Single-cell RNA-sequencing Study Research Articles

Exploring copper metabolism-induced cell death in gastric cancer: a single-cell RNA sequencing study and prognostic model development

BackgroundGastric cancer (GC) is the third leading cause of cancer-related deaths globally. Despite advancements in treatment, the overall 5-year survival rate remains below 30%, particularly in advanced stages. Copper metabolism, vital for various cellular processes, has been linked to cancer progression, but its role in GC, especially at the single-cell level, is not well understood.ObjectiveThis study aims to investigate copper metabolism in GC by integrating single-cell RNA sequencing (scRNA-seq) data and developing a prognostic model based on copper metabolism-related gene (CMRG) expression. The study explores how copper metabolism affects the tumor microenvironment and identifies potential therapeutic targets.MethodsscRNA-seq data from gastric cancer and normal tissues were analyzed using the Seurat package. Principal Component Analysis (PCA) and Uniform Manifold Approximation and Projection (UMAP) were used for dimensionality reduction and clustering. Non-negative matrix factorization (NMF) was employed for T cell subpopulation analysis. A high-dimensional weighted gene co-expression network analysis (HdWGCNA) identified key molecular features. LASSO regression and Random Survival Forest (RSF) techniques were used to create and validate a prognostic model. Survival analysis, immune microenvironment assessment, and drug sensitivity analysis were conducted.ResultsSixteen cell clusters and nine distinct cell types were identified, with T cells showing significant roles in cell communication. The NMF analysis of CD8 +T cells revealed five copper metabolism-related subtypes. The prognostic model based on nine CMRGs indicated significant survival differences between high- and low-risk groups. High-risk patients showed shorter survival times, increased immune cell infiltration, and altered immune responses. Drug sensitivity analysis suggested higher efficacy of certain drugs in high-CMRG patients.

Dendritic cell effector mechanisms and tumor immune microenvironment infiltration define TLR8 modulation and PD-1 blockade.

The potent immunostimulatory effects of toll-like receptor 8 (TLR8) agonism in combination with PD-1 blockade have resulted in various preclinical investigations, yet the mechanism of action in humans remains unknown. To decipher the combinatory mode of action of TLR8 agonism and PD-1 blockade, we employed a unique, open-label, phase 1b pre-operative window of opportunity clinical trial ( NCT03906526 ) in head and neck squamous cell carcinoma (HNSCC) patients. Matched pre- and post-treatment tumor biopsies from the same lesion were obtained. We used single-cell RNA sequencing and custom multiplex staining to leverage the unique advantage of same-lesion longitudinal sampling. Patients receiving dual TLR8 agonism and anti-PD-1 blockade exhibited marked upregulation of innate immune effector genes and cytokines, highlighted by increased CLEC9A+ dendritic cell and CLEC7A/SYK expression. This was revealed via comparison with a previous cohort from an anti-PD-1 blockade monotherapy single-cell RNA sequencing study. Furthermore, in dual therapy patients, post-treatment mature dendritic cells increased in adjacency to CD8 + T-cells. Increased tumoral cytotoxic T-lymphocyte densities and expanded CXCL13 + CD8 + T- cell populations were observed in responders, with increased tertiary lymphoid structures (TLSs) across all three patients. This study provides key insights into the mode of action of TLR8 agonism and anti-PD-1 blockade immune targeting in HNSCC patients.

Identifying prognostic biomarkers in oral squamous cell carcinoma: an integrated single-cell and bulk RNA sequencing study on mitophagy-related genes

Oral squamous cell carcinoma (OSCC) has an extremely poor prognosis. Recent studies have suggested that mitophagy-related genes (MRGs) are closely correlated with the development and occurrence of cancer, but the role they play in oral cancer has not yet been explained.We conducted a comprehensive analysis of integrated single-cell and bulk RNA sequencing (RNA-seq) data retrieved from Gene Expression Omnibus (GEO) datasets and The Cancer Genome Atlas (TCGA) database. Multiple methods were combined to provide a comprehensive understanding of the genetic expression patterns and biology of OSCC, such as analysis of pseudotime series, CellChat cell communication, immune infiltration, Gene Ontology (GO), LASSO Cox regression, gene set variation analysis (GSVA), Kyoto Encyclopedia of Genes and Genomes (KEGG), gene set enrichment analysis (GSEA), Tumor Mutation Burden (TMB) and drug sensitivity assessments. The findings of this study demonstrated significantly greater activity of MRGs in NK cells than in other cells in OSCC. A reliable prognostic model was developed using 12 candidate genes strongly associated with mitochondrial autophagy. T stage, N stage and risk score were revealed as independent prognostic factors. Distinctively enriched pathways and immune cells were observed in different risk groups. Notably, low-risk patients were more responsive to chemotherapy. In addition, a nomogram model with excellent predictive ability was established by combining the risk scores and clinical features. The activity of MRGs suggest the potential for the development of new targeted therapies. The construction of a robust prognostic model also provides reference value for individualized prediction and clinical decision-making in patients with OSCC.

Unraveling Divergent Transcriptomic Profiles: A Comparative Single-Cell RNA Sequencing Study of Epithelium, Gingiva, and Periodontal Ligament Tissues.

The periodontium comprising periodontal ligament (PDL), gingiva, and epithelium play crucial roles in maintaining tooth integrity and function. Understanding tissue cellular composition and gene expression is crucial for illuminating periodontal pathophysiology. This study aimed to identify tissue-specific markers via scRNA-Seq. Primary human PDL, gingiva, and epithelium tissues (n = 7) were subjected to cell hashing and sorting. scRNA-Seq library preparation using 10× Genomics protocol and Illumina sequencing was conducted. The analysis was performed using Cellranger (v3.1.0), with downstream analysis via R packages Seurat (v5.0.1) and SCORPIUS (v1.0.9). Investigations identified eight distinct cellular clusters, revealing the ubiquitous presence of epithelial and gingival cells. PDL cells evolved in two clusters with numerical superiority. The other clusters showed varied predominance regarding gingival and epithelial cells or an equitable distribution of both. The cluster harboring most cells mainly consisted of PDL cells and was present in all donors. Some of the other clusters were also tissue-inherent, while the presence of others was environmentally influenced, revealing variability across donors. Two clusters exhibited genetic profiles associated with tissue development and cellular integrity, respectively, while all other clusters were distinguished by genes characteristic of immune responses. Developmental trajectory analysis uncovered that PDL cells may develop after epithelial and gingival cells, suggesting the inherent PDL cell-dominated cluster as a final developmental stage. This single-cell RNA sequencing study delineates the hierarchical organization of periodontal tissue development, identifies tissue-specific markers, and reveals the influence of environmental factors on cellular composition, advancing our understanding of periodontal biology and offering potential insights for therapeutic interventions.

OA26 Paired bulk and single cell analysis of transitional B cells in systemic sclerosis highlights defective peripheral tolerance

Abstract Background/Aims Transitional B cells comprise a distinct population of B cells that have recently migrated to the periphery from the bone marrow. In systemic sclerosis (SSc), there are differences in the number and function of transitional B cells compared with healthy controls (HCs). In addition, we have previously shown that transitional B cells evade peripheral tolerance from SSc patients who are seropositive for anti-topoisomerase I autoantibodies (ATA). To investigate mechanisms that underpin this defect, we performed a paired bulk and single-cell RNA sequencing study of transitional B cell subsets in treatment-naïve ATA-positive patients with diffuse cutaneous SSc (dcSSc) and HCs. Methods CD19+CD24hiCD38hi transitional B cells (5000) were sorted from four HCs and four treatment-naïve ATA-positive dcSSc patients. Single-cell RNA-sequencing was performed on the sorted B cells and the data was analysed using Seurat in RStudio. Paired bulk RNA-sequencing of the sorted transitional T1 (CD19+CD24hiCD38hi CD27-IgMhiIgDmed) and T2 (CD19+CD24hiCD38hiCD27-IgMhiIgDhi) subsets was undertaken in a subset of this cohort. Results From the sorted CD19+CD24hiCD38hi lymphocytes single cell transcriptomics identified six B cell clusters in SSc patients and HCs with differential expression of key genes such as CD19 and TCL1A. This included a cluster of marginal zone precursor cells expressing PLD4 and MZB1 which is expanded in SSc. We also identified an interesting memory-like B cell cluster which was diminished in the SSc group and expresses genes such as TNFRSF13B (BAFF), AICDA (AID) and S100A10. In the HCs, this cluster also expresses genes such as CD27 and IGHA1 which are absent in SSc indicating that B cell development is impaired in the disease. Subsequent pathway analyses provided evidence for the molecular bases of defective tolerance in SSc patients. This appears to be related to dysregulated NF-κB signalling and aberrant extrafollicular B cell development as evidenced through pseudotime trajectory analysis. Differential gene expression analyses using bulk RNA-sequencing of sorted T1 and T2 transitional B cells further identified five candidate gene signatures such as MYC-targets and DNA repair which are significantly enriched in the T2 subset. This latter finding is in congruence with the single cell data and provides further evidence to support the existence of a peripheral tolerance/selection checkpoint at the transitional B cell stage. Conclusion We have identified four transitional B cell subsets and have characterised their transcriptomic profile in SSc. Additionally, we provide new evidence for a potential breach in peripheral tolerance of SSc patients. Further studies are underway to identify key pathways that underpin B cell dysregulation in SSc and explore the relevance of such findings for better targeted therapies. Disclosure C. Beesley: Grants/research support; C.B. is supported through Versus Arthritis (grant number 558800). N. Goldman: None. D. Abraham: None. C. Denton: None. R. Mageed: None. V.H. Ong: None.

HEG1 Protects Against Atherosclerosis by Regulating Stable Flow-Induced KLF2/4 Expression in Endothelial Cells.

Atherosclerosis preferentially occurs in arterial regions of disturbed blood flow, and stable flow (s-flow) protects against atherosclerosis by incompletely understood mechanisms. Our single-cell RNA-sequencing data using the mouse partial carotid ligation model was reanalyzed, which identified Heart-of-glass 1 (HEG1) as an s-flow-induced gene. HEG1 expression was studied by immunostaining, quantitive polymerase chain reaction, hybridization chain reaction, and Western blot in mouse arteries, human aortic endothelial cells (HAECs), and human coronary arteries. A small interfering RNA-mediated knockdown of HEG1 was used to study its function and signaling mechanisms in HAECs under various flow conditions using a cone-and-plate shear device. We generated endothelial-targeted, tamoxifen-inducible HEG1 knockout (HEG1iECKO) mice. To determine the role of HEG1 in atherosclerosis, HEG1iECKO and littermate-control mice were injected with an adeno-associated virus-PCSK9 [proprotein convertase subtilisin/kexin type 9] and fed a Western diet to induce hypercholesterolemia either for 2 weeks with partial carotid ligation or 2 months without the surgery. S-flow induced HEG1 expression at the mRNA and protein levels in vivo and in vitro. S-flow stimulated HEG1 protein translocation to the downstream side of HAECs and release into the media, followed by increased messenger RNA and protein expression. HEG1 knockdown prevented s-flow-induced endothelial responses, including monocyte adhesion, permeability, and migration. Mechanistically, HEG1 knockdown prevented s-flow-induced KLF2/4 (Kruppel-like factor 2/4) expression by regulating its intracellular binding partner KRIT1 (Krev interaction trapped protein 1) and the MEKK3-MEK5-ERK5-MEF2 pathway in HAECs. Compared with littermate controls, HEG1iECKO mice exposed to hypercholesterolemia for 2 weeks and partial carotid ligation developed advanced atherosclerotic plaques, featuring increased necrotic core area, thin-capped fibroatheroma, inflammation, and intraplaque hemorrhage. In a conventional Western diet model for 2 months, HEG1iECKO mice also showed an exacerbated atherosclerosis development in the arterial tree in both sexes and the aortic sinus in males but not in females. Moreover, endothelial HEG1 expression was reduced in human coronary arteries with advanced atherosclerotic plaques. Our findings indicate that HEG1 is a novel mediator of atheroprotective endothelial responses to flow and a potential therapeutic target.

Systematic analysis of apoptosis-related genes in the prognosis of lung squamous cell carcinoma: a combined single-cell RNA sequencing study.

Lung squamous cell carcinoma (LUSC) has a poor prognosis and lacks appropriate diagnostic and treatment strategies. Apoptosis dysregulation is associated with tumor occurrence and drug resistance, but the prognostic value of apoptosis-related genes (ARGs) in LUSC remains unclear. Using univariate Cox regression, least absolute shrinkage and selection operator (LASSO) regression, and multivariate Cox regression analysis based on differentially expressed ARGs, we constructed an ARG-related prognostic model for LUSC survival rates. We conducted correlation analysis of prognostic ARGs by incorporating the dataset of normal lung tissue from the Genotype-Tissue Expression (GTEx) database. We then constructed a risk model, and the predictive ability of the model was evaluated using receiver operating characteristic (ROC) curve analysis. Non-small cell lung cancer (NSCLC) single-cell RNA sequencing (scRNA-seq) data were downloaded from the Gene Expression Omnibus (GEO) database. Subsequently, these data were subjected to single-cell analysis. Cell subgroups were determined and annotated by dimensionality reduction clustering, and the cell subgroups in disease development were identified via pseudotemporal analysis with the Monocle 2 algorithm. We identified four significantly prognostic ARGs and constructed a stable prognostic risk model. Kaplan-Meier curve analysis showed that the high-risk group had a poorer prognosis (P<0.05). Furthermore, the ROC analysis of 3-, 5- and 7-year survival rates confirmed that the model had good predictive value for patients with LUSC. Single-cell RNA sequencing showed the prognostic ARGS were enriched in epithelial cells, smooth muscle cells, and T cells. Pseudotime analysis was used to infer the differentiation process and time sequence of cells. This study identified ARGs that are associated with prognosis in LUSC, and a risk model based on these prognostic genes was constructed that could accurately predict the prognosis of LUSC. Single-cell sequencing analysis provided new insights into the cellular-level development of tumors. These findings provide more guidance for the diagnosis and treatment of patients with LUSC.

The DNA Sensor AIM2 Determines B Cell Fate after BCR Activation in Chronic Graft-Vs-Host Disease

Prevention and treatment of chronic graft-vs-host disease (cGVHD), a debilitating late immune toxicity in patients after allogeneic hematopoietic stem cell transplantation (allo-HCT), who are otherwise cured of hematological malignancies, remains a major challenge. T cells incite and promote cGVHD through coordinated T-B cell responses. Alloantibody production ( Srinivasan et al, Blood 2012, Jin et al, Blood 2016) and aberrant B Cell Receptor (BCR) signaling ( Allen et al, Blood 2014, Flynn et al, Blood 2015) play fundamental roles in the pathology of cGVHD. B cell-intrinsic pathways in cGVHD remain incompletely understood, limiting our therapeutic capacity for patients. In a single cell RNA sequencing study, we found that transcript levels of the DNA sensor Absent in Melanoma 2 (AIM2) were significantly increased in CD27+ BCR-activated B cell clusters in circulating as well as in tissue-resident cGVHD B cells ( Poe et al, JCI Insight 2023). After confirming AIM2 expression is restricted to the nucleus, we also previously showed that AIM2-expressing B cells were augmented after BCR and interferon-gamma (IFNγ) and/or Toll-like receptor (TLR)-7 or 9 stimulation ( Visentin J, Blood 2022;140 Supp 1), suggesting a context-dependent role for AIM2 after allo-HCT. Our new findings now afford the hypothesis that an AIM2-BCR signaling axis determines B cell fate after allo-HCT. To address this hypothesis, we first examined whether AIM2 modulates the promotion of antibody-secreting cells (ASCs) after BCR activation using a hapten-carrier immunization model. We compared numbers of hapten-specific ASCs from immunized WT and AIM2-KO mouse splenocytes using enzyme-linked immunosorbent spot (ELISPOT). We compared numbers of ASCs on day 3 post-immunization, when early germinal center (GC) formation is known to occur. We found that hapten-specific IgM ASCs were significantly increased in WT splenocytes compared to AIM2-KO (p=0.002). By contrast, when we looked at day 8, when mature GC formation and rapid ASC proliferation is known to occur, we found that AIM2-KO immunized mice had significantly lower numbers of hapten-specific IgM (p=0.005) and IgG (p=0.004) ASCs in AIM2-expressing mice (Figure 1a). Our results suggest a role for AIM2 in regulating the survival of short-lived plasmablast-like ASCs after high affinity antigen encounter and maturation of germinal centers. To further investigate whether AIM2 affects expansion of GC-derived (GL7+CD95+) ASCs, we stimulated B cells purified from wildtype (WT) or global AIM2-deficient (AIM2-KO) mice in vitro with lipopolysaccharide (LPS) and IL-4 +/- BCR stimulation. We found that AIM2-deficient B cells, especially GC-like cell populations, expanded significantly in an anti-IgM concentration-dependent manner up to 12-fold (p=0.03) after 72 hr (Figure 1b). By contrast, B cell expansion was constrained in AIM2-sufficient B cells. This expansion was unlikely to be due to differences in proliferation alone, as we did not detect differential Ki67 expression in stimulated B cells with or without AIM2. Together, these data suggest a role for AIM2 in modulating BCR signaling. Finally, we examined the role of AIM2 in BCR-mediated cell death after chronic antigen exposure using our previously described bone marrow transplant (BMT) model, where recipients of bone marrow and splenocytes (BM+Spl) exhibit cGVHD manifestations ( Poe et al, JCI Insight 2018, Jia et al, Blood 2021). We have previously generated C57BL/6J mice with conditional deficiency of AIM2 only in B cells (AIM2 fl/flMb1 Cre/+, CKO), and used these in our BMT model as donors. At day 58 post BMT, we performed ex vivo stimulation of splenic B cells from BMT recipients. While B cells from CKO BM+Spl recipients expanded ex vivo in an anti-IgM concentration-dependent manner, B cells from AIM2-sufficient BM+Spl recipients demonstrated limited responsiveness ex vivo to BCR stimulation. This extends our in vitro observations by suggesting a role for AIM2 in B cell tolerance after chronic antigen exposure. Ongoing experiments will determine if B cell-intrinsic AIM2 contributes to cGVHD pathogenesis. Thus far, our data reveal a novel role for AIM2 as a B cell survival checkpoint after BCR activation. Further delineation of context-dependent and synergistic AIM2-BCR signaling after allo-HCT will lead to strategies that block the development of cGVHD without adversely affecting anti-tumor activity.

Self-recruited neutrophils trigger over-activated innate immune response and phenotypic change of cardiomyocytes in fulminant viral myocarditis

Fulminant myocarditis (FM) is a life-threatening inflammatory disease. However, the mechanisms underlying its acute onset are unknown. By dynamic cardiac function measurement, we discovered that the initiation of sudden hemodynamic collapse was on day 4 in the mouse model of FM. Single-cell RNA-sequencing study revealed that healthy cardiomyocytes (CMs) lost their contractile and metabolic function and differentiated into pro-angiogenic and pro-inflammatory CMs. Meanwhile, neutrophils, the most expanded immune cells, exhibited a unique developmental trajectory only after migrating to the heart, where they continuously attracted peripheral neutrophils via Cxcl2/Cxcl3, resulting in the acute accumulation of neutrophils in the heart. Well-differentiated cardiac-infiltrating neutrophils, rather than viruses, induced phenotypic changes in CMs. Moreover, neutrophils could amplify cytokine storm by recruiting and activating pro-inflammatory monocytes. Blockade of the self-recruiting loop of neutrophils by targeting the Cxcl2/Cxcl3-Cxcr2 axis substantially alleviated FM in mice. Collectively, we provide a comprehensive single-cell atlas of immune cells and CMs in FM, elucidate the disease pathogenesis, and suggest potential therapeutic strategies.

Differences in the peripheral immune cell landscape in atherosclerosis – Insights from the LURIC single cell RNA-sequencing study (LURNA)

Differences in the peripheral immune cell landscape in atherosclerosis – Insights from the LURIC single cell RNA-sequencing study (LURNA)

Fibroblast activation in response to TGFβ1 is modulated by co-culture with endothelial cells in a vascular organ-on-chip platform.

Background: Tissue fibrosis is a major healthcare burden that affects various organs in the body for which no effective treatments exist. An underlying, emerging theme across organs and tissue types at early stages of fibrosis is the activation of pericytes and/or fibroblasts in the perivascular space. In hepatic tissue, it is well known that liver sinusoidal endothelial cells (EC) help maintain the quiescence of stellate cells, but whether this phenomenon holds true for other endothelial and perivascular cell types is not well studied. Methods: The goal of this work was to develop an organ-on-chip microvascular model to study the effect of EC co-culture on the activation of perivascular cells perturbed by the pro-fibrotic factor TGFβ1. A high-throughput microfluidic platform, PREDICT96, that was capable of imparting physiologically relevant fluid shear stress on the cultured endothelium was utilized. Results: We first studied the activation response of several perivascular cell types and selected a cell source, human dermal fibroblasts, that exhibited medium-level activation in response to TGFβ1. We also demonstrated that the PREDICT96 high flow pump triggered changes in select shear-responsive factors in human EC. We then found that the activation response of fibroblasts was significantly blunted in co-culture with EC compared to fibroblast mono-cultures. Subsequent studies with conditioned media demonstrated that EC-secreted factors play at least a partial role in suppressing the activation response. A Luminex panel and single cell RNA-sequencing study provided additional insight into potential EC-derived factors that could influence fibroblast activation. Conclusion: Overall, our findings showed that EC can reduce myofibroblast activation of perivascular cells in response to TGFβ1. Further exploration of EC-derived factors as potential therapeutic targets in fibrosis is warranted.

40-OR: Insulin Stabilized Arterial Plaque by Regulating Vascular Smooth Muscle Cell Metabolism and Differentiation

The main pathology of cardiovascular diseases (CVD) in insulin resistance and diabetes is an acceleration of atherosclerosis and increased risk for the development of unstable plaques. Insulin’s effect on vascular smooth muscle cell (VSMC) has been attributed to be pro-atherogenic due to its actions to increase migration and proliferation. We reported that insulin receptors (IR) are responsible for increasing arterial intimal hyperplasia after wire injury. Surprisingly, mice with VSMC specific deletion of IR (Myh11IRKO/ApoE-/-), atherosclerosis was increased with plaques exhibited unstable pathologies of less VSMC and ECM, but more macrophages and necrosis than ApoE-/- mice. To characterize these unexpected findings in the arterial plaques of Myh11IRKO/ApoE-/- mice, we performed single cell RNA sequencing study of VSMC comparing 4 groups of mice including ApoE-/- and MyH11IRKO/ApoE-/- on normal chow or high fat diet (HFD). Bioinformatics analysis demonstrated that VSMC can be separated into 21 distinctive clusters with those expression enriched for contractile and mitochondrial genes separated from clusters which expressed heat shock protein (HSP) or inflammatory genes. Aorta of HFD insulin resistant WT ApoE-/- mice exhibited significantly increases of multiple clusters of inflammatory VSMCs and, surprisingly, also those clusters enriched for mitochondrial metabolism. However, in Myh11IRKO/ApoE-/- mice on HFD, aortic VMSC clusters with contractile and mitochondrial genes were significantly reduced, but distinct VMSC clusters with expressions of HSP or inflammatory genes were greatly increased. These novel findings, along with metabolic studies in cultured VSMC, indicated that insulin’s actions on VSMC metabolism to reduce the transition of VSMCs subsets from mitochondria enriched VSMCs to stressed and inflammatory VSMCs, which is critical to increase plaque stability and decrease CVD risks in people with insulin resistance and diabetes. Disclosure Q.Li: None. J.Fu: None. K.Park: None. I.Wu: None. G.L.King: Research Support; Janssen Research &amp; Development, LLC.

Single-cell RNA sequencing reveals peripheral blood mononuclear immune cell landscape associated with operational tolerance in a kidney transplant recipient

Operational tolerance (OT) after kidney transplantation is defined as stable graft acceptance without the need for immunosuppression therapy. However, it is not clear which cellular and molecular pathways are driving tolerance in these patients. In this first-of-its-kind pilot study, we assessed the immune landscape associated with OT using single-cell analyses. Peripheral mononuclear cells from a kidney transplant recipient with OT (Tol), 2 healthy individuals (HC), and a kidney transplant recipient with normal kidney function on standard-of-care immunosuppression (SOC) were evaluated. The immune landscape of the Tol was drastically different from that of SOC and emerged closer to the profile of HC. TCL1A+ naive B cells and LSGAL1+ regulatory T cells (Tregs) were in higher proportions in Tol. We were unable to identify the Treg subcluster in SOC. The ligand-receptor analysis in HC and Tol identified interactions between B cells, and Tregs that enhance the proliferation and suppressive function of Tregs. SOC reported the highest proportion of activated B cells with more cells in the G2M phase. Our single-cell RNA sequencing study identified the mediators of tolerance; however, it emphasizes the requirement of similar investigations on a larger cohort to reaffirm the role of immune cells in tolerance.

Epithelial cells activate fibroblasts to promote esophageal cancer development.

Esophageal squamous-cell carcinoma (ESCC) develops through multistage epithelial cancer formation, i.e., from normal epithelium, low- and high-grade intraepithelial neoplasia to invasive carcinoma. However, how the precancerous lesions progress to carcinoma remains elusive. Here, we report a comprehensive single-cell RNA sequencing and spatial transcriptomic study of 79 multistage esophageal lesions from 29 patients with ESCC. We reveal a gradual and significant loss of ANXA1 expression in epithelial cells due to its transcription factor KLF4 suppression along the lesion progression. We demonstrate that ANXA1 is a ligand to formyl peptide receptor type 2 (FPR2) on fibroblasts that maintain fibroblast homeostasis. Loss of ANXA1 leads to uncontrolled transformation of normal fibroblasts into cancer-associated fibroblasts (CAFs), which can be enhanced by secreted TGF-β from malignant epithelial cells. Given the role of CAFs in cancer, our study underscores ANXA1/FPR2 signaling as an important crosstalk mechanism between epithelial cells and fibroblasts in promoting ESCC.

Zinc Supplementation Induced Transcriptional Changes in Primary Human Retinal Pigment Epithelium: A Single-Cell RNA Sequencing Study to Understand Age-Related Macular Degeneration.

Zinc supplementation has been shown to be beneficial to slow the progression of age-related macular degeneration (AMD). However, the molecular mechanism underpinning this benefit is not well understood. This study used single-cell RNA sequencing to identify transcriptomic changes induced by zinc supplementation. Human primary retinal pigment epithelial (RPE) cells could mature for up to 19 weeks. After 1 or 18 weeks in culture, we supplemented the culture medium with 125 µM added zinc for one week. RPE cells developed high transepithelial electrical resistance, extensive, but variable pigmentation, and deposited sub-RPE material similar to the hallmark lesions of AMD. Unsupervised cluster analysis of the combined transcriptome of the cells isolated after 2, 9, and 19 weeks in culture showed considerable heterogeneity. Clustering based on 234 pre-selected RPE-specific genes divided the cells into two distinct clusters, we defined as more and less differentiated cells. The proportion of more differentiated cells increased with time in culture, but appreciable numbers of cells remained less differentiated even at 19 weeks. Pseudotemporal ordering identified 537 genes that could be implicated in the dynamics of RPE cell differentiation (FDR < 0.05). Zinc treatment resulted in the differential expression of 281 of these genes (FDR < 0.05). These genes were associated with several biological pathways with modulation of ID1/ID3 transcriptional regulation. Overall, zinc had a multitude of effects on the RPE transcriptome, including several genes involved in pigmentation, complement regulation, mineralization, and cholesterol metabolism processes associated with AMD.

Adult hippocampal neurogenesis in Alzheimer's disease: A roadmap to clinical relevance.

Adult hippocampal neurogenesis (AHN) drops sharply during early stages of Alzheimer's disease (AD), via unknown mechanisms, and correlates with cognitive status in AD patients. Understanding AHN regulation in AD could provide a framework for innovative pharmacological interventions. We here combine molecular, behavioral, and clinical data and critically discuss the multicellular complexity of the AHN niche in relation to AD pathophysiology. We further present a roadmap toward a better understanding of the role of AHN in AD by probing the promises and caveats of the latest technological advancements in the field and addressing the conceptual and methodological challenges ahead.

Cross center single-cell RNA sequencing study of the immune microenvironment in rapid progressing multiple myeloma

Despite advancements in understanding the pathophysiology of Multiple Myeloma (MM), the cause of rapid progressing disease in a subset of patients is still unclear. MM’s progression is facilitated by complex interactions with the surrounding bone marrow (BM) cells, forming a microenvironment that supports tumor growth and drug resistance. Understanding the immune microenvironment is key to identifying factors that promote rapid progression of MM. To accomplish this, we performed a multi-center single-cell RNA sequencing (scRNA-seq) study on 102,207 cells from 48 CD138- BM samples collected at the time of disease diagnosis from 18 patients with either rapid progressing (progression-free survival (PFS) < 18 months) or non-progressing (PFS > 4 years) disease. Comparative analysis of data from three centers demonstrated similar transcriptome profiles and cell type distributions, indicating subtle technical variation in scRNA-seq, opening avenues for an expanded multicenter trial. Rapid progressors depicted significantly higher enrichment of GZMK+ and TIGIT+ exhausted CD8+ T-cells (P = 0.022) along with decreased expression of cytolytic markers (PRF1, GZMB, GNLY). We also observed a significantly higher enrichment of M2 tolerogenic macrophages in rapid progressors and activation of pro-proliferative signaling pathways, such as BAFF, CCL, and IL16. On the other hand, non-progressive patients depicted higher enrichment for immature B Cells (i.e., Pre/Pro B cells), with elevated expression for markers of B cell development (IGLL1, SOX4, DNTT). This multi-center study identifies the enrichment of various pro-tumorigenic cell populations and pathways in those with rapid progressing disease and further validates the robustness of scRNA-seq data generated at different study centers.

The DNA-Sensor AIM2 Promotes BCR-Activated B Cells in Chronic Graft-Versus-Host-Disease

Chronic graft-versus-host-disease (cGVHD) remains a common and poorly understood cause of late non-relapse morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HCT). T cells and IgG-producing B cells mediate cGVHD development and perpetuation, but molecular mechanisms driving immune pathology require further study. We have shown that B cells from cGVHD patients are B Cell Receptor (BCR)-activated and primed for survival. In a mouse model of bone-marrow transplantation (BMT) with cGVHD manifestations, BCR signaling is promoted by alloantigens and BAFF (Jia et al., Blood 2021). BCR signaling is a viable therapeutic target in cGVHD, but other targetable pathways that promote pathological BCR-activated B cells remain largely unknown. Our single cell RNA-sequencing study of B cells from the peripheral blood (PB) of HCT patients identified 'Absent in Melanoma 2' (AIM2) as a prime candidate for further study (ASH abstract 2019, Poe et al., manuscript submitted). AIM2 was a signature gene in a BCR-activated subset after HCT, and AIM2 transcripts were significantly increased in a pre-germinal center (GC) B cell subset in cGVHD vs. no cGVHD patients (adjusted p=0.049). AIM2 incites inflammasome-mediated pyroptotic cell death in myeloid cells, but the function of AIM2 in B cells is largely unknown. AIM2 is expressed by CD27+ antigen (Ag)-experienced B cells in healthy individuals (HI) (Svensson et al., PlosOne 2017) and emerging evidence suggests that AIM2 promotes antibody production (Yang et al., Signal Transduct Target Ther 2021). We hypothesize that AIM2 promotes pathological BCR-activated B cells and anti-host IgG production in cGVHD. Using intracellular flow cytometry (IFC) and PB samples from HI (n=27), we first confirmed that the AIM2 protein is mainly expressed by CD27+ Ag-experienced B cells. Specifically, 88% and 99% of the IgD+ pre- and IgD- post-GC CD27+ B cells expressed AIM2, respectively. In HCT, we found a significantly higher proportion of AIM2+ cells in the circulating CD27+ IgD- post-GC B cells from cGVHD patients (n=7) vs. no cGVHD patients (n=5) (p=0.02). We also found significant overexpression of AIM2 transcripts in B cells from mice with cGVHD manifestations vs. no cGVHD mice (n=5/group, p=0.03, Figure 1A). To determine if Ag-activation together with other extrinsic factors typically encountered by B cells after HCT induced AIM2 expression by purified CD27- B cells from HI, we used IFC. The proportion of AIM2+ CD27- B cells increased after BCR +/- Toll-like receptor 7 or 9 stimulation only when IFNγ was present. Next, we studied the function of AIM2 in B cells. Unlike myeloid cells, CD27+ B cells from HI did not undergo pyroptotic cell death after stimulation with double stranded DNA. This lack of pyroptosis was not due to a deficiency in the inflammasome components and B cells had the capacity to undergo pyroptosis after exposure to the NLRP1/3 activator, nigericin. To assess effects of AIM2 on specific IgG production, we used AIM2 KO mice in a hapten-carrier immunization model (NP-KLH + alum i.p.). AIM2-KO mice had larger splenic GC vs. WT controls at day 8 post-immunization (p<0.0001). GC enlargement paradoxically associated with lower anti-NP IgG levels (p=0.03). Finally, having previously shown that BM+Spl recipients have a significantly higher proportion of GL7+ BCR-activated blood B cells (Jia et al., Blood 2021), we asked whether AIM2 promoted GL7+ B cells in cGVHD. To address this, we used AIM2-floxed mice, kindly supplied by Genentech, to breed AIM2fl/fl Mb1Cre/+ conditional KO mice (AIM2-CKO), deficient in AIM2 only in B cells. We then used these AIM2-CKO vs. AIM2fl/fl Mb1+/+ control littermates mice which are AIM2-sufficient as BMT donors. At day 24 post-BMT, recipients of BM+Spl from AIM2-CKO mice had a significantly lower percentage of mature (CD19+ CD93-) GL7+ B cells in PB vs. recipients of BM+Spl from the AIM2-sufficient littermates (n=10/group, p=0.004, Figure 1B). This was not due to baseline B cell differences in donor CKO mice. These results are being confirmed at later time points in ongoing, additional experiments. Our data show that AIM2 promotes IgG-producing B cells and circulating GL7+ B cells, suggesting that AIM2 plays a novel inflammasome-independent role in cGVHD B cell pathology. We are actively investigating how AIM2 effects alloantibody production and cGVHD development, so that therapeutic targets can be identified. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single-Cell RNA-Sequencing of 370 Bone Marrow and Peripheral Blood Immune Cell Samples from Patients with MGUS and Smoldering Multiple Myeloma Reveal Significant Immune Dysregulation at the MGUS Stage, and Novel Interactions between Tumor and Immune Cells

Introduction A patient's risk of progression from Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM) to full-blown Multiple Myeloma (MM) is the result of complex interactions between the tumor burden, alterations of tumor -omics, and tumor-extrinsic factors such as immune dysregulation. Prior studies have suggested that immune dysregulation can be observed as early as at the MGUS stage, but immune alterations in patients with MGUS have not been comprehensively studied at scale. Furthermore, it is unclear which of the immune cell populations that are altered in disease may directly interact with the tumor, as opposed to responding to general changes in the milieu. Methods Here, we present results from the largest single-cell RNA-sequencing cohort (n=370) of bone marrow CD138-negative (n=243) and peripheral blood mononuclear cell samples (n=127) from patients with MGUS and SMM. Specifically, we profiled 51 samples from patients with MGUS, 226 samples from patients with SMM, 28 samples from patients with MM, and 61 samples from healthy donors. Bone marrow samples were subjected to magnetic bead enrichment for CD138 (Miltenyi Biotec) prior to library preparation. Libraries were prepared with the Chromium Single-cell 5' kit by 10X Genomics and sequenced on NovaSeq S4 flow cells at the Genomics Platform of the Broad Institute of MIT and Harvard (Cambridge, MA). CellRanger v.6.0.1. was used to extract FASTQ files and produce count matrices. Droplets with more than 15% mitochondrial gene expression or fewer than 200 genes per cell were filtered out, doublets were identified using Scrublet, SCDS, and scDblFinder, and samples were integrated using Harmony. Excluding plasma cells, immune cells isolated from both the CD138-positive and CD138-negative fractions were included in this analysis. Results Overall, we profiled 2,327,008 immune cells, including T-cells, NK cells, B-cells, Monocytes, Dendritic cells, megakaryocytes, and progenitor cells. To identify immune cell populations that are more likely to interact with tumor cells, we compared the abundance of populations between matched bone marrow CD138-positive and CD138-negative fractions in patients and healthy donors. In healthy donors, we observed a significant enrichment of CD14+ monocyte subpopulations and CD168+ macrophages expressing complement factor C1q in the CD138-positive fractions, which is in line with their role in promoting and maintaining plasma cell differentiation. In patients, where decreased abundance of CD14+ monocytes is observed, this interaction was no longer significant, although macrophages were still enriched in the CD138-positive fraction. Consistent with prior publications, megakaryocytes were enriched in the CD138-positive fraction of patients, an interaction that has been shown to promote myeloma progression in vitro and in vivo. Of note, pro-B-cells, pre-B-cells, and memory B-cells were enriched in the CD138-positive fraction of patients, raising questions about their potential involvement in the clonal population. Lastly, Granzyme K-expressing CD8+ T-cells were significantly enriched in the CD138-positive fraction of patients, suggesting an interaction between this population and tumor cells. Remarkably, despite their early stage, patients with MGUS had on average fewer pDCs, progenitor cells, and CD14+ Monocytes, and more CD4+ T-cells (including Tregs), Granzyme B-expressing CD8+ T-cells and CD56dim NK cells, compared to healthy donors. These results suggest that bone marrow immune cell composition can be significantly altered at the MGUS stage. Discussion In the largest to date single-cell RNA-sequencing study of bone marrow and peripheral blood immune cells from patients with MGUS and SMM, we have comprehensively mapped alterations in bone marrow immune cell composition in patients with MGUS, and identified immune cell populations that are more likely to interact directly with tumor cells in the bone marrow microenvironment.

O.16 Single cell RNA sequencing study of FAPS obtained from muscle samples of DMD patients reveals new pathogenic pathways of the muscle degeneration process

O.16 Single cell RNA sequencing study of FAPS obtained from muscle samples of DMD patients reveals new pathogenic pathways of the muscle degeneration process