High-resolution profiling of osteocyte transcriptomes via single-nucleus RNA sequencing.

Applications of single-cell and bulk RNA sequencing in onco-immunology

Immune-based risk score derived from bulk and single-cell transcriptomics enhances prognostic stratification in MDS

Single-nuclei RNA-sequencing fails to detect molecular dysregulation in the preeclamptic placenta.

SUMMARYSepsis survivors frequently develop long-term immune dysfunction, but the epigenetic mechanisms underlying persistent myeloid suppression remain unclear. Myeloid-derived suppressor cells (MDSCs), whose function is shaped by host age and sex, are key contributors to post-sepsis immune dysregulation. Here, we present a high-resolution epigenetic map targeting gene promoters of MDSCs after sepsis using MAPit-FENGC, a single-molecule assay that simultaneously profiles DNA methylation and chromatin accessibility. In a clinically relevant murine model including young and older adult male and female mice, splenic MDSCs were isolated for MAPit-FENGC and single-cell RNA sequencing. Unsupervised clustering identified nine promoter classes reflecting chromatin dynamics: age- and sex-dependent sepsis-induced opening (Classes 1–4), persistent closure with varying levels of DNA methylation (Classes 5–7), and constitutive openness post-sepsis (Classes 8, 9). Transcriptomic profiling corroborated these promoter states, linking accessibility with gene expression. These findings establish how epigenetic reprogramming of MDSCs may shape age- and sex-specific immune trajectories in sepsis survivors.

Mathys et al. conducted the first single-nucleus RNA-seq (snRNA-seq) study of Alzheimer’s disease (AD) (Mathys et al., 2019). With bulk RNA-seq, changes in gene expression across cell types can be lost, potentially masking the differentially expressed genes (DEGs) across different cell types. Through the use of single-cell techniques, the authors benefitted from increased resolution with the potential to uncover cell type-specific DEGs in AD for the first time. However, there were limitations in both their data processing and quality control and their differential expression analysis. Here, we correct these issues and use best-practice approaches to snRNA-seq differential expression, resulting in 549 times fewer DEGs at a false discovery rate of 0.05. Thus, this study highlights the impact of quality control and differential analysis methods on the discovery of disease-associated genes and aims to refocus the AD research field away from spuriously identified genes.

Salivary glands are composed of several types of cells, and each cell type is predicted to be involved in the carcinogenesis of different types of cancers including Adenoid cystic carcinoma (ACC), Acinic cell carcinoma (AciCC), salivary duct carcinoma (SDC), myoepithelial carcinoma (MECA) and other histology. In this study, we performed single nucleus RNA-seq on three human salivary gland samples to clarify the gene expression profile of each complex cellular component of the salivary glands and related these expression patterns to expression found in salivary gland cancers (SGC) to infer cell of origin. By single nucleus RNA-seq, total 13,643 salivary gland cells were stratified into four clusters: acinar cells, ductal cells 1, ductal cells 2, and myoepithelial cells/stromal cells, with differentially expressed genes of each group. The localization of each cell group was anatomically validated by IHC of each cluster marker gene, and one group of ductal cells was found to represent intercalated ductal cells labeled with HES1. Gene ontology analysis for this group included “epithelial cell differentiation” confirming the intercalated duct. Furthermore, to elucidate the potential etiologic relationship between normal salivary glands cell type and salivary gland carcinoma histology, we compared the expression of each cluster marker to SGC public RNA-seq data. In comparison between AciCC and ACC, we used normalized RNA-seq data of 54 ACC and 21 AciCC. ACC had significantly less expression of acinar cells markers (STATH, CST2, and LPO) compared to other markers expression(***P = 4 × 10−51), unlike AciCC (P = 0.54), suggesting a possible contribution of acinar cells to the carcinogenesis of AciCC. In another cohort, we compared 16 SDC, and 38 MECA with publicly available RNA-seq data. Remarkably, HES1 and ELF3, each of two different ductal cell markers, were significantly upregulated in SDC consistent with origin of SDC from ductal cells (P=0.001, P=0.0007, respectively). Consistent with bulk RNA-seq results, expression of HES1 in IHC using clinical human SDC and MECA samples was higher in SDC than MECA, suggesting that the expression of the derived cells is involved in salivary gland carcinogenesis. Cell type expressions in specific SGC histology are similar to those found in normal salivary gland populations, indicating a potential etiologic relationship. On the other hand, there was no significant difference in myoepithelial marker ACTA2 expression between SDC and MECA, suggesting that the markers of origin are not always highly expressed, depending on cancer subtypes. Although publicly available SGC RNA-seq data used in this study are limited, the results remarkably reflect the diverse modes of carcinogenesis of SGC. These findings might provide a better understanding of the molecular basis of SGC and treatment strategies for SGC. Citation Format: Takuya Nakagawa, Chanond Nasamran, Jingjing Hu, Prakriti Sen, Theresa Guo, Kathleen Fisch, Joseph Califano. Single nucleus RNA-sequencing using normal human salivary glands revealed a potential etiologic relationship between each salivary gland cell and salivary gland malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1076.

Combination of Dasatinib and Quercetin alleviates heat stress-induced cognitive deficits in aged and young adult male mice

Despite many clinical trials, outcome for osteosarcoma (OS) patients remains poor, especially for those with metastatic or relapsed/refractory disease. We leveraged network-based systems biology approaches to discover Master Regulator (MR) proteins representing pharmacologically accessible, mechanistic determinants of OS cell state, and to dissect tumor transcriptional heterogeneity. MRs were identified by interrogating an OS regulatory network—generated from 87 RNAseq profiles in the TARGET OS cohort—with gene expression signatures from 149 diagnostic OS samples, using the VIPER algorithm. RNAseq profiles were generated in four sarcoma cell lines following perturbation with ~400 oncology drugs, and used to identify drugs capable of inverting MR activity profiles with the OncoTreat algorithm. Unsupervised, protein activity-based clustering of the samples identified two clusters, characterized by a significant overall survival difference (76% vs 38%, log-rank p-val 0.0041). Patients in the high-risk cluster presented aberrant activity of proteins involved in cell cycle (FOXM1, CENPF, TOP2A) and epigenetic remodeling (EZH2, KDM1A), while those in the low-risk cluster had aberrant activity of proteins involved in immune response (VAV1, CD86, CEBPE), interferon gama signaling (IRF5, MNDA), and senescence control (CREG1, TFEC). We further identified patients with metastatic disease and particularly poor outcome (<15% survival) showing high activity in stem cell-related proteins NANOG, ZSCAN4, and LIN28A. To address intra-tumor heterogeneity and immune infiltration (40-60% in the samples) in bulk RNAseq data, we inferred protein activity in previously published single-cell RNAseq profiles from 11 OS patients, using regulatory networks also produced by single-cell analysis. We identified three tumor subpopulations, co-existing in all the samples, with lineage similarity to osteoblasts, chondroblasts, and fibroblasts. The most active MR proteins (p-val < 10E-30) were highly distinct across these subpopulations: NANOG, REL, IGF2, TOP2A (osteoblasts), SSTR3, WNT1, KDM6B, EZH2 (chondroblasts), and NOD1, ERBB3, PRKDC, INSR (fibroblasts). Pharmacologically accessible MRs significantly active across the three subpopulations and in subsets of bulk RNAseq samples included EZH2, CDK2, TOP2A, HDAC5, and PRKDC. OncoTreat analysis predicted sensitivity to camptothecin, seliciclib, galunisertib, and rigosertib in both fibroblast- and chondroblast-like subpopulations. In conclusion, using network-based systems biology approaches in OS, we identified pharmacologically accessible MR subtypes differentiating survival at the bulk tissue level, and defining three distinct tumor subpopulations at the single-cell level with unique predicted drug sensitivities. Citation Format: Somnath Tagore, Jovana Pavisic, Aaron T. Griffin, Katherine Janeway, Andrew L. Kung, Filemon Dela Cruz, Alejandro Sweet-Cordero, Inge Behroozfard, Stanley Leung, Alex Lee, Darrell Yamashiro, Julia Glade Bender, Andrea Califano. A systems biology approach to defining tumor heterogeneity and prognostic and targetable master regulator protein signatures from bulk and single-cell RNAseq in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 488.

Pneumonia is a common comorbidity in patients with severe traumatic brain injury (TBI), and is associated with increased morbidity and mortality. In this study, we established a model of intratracheal Klebsiella pneumoniae administration in young adult male and female mice, at 4 days following an experimental TBI, to investigate how K. pneumoniae infection influences acute post-TBI outcomes. A dose-response curve determined the optimal dose of K. pneumoniae for inoculation (1 x 10^6 colony forming units), and administration at 4 days post-TBI resulted in transient body weight loss and sickness behaviors (hypoactivity and acute dyspnea). K. pneumoniae infection led to an increase in pro-inflammatory cytokines in serum and bronchoalveolar lavage fluid at 24 h post-infection, in both TBI and sham (uninjured) mice. By 7 days, when myeloperoxidase + neutrophil numbers had returned to baseline in all groups, lung histopathology was observed with an increase in airspace size in TBI + K. pneumoniae mice compared to TBI + vehicle mice. In the brain, increased neuroinflammatory gene expression was observed acutely in response to TBI, with an exacerbated increase in Ccl2 and Hmox1 in TBI + K. pneumoniae mice compared to either TBI or K. pneumoniae alone. However, the presence of neuroinflammatory immune cells in the injured brain, and the extent of damage to cortical and hippocampal brain tissue, was comparable between K. pneumoniae and vehicle-treated mice by 7 days. Examination of the fecal microbiome across a time course did not reveal any pronounced effects of either injury or K. pneumoniae on bacterial diversity or abundance. Together, these findings demonstrate that K. pneumoniae lung infection after TBI induces an acute and transient inflammatory response, primarily localized to the lungs with some systemic effects. However, this infection had minimal impact on secondary injury processes in the brain following TBI. Future studies are needed to evaluate the potential longer-term consequences of this dual-hit insult.

BackgroundDespite affecting millions of people worldwide, psychiatric disorders are inadequately treated. Understanding how adversity, one of the major risk factors for these disorders, raises risk for psychopathology is essential for identifying new treatments. The orbitofrontal cortex (OFC), a sub-region of prefrontal cortex (PFC), is involved in higher cognitive processes and is highly vulnerable to adverse experiences. However, the impacts of adversity on various cell-types across the cortical layers of the OFC are largely unknown. Here, we interrogated human postmortem OFC (BA11) samples to unravel adversity-induced cell-type-specific transcriptomic changes in psychiatric disorders. Explorations of single-cell epigenomic, as well as, spatial transcriptomic alterations within the same samples are currently underway.Aims & ObjectivesThe primary objective of this study is to disentangle adversity-induced cell-type- and cortical layer-specific molecular changes in the OFC of psychiatric patients. The overarching goal is to determine if adversity is associated with a transdiagnostic subtype of psychiatric disorders that may benefit from targeted therapy.MethodWe examined 86 individuals (n=32 controls vs n=54 cases with mixed major psychiatric disorders: schizophrenia, major depression and bipolar disorder) with or without history of profound psychological adversity exposure (n=23 with severe adversity and n=31 without). To capture the cell-type-specific effects, we generated single-nucleus Assay for Transposase-Accessible Chromatin sequencing (snATAC-seq) and single-nucleus RNA sequencing (snRNA-seq) datasets using 10x Chromium (10x Genomics, USA). To dissect spatial heterogeneity, we produced spatial transcriptomic dataset using Visium (10x Genomics, USA). We performed compositional and differential gene expression analyses, followed by gene ontology and disease enrichment analyses, on the snRNA-seq data.ResultsAfter filtering out low-quality nuclei from the snRNA-seq dataset, about ~800,000 nuclei from 15 different cell types were retained. The proportions of inhibitory neurons and astrocytes were significantly lower in individuals exposed to adversity vs those without adversity, while the proportion of oligodendrocytes was higher (Log2FoldChange of -0.51, -1.02 and 0.33, respectively). We identified a large number of differentially expressed genes (DEGs) in oligodendrocytes and reelin-expressing inhibitory neurons (160 and 265, respectively) only when the contrast was performed among cases based on their history of adversity. Overall, the DEGs showed significant enrichment of psychopathology-associated metabolic processes and phenotypes, including synaptic signaling and inflammatory responses.Discussion & ConclusionsThese results highlight the striking cell-type-specific impacts of adverse experiences on gene expressions and cellular abundances in the OFC of individuals with psychiatric disorders. The glial cells such as astrocytes and oligodendrocytes, which are known to be involved in stress response, as well as inhibitory neuronal subtypes, known to be affected in stress-related disorders, showed marked impacts of adversity in the context of psychopathology. In general, synaptic signaling, transmembrane transports, energy metabolism and immune response pathways were affected, all of which are implicated in psychopathology.Experiments and analyses are underway to further disentangle the regulatory changes affecting transcription by delineating epigenomic modifications and gene co-expression networks within these cell types and to unmask the changes in transcript and cellular abundances across the cortical layers. The study will thus help decompose adversity-induced transcriptomic and gene regulatory changes into cell-type-specific alterations across cortical layers.

BackgroundSingle-cell RNA-seq has emerged as an innovative technology used to study complex tissues and characterize cell types, states, and lineages at a single-cell level. Classification of bulk tumors by their individual cellular constituents has also created new opportunities to generate single-cell atlases for many organs, cancers, and developmental models. Despite the tremendous promise of this technology, recent evidence studying epithelial tissues and diverse carcinomas suggests the methods used for tissue processing, cell disaggregation, and preservation can significantly bias gene expression and alter the observed cell types. To determine whether sarcomas – tumors of mesenchymal origin – are subject to the same technical artifacts, we profiled patient-derived tumor explants (PDXs) propagated from three aggressive subtypes: osteosarcoma (OS), Ewing sarcoma (ES), desmoplastic small round cell tumor (DSRCT). Given the rarity of these sarcoma subtypes, we explored whether single-nuclei RNA-seq from more widely available archival frozen specimens could accurately be identified by gene expression signatures linked to tissue phenotype or pathognomonic fusion proteins.ResultsWe systematically assessed dissociation methods across different sarcoma subtypes. We compared gene expression from single-cell and single-nucleus RNA-sequencing of 125,831 whole-cells and nuclei from ES, DSRCT, and OS PDXs. We detected warm dissociation artifacts in single-cell samples and gene length bias in single-nucleus samples. Classic sarcoma gene signatures were observed regardless of the dissociation method. In addition, we showed that dissociation method biases could be computationally corrected.ConclusionsWe highlighted transcriptional biases, including warm dissociation and gene-length biases, introduced by the dissociation method for various sarcoma subtypes. This work is the first to characterize how the dissociation methods used for sc/snRNA-seq may affect the interpretation of the molecular features in sarcoma PDXs.

Ischemic stroke is a major cause of death and disability worldwide. Translation into the clinical setting of neuroprotective agents showing promising results in pre-clinical studies has systematically failed. One possible explanation is that the animal models used to test neuroprotectants do not properly represent the population affected by stroke, as most of the pre-clinical studies are performed in healthy young male mice. Therefore, we aimed to determine if the response to cerebral ischemia differed depending on age, sex and the presence of comorbidities. Thus, we explored proteomic and transcriptomic changes triggered during the hyperacute phase of cerebral ischemia (by transient intraluminal middle cerebral artery occlusion) in the brain of: (1) young male mice, (2) young female mice, (3) aged male mice and (4) diabetic young male mice. Moreover, we compared each group's proteomic and transcriptomic changes using an integrative enrichment pathways analysis to disclose key common and exclusive altered proteins, genes and pathways in the first stages of the disease. We found 61 differentially expressed genes (DEG) in male mice, 77 in females, 699 in diabetics and 24 in aged mice. Of these, only 14 were commonly dysregulated in all groups. The enrichment pathways analysis revealed that the inflammatory response was the biological process with more DEG in all groups, followed by hemopoiesis. Our findings indicate that the response to cerebral ischemia regarding proteomic and transcriptomic changes differs depending on sex, age and comorbidities, highlighting the importance of incorporating animals with different phenotypes in future stroke research studies.

The Ehlers‐Danlos syndromes (EDS) are inherited connective tissue diseases with primary manifestations that affect the skin and the musculoskeletal system. However, the effects of EDS on the respiratory system are not well understood and are described in the literature as sporadic case reports. We performed histological, histomorphometric, and the first in‐depth characterization of respiratory system function in a mouse model of classical EDS (cEDS) with haploinsufficiency of type V collagen (Col5a1+/−). In young adult male and female mice, lung histology showed reduced alveolar density, reminiscent of emphysematous‐like changes. Respiratory mechanics showed a consistent increase in respiratory system compliance accompanied by increased lung volumes in Col5a1+/− compared to control mice. Flow–volume curves, generated to mimic human spirometry measurements, demonstrated larger volumes throughout the expiratory limb of the flow volume curves in Col5a1+/− compared to controls. Some parameters showed a sexual dimorphism with significant changes in male but not female mice. Our study identified a clear respiratory phenotype in the Col5a1+/− mouse model of EDS and indicated that intrinsic respiratory and lung changes may exist in cEDS patients. Their potential impact on the respiratory function during lung infections, other respiratory disease processes, or insults may be significant and justify further clinical evaluation.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by a decline in cognitive abilities, memory loss, and heightened neuroinflammation. The presence of the APOE4 allele is the most significant genetic risk factor for AD, known to accelerate the onset and progression of the disease by interacting with hallmark features such as beta-amyloid plaques and neurofibrillary tau tangles. Additionally, APOE4 directly impairs microglial function in AD leading to neuroinflammation, further contributing to neurodegeneration. While the relationship between AD and dietary habits or comorbid conditions, particularly hypertension, remains unclear, we hypothesize that high dietary salt intake will exacerbate neuroinflammation within the hippocampus of APOE4-expressing mice, providing a potential mechanistic link between diet, hypertension, and Alzheimer’s disease. To test this hypothesis, we utilized mice that exclusively expressed human APOE4 or the control APOE3 in the brain. Young adult male and female mice, aged 5–7 months old (n=18 in each group), were given a 4% NaCl (high salt) or a 0.1% NaCl (low salt) diet for 4 weeks. Using design-based stereology, we evaluated the total count of Iba1 positive microglia in the hippocampus of APOE4 expressing mice and the control APOE3 mice. Preliminary results show APOE4 mice exhibit significant neuroinflammation in the hippocampus when compared to APOE3 mice. However, the APOE4 low-salt diet was seen to be beneficial. Further work is needed, and results will be discussed.

Remodeling of the extracellular matrix (ECM) is required for the proper healing, strengthening, and maintenance of tendon tissue. There are well documented sex differences in tendon injury rates and healing outcomes, often attributed to either innate differences in tissue structure and resident cell signaling or the influence of sex hormones. However, these factors are rarely decoupled. Estrogen (17β-estradiol) and progesterone (P4) receptors are expressed in both male and female tendons and thus could participate in the remodeling process, but studies are extremely limited. Therefore, the objective of this work was to address whether biological sex differences are present in tendon remodeling and to determine the individual and combined roles of estrogen and progesterone in the remodeling process. We utilized an explant model of the flexor digitorum longus tendon harvested from young adult male and female mice to examine cell-mediated remodeling without disruption to the native environment. We found sex differences in tendon remodeling in the absence of hormonal stimulation, revealing how biological sex alone influences tendon health. We also demonstrate that the response to exogenous hormone delivery is sex-dependent, and that progesterone and estrogen serve complimentary yet independent roles. Overall, this work presents the first examination of sex-dependent matrix turnover in response to hormones and underscores the critical need for additional research in this area.