. Moreover, HOTT uniquely allows for perturbations in either patient tumors or their microenvironments, or both, to verify PTPRZ1 function. Tumor knockdown of PTPRZ1 confirmed its role in promoting tumor migration and maintaining progenitor identity. Surprisingly, environmental PTPRZ1 knockdown in human cortical organoids before tumor transplantation inhibited tumor migration and promoted differentiation, even without direct tumor manipulation. Overall, this fundamental result demonstrates that a single treatment delivered to the tumor and its TME can exert opposite effects on the tumor. Consequently, it underscores the necessity of studying human glioblastoma within a human microenvironment context, such as HOTT, especially when evaluating therapeutic efficacy in GBM, as effective treatments must address both the tumor and its complex TME, rather than the tumor alone.

Transcriptomic analysis of the 12 major human breast cell types reveals mechanisms of cell and tissue function.

A fundamental question in biology, central to our understanding of cancer and other pathologies, is determining how different cell types coordinate to form and maintain tissues. Recognizing the distinct features and capabilities of the cells that compose these tissues is critical. Unfortunately, the complexity of tissues often hinders our ability to distinguish between neighboring cell types and, in turn, scrutinize their transcriptomes and generate reliable and tractable cell models for studying their inherently different biologies. We have recently introduced a novel method that permits the identification and purification of the 12 cell types that compose the human breast-nearly all of which could be reliably propagated in the laboratory. Here, we explore the nature of these cell types. We sequence mRNAs from each purified population and investigate transcriptional patterns that reveal their distinguishing features. We describe the differentially expressed genes and enriched biological pathways that capture the essence of each cell type, and we highlight transcripts that display intriguing expression patterns. These data, analytic tools, and transcriptional analyses form a rich resource whose exploration provides remarkable insights into the inner workings of the cell types composing the breast, thus furthering our understanding of the rules governing normal cell and tissue function.

Pan-Cancer Single-Cell Transcriptomic Analysis Reveals Divergent Expression of Embryonic Proangiogenesis Gene Modules in Tumorigenesis.

Angiogenesis is indispensable for the sustained survival and progression of both embryonic development and tumorigenesis. This intricate process is tightly regulated by a multitude of pro-angiogenic genes. The presence of gene modules facilitating angiogenesis has been substantiated in both embryonic development and the context of tumor proliferation. However, it remains unresolved whether the pro-angiogenic gene modules expressed during embryonic development also exist in tumors. This study performed a pan-cancer single-cell RNA sequencing (scRNA-seq) analysis on samples from 332 patients across seven cancer types: thyroid carcinoma, lung cancer, breast cancer, hepatocellular carcinoma, colorectal cancer, ovarian carcinoma, and prostate adenocarcinoma. Data processing was carried out using the Seurat R package, with rigorous quality control to filter high-quality cells and mitigate batch effects across datasets. We used principal component analysis (PCA), shared nearest neighbor graph-based clustering, and Uniform Manifold Approximation and Projection (UMAP) to visualize cell types and identify distinct cell clusters. Myeloid cell subpopulations were further analyzed for the expression of embryonic pro-angiogenic gene modules (EPGM) and tumor pro-angiogenic gene modules (TPGM). The analysis identified nine major cell types within the tumor microenvironment, with myeloid cells consistently exhibiting elevated expression of both tumor pro-angiogenic gene modules (TPGM) and EPGM across all tumor types. In particular, myeloid cells, including macrophages and monocytes, showed high EPGM expression, indicating an active role of embryonic pro-angiogenesis pathways in tumors. A subset analysis revealed 20 distinct myeloid subtypes with varying EPGM and TPGM expression across different cancers. Treatment and disease stage influenced these gene expressions, with certain subtypes, such as HSPAhi/STAT1+ macrophages in breast cancer, displaying reduced pro-angiogenic gene activity post-treatment. This study provides evidence that tumors may exploit EPGM to enhance vascularization and support sustained growth, as evidenced by the elevated EPGM expression in tumor-associated myeloid cells. The consistent presence of EPGM in TAMs across multiple cancer types suggests a conserved mechanism wherein tumors harness embryonic angiogenic pathways to facilitate their progression. Distinct EPGM expression patterns in specific myeloid cell subsets indicate potential therapeutic targets, particularly in cases where EPGM activation contributes to resistance against anti-angiogenic therapies. These findings shed new light on the molecular mechanisms underlying tumor angiogenesis and highlight the prognostic relevance of EPGM expression in cancer, underscoring its potential as a biomarker for clinical applications.

Nanoscale Optical Imaging, Reconstruction, and Spatial Analysis of Whole Mouse Glomeruli.

Renal glomeruli have traditionally been studied by micrometer-scale optical microscopy to interrogate overall physiology or molecular distributions and by nanoscale electron microscopy to interrogate the ultrastructure of thin sections. While these approaches are powerful, they have been limited in their ability to obtain detailed views of the glomeruli as holistic 3D functional units. To fill this knowledge gap, we have developed a novel pipeline for imaging, reconstructing, and analyzing whole mouse glomeruli at 100 nm resolution using super-resolution fluorescence microscopy. This pipeline integrates both manual and machine learning approaches to annotate and analyze glomerular structures. Using this method, we created 18 detailed glomerulus models, from a range of healthy, aged, and model diseased mice, that outline all major structures and cell types. These models have been made publicly accessible in an online repository, providing a valuable resource for further studies. Our results also uncovered a diverse set of novel phenotypes including nuclear enlargement in all glomerular cell types in aging and disease, as well as an aging-related pattern of regional thickening of the Bowman's capsule basement membrane near the tubular-glomerular junction.

Single-cell transcriptomic and proteomic analysis of Parkinson's disease brains.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder, and recent evidence suggests that pathogenesis may be in part mediated by inflammatory processes, the molecular and cellular architectures of which are largely unknown. To identify and characterize selectively vulnerable brain cell populations in PD, we performed single-nucleus transcriptomics and unbiased proteomics to profile the prefrontal cortex from postmortem human brains of six individuals with late-stage PD and six age-matched controls. Analysis of nearly 80,000 nuclei led to the identification of eight major brain cell types, including elevated brain-resident T cells in PD, each with distinct transcriptional changes in agreement with the known genetics of PD. By analyzing Lewy body pathology in the same postmortem brain tissues, we found that α-synuclein pathology was inversely correlated with chaperone expression in excitatory neurons. Examining cell-cell interactions, we found a selective abatement of neuron-astrocyte interactions and enhanced neuroinflammation. Proteomic analyses of the same brains identified synaptic proteins in the prefrontal cortex that were preferentially down-regulated in PD. By comparing this single-cell PD dataset with a published analysis of similar brain regions in Alzheimer's disease (AD), we found no common differentially expressed genes in neurons but identified many shared differentially expressed genes in glial cells, suggesting that the disease etiologies, especially in the context of neuronal vulnerability, in PD and AD are likely distinct.