Single-cell RNA Sequencing Studies Research Articles

Ontogenetic rules for the molecular diversification of hypothalamic neurons.

The hypothalamus is an evolutionarily conserved endocrine interface that, among other roles, links central homeostatic control to adaptive bodily responses by releasing hormones and neuropeptides from its many neuronal subtypes. In its preoptic, anterior, tuberal and mammillary subdivisions, a kaleidoscope of magnocellular and parvocellular neuroendocrine command neurons, local-circuit neurons, and neurons that project to extrahypothalamic areas are intermingled in partially overlapping patches of nuclei. Molecular fingerprinting has produced data of unprecedented mass and depth to distinguish and even to predict the synaptic and endocrine competences, connectivity and stimulus selectivity of many neuronal modalities. These new insights support eminent studies from the past century but challenge others on the molecular rules that shape the developmental segregation of hypothalamic neuronal subtypes and their use of morphogenic cues for terminal differentiation. Here, we integrate single-cell RNA sequencing studies with those of mouse genetics and endocrinology to describe key stages of hypothalamus development, including local neurogenesis, the direct terminal differentiation of glutamatergic neurons, transition cascades for GABAergic and GABAergic cell-derived dopamine cells, waves of local neuronal migration, and sequential enrichment in neuropeptides and hormones. We particularly emphasize how transcription factors determine neuronal identity and, consequently, circuit architecture, and whether their deviations triggered by environmental factors and hormones provoke neuroendocrine illnesses.

The role of specialized cell cycles during erythroid lineage development: insights from single-cell RNA sequencing.

Early erythroid progenitors known as CFU-e undergo multiple self-renewal cell cycles. The CFU-e developmental stage ends with the onset of erythroid terminal differentiation (ETD). The transition from CFU-e to ETD is a critical cell fate decision that determines erythropoietic rate. Here we review recent insights into the regulation of this transition, garnered from flow cytometric and single-cell RNA sequencing studies. We find that the CFU-e/ETD transition is a rapid S phase-dependent transcriptional switch. It takes place during an S phase that is much shorter than in preceding or subsequent cycles, as a result of globally faster replication forks. Furthermore, it is preceded by cycles in which G1 becomes gradually shorter. These dramatic cell cycle and S phase remodeling events are directly linked to regulation of the CFU-e/ETD switch. Moreover, regulators of erythropoietic rate exert their effects by modulating cell cycle duration and S phase speed. Glucocorticoids increase erythropoietic rate by inducing the CDK inhibitor p57KIP2, which slows replication forks, inhibiting the CFU-e/ETD switch. Conversely, erythropoietin promotes induction of ETD by shortening the cycle. S phase shortening was reported during cell fate decisions in non-erythroid lineages, suggesting a fundamentally new developmental role for cell cycle speed.

Dissecting the Heterogeneity of Human Thoracic Aortic Aneurysms Using Single-Cell Transcriptomics.

Thoracic aortic aneurysm is a life-threatening condition caused by weakening of the thoracic aorta wall, often developing silently until dissection or rupture occurs. Despite substantial efforts in the past decade, there have been no significant therapeutic advances to prevent or clinically manage diverse forms of thoracic aortic aneurysm and dissection with the only effective treatment being surgical repair. There is an urgent need to understand intra- and inter-aneurysmal heterogeneity underlying thoracic aortic aneurysm and dissection pathogenesis. The human aortic wall consists of many cell types and exhibits significant regional heterogeneity. High-throughput single-cell RNA sequencing has emerged as the principal tool to reveal the complexity in human tissues and clinical specimens. Recent single-cell RNA sequencing studies of different aortic cell populations both in vivo and in vitro began to dissect this complexity and have provided valuable information. In this review, we summarize these findings and discuss the potential applications of single-cell transcriptomics and related high-content technologies in human thoracic aortic aneurysm and dissection research, as well as the challenges associated with it.

Abstract 4013: Functional heterogeneity of cancer-associated stromal subtypes in the prostate cancer microenvironment

Abstract The stromal tumor microenvironment plays a central role in the development, progression and therapy resistance of prostate cancer (PCa), the second leading cause of cancer-related death in males in western nations. Functional and single cell RNA sequencing (scRNA-seq) studies unequivocally demonstrate the coexistence of functionally and spatially distinct fibroblast and mural cell states within the tumor microenvironment of various non-prostate tumors. Stromal cellular heterogeneity in PCa however remains poorly understood. This study therefore aimed to (1) comprehensively analyze stromal cell states within the PCa microenvironment and (2) to identify and characterize onco-supportive fibromuscular entities in respect to their potential clinical exploitation. Thus, we performed bulk tissue scRNA-seq on biopsy cores from benign and cancerous prostate tissue of 4 different patients. Analyses presented here focused on the fibroblast and mural cell clusters, which each delineated further into 2 subclusters. One fibroblast subcluster showed strong similarity to published signatures of inflammatory cancer-associated fibroblasts (iCAF). A closely related but distinct fibroblast subcluster exhibited hallmarks of benign interstitial fibroblasts with enrichment of GO terms such as antigen processing and presentation. Based on expression signatures and spatial localization via immunohistochemistry, the 2 mural cell subclusters (MCAM+) were identified as vascular smooth muscle cells (vSMCs) and dedifferentiated prostate SMCs/myofibroblasts. Notably, the gene signature of this latter subcluster correlated with increasing Gleason score and decreased disease-specific survival. On the basis of scRNA-seq data from this and independent datasets, panels of subcluster-specific markers were selected for validation studies via FACS, multiplex immunohistochemistry and quantitative real time PCR. Thus far, immunohistochemical studies validated not only the in vivo presence of the 4 distinct cellular states but also revealed their distinct spatial localization. We will present molecular and functional data from these ongoing analyses in (i) immortalized fibroblast and smooth muscle cell lines, (ii) heterogeneous ex vivo outgrowth cultures of primary PCa fibroblasts/myofibroblasts and (iii) primary cells from single cell suspensions of PCa biopsies enzymatically digested using an in-house approach optimized for maximal recovery of fibromuscular cells. These studies aim to evaluate the specificity, robustness and suitability of the chosen markers to delineate distinct tumor-relevant stromal cell subtypes enabling their functional contribution to PCa pathophysiology and clinical relevance to be assessed in future experiments. Citation Format: Elisabeth Damisch, Elena Brunner, Sieghart Sopper, Isabel Heidegger, Andreas Pircher, Georgios Fotakis, Zlatko Trajanoski, Sandra Theresa Deichsler, Georg Schäfer, Natalie Sampson. Functional heterogeneity of cancer-associated stromal subtypes in the prostate cancer microenvironment [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 4013.

Abstract 3539: IL-12 overcomes T-cell immune suppression by the B-ALL microenvironment

Abstract Leukemia remains one of the leading causes of disease-related deaths in children. Recent clinical advancements in both cell-directed and antibody therapies for B-cell leukemias demonstrate the importance of the immune system in eliciting anti-leukemia activity. Yet, there are still patients who suffer relapse with these treatments, which underscores the need to better understand immune evasion by leukemia cells and the need to develop more effective therapies. Although IL-12 has been well-studied in the context of pathogenic infections and immune cells, we previously published that IL-12 potently promotes B-ALL clearance in immune competent mice via increased T-cell activation. To further characterize the role of IL-12 in leukemia control, we examined the bone marrow of immune competent mice with B-ALL using an extensive flow cytometry panel. From these studies, we observed both an increase in T-cell and dendritic cell (DC) numbers and augmented DC activation in rIL-12p70 treated WT mice. Furthermore, rIL-12p70 treatment of WT mice with B-ALL exhibited increased GM-CSF, IL-2, IL-1β, CXCL2, CCL12, and IFN-gamma levels in the bone marrow microenvironment. In these experiments, we also analyzed gene expression profiles in T-cells. The expression of exhaustion-associated genes, including Lag3, Pcdc1, and Tigit, was higher in T-cells recovered from untreated mice with B-ALL relative to those from mice without leukemia. These data are consistent with observations made from single cell RNA-sequencing studies of children with B-ALL, where it was revealed that immune cell exhaustion was present at higher levels in those with measurable disease after induction therapy. Treatment of mice with IL-12 abrogated the leukemia induced increases in exhaustion-associated gene expression. Mechanistically, we observed that when T-cells are stimulated in vitro in B-ALL supernatant there is a reduction in the surface expression of the T-cell activation marker, CD44, and marker of degranulation, CD107b. Jurkat T-cells expressing NFAT or NF-kB reporter plasmids were used to assess activation of T-cell signaling pathways. In addition to suppressing CD44 and CD107b, we also found that B-ALL-secreted factors also suppress the activation of signaling pathways downstream of the TCR. Considering clinical implications of this discovery, we further explored IL-12 ex vivo in combination with the clinically approved bi-specific T-cell engager, blinatumomab. Notably, B-ALL-secreted factors reduced blinatumomab-mediated T-cell lysis of target cells, which was restored with recombinant IL-12 treatment. Overall, this data shows the potential for IL-12 to be used as an immunotherapeutic in conjunction with other current therapies to overcome T-cell suppression by B-ALL. Citation Format: Rae Hunter, Kathleen Imbach, Jodi Dougan, Priscilla Do, Ashley Townsel, Erik Dreaden, Gregory Gibson, Curtis Henry, Christopher Porter. IL-12 overcomes T-cell immune suppression by the B-ALL microenvironment [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 3539.

Estimation of tumor cell total mRNA expression in 15 cancer types predicts disease progression

Single-cell RNA sequencing studies have suggested that total mRNA content correlates with tumor phenotypes. Technical and analytical challenges, however, have so far impeded at-scale pan-cancer examination of total mRNA content. Here we present a method to quantify tumor-specific total mRNA expression (TmS) from bulk sequencing data, taking into account tumor transcript proportion, purity and ploidy, which are estimated through transcriptomic/genomic deconvolution. We estimate and validate TmS in 6,590 patient tumors across 15 cancer types, identifying significant inter-tumor variability. Across cancers, high TmS is associated with increased risk of disease progression and death. TmS is influenced by cancer-specific patterns of gene alteration and intra-tumor genetic heterogeneity as well as by pan-cancer trends in metabolic dysregulation. Taken together, our results indicate that measuring cell-type-specific total mRNA expression in tumor cells predicts tumor phenotypes and clinical outcomes.

Immuno-senescence and elevated central carbon metabolism in hematopoietic stem cell compartment upon aging.

e24030 Background: Aging is associated with decline of hematopoiesis, compromised innate immunity, and propensity for malignancies. Aging of the hematopoietic stem and progenitor cell (HSPC) compartment is caused by accumulation of senescent cells and elimination of the latter rejuvenates innate immunity in animal models. Methods: HSC (CD34+) were isolated from the bone marrow of healthy human subjects (age range 20 – 74 years; n = 18) and separated by FACS in 3 distinct subsets with high, intermediate, and low glucose uptake (GU) capacity (GUhigh, GUinter, GUlow). Single-cell RNA-sequencing (scRNAseq) studies, followed by gene ontology (GO) analysis of biological processes, analysis of cell cycle, and gene set enrichment analysis (GSEA) were performed. Results: While the percentages of GUinter and GUlow subpopulations remain relatively constant with age, the proportion of GUhigh subset increases continuously with age (from 0.2% in adults < 30 years to 6,7% in > 60 years). GO analysis revealed that the GUhigh subset showed a significantly higher expression of genes involved in myeloid development, cell cycle arrest, inflammation and stress response, apoptosis, and significantly lower expressions of genes involved in lymphoid development. GSEA confirmed that the gene sets for cell cycle arrest, senescence associated secretory phenotype (SASP), anti-apoptosis/survival and P53 pathways are significantly up-regulated in the GUhigh subset. The latter shows therefore all deregulations that are inherent hallmarks of senescence. Most of the molecular deregulations are similar to those found in murine models of aging, but there are significant differences. In addition, cell cycle analysis revealed that the proportion of cells in the G1 phase was significantly reduced in the GUhigh subset. Comparing the frequencies of old to young GUhigh, GUlow cells along the cell cycle progression ranks showed a significant depletion of cells in late G1 phase among GUhigh cells but not in the other subsets. The frequencies of non-cycling cells in the GUlowsubset have remained high (> 70%) irrespective of age, thus providing evidence for the quiescence status of the GUlow subset, as well as for persistence of HSPC with “young phenotype”. Conclusions: Our series of proteome and single cell transcriptome analyses have shown that the GUhigh subpopulation is highly enriched in senescent HSPCs. This property may be exploited to enrich, visualize, and trace senescence in human bone marrow. Cell cycle analysis has revealed a significantly shortened G1 phase that is linked to dedifferentiated state in the senescent HSPC. Our results indicate a dependency of senescent HSPCs on elevated glucose metabolism as well as on anti-apoptotic factors for survival. These may represent ideal targets for elimination of senescent HSPC to restore innate immunity as demonstrated in murine models.

Mesothelial cell-derived antigen-presenting cancer-associated fibroblasts induce expansion of regulatory Tcells in pancreatic cancer.

Recent studies have identified a unique cancer-associated fibroblast (CAF) population termed antigen-presenting CAFs (apCAFs), characterized by the expression of major histocompatibility complex class II molecules, suggesting a function in regulating tumor immunity. Here, by integrating multiple single-cell RNA-sequencing studies and performing robust lineage-tracing assays, we find that apCAFs are derived from mesothelial cells. During pancreatic cancer progression, mesothelial cells form apCAFs by downregulating mesothelial features and gaining fibroblastic features, a process induced by interleukin-1 and transforming growth factor β. apCAFs directly ligate and induce naive CD4+ Tcells into regulatory Tcells (Tregs) in an antigen-specific manner. Moreover, treatment with an antibody targeting the mesothelial cell marker mesothelin can effectively inhibit mesothelial cell to apCAF transition and Treg formation induced by apCAFs. Taken together, our study elucidates how mesothelial cells may contribute to immune evasion in pancreatic cancer and provides insight on strategies to enhance cancer immune therapy.

OP0110 CELL-TYPE-SPECIFIC TRANSCRIPTOME ARCHITECTURE UNDERLYING THE ESTABLISHMENT AND EXACERBATION OF SYSTEMIC LUPUS ERYTHEMATOSUS

BackgroundSystemic lupus erythematosus (SLE) is a complex autoimmune disease with unknown etiology involving multiple immune cells and has diverse clinical phenotypes. This heterogeneous nature has hampered a better understanding of SLE pathogenesis and the development of effective therapeutic agents. While recent single-cell RNA sequencing studies of SLE identified several important cell subpopulations, they were limited by sparse expression information at single-cell level and small sample sizes.ObjectivesThis study aimed to elucidate the dysregulated gene expression pattern linked to multiple clinical statuses of SLE with a fine cellular resolution and higher sensitivity. We also attempted to resolve a complex interaction between risk variants and the transcriptome dysregulation seen in SLE patients.MethodsWe conducted a large-scale bulk transcriptome study of 6,386 RNA-sequencing data including 27 purified immune cell types in peripheral blood from 136 SLE and 89 healthy donors in the Immune Cell Gene Expression Atlas from the University of Tokyo (ImmuNexUT) cohort1. At enrollment, SLE patients had diverse clinical manifestations (disease activity, organ involvement and treatment profiles) and 22 patients were re-evaluated after belimumab treatment.ResultsWe first profiled two distinct cell-type-specific transcriptomic signatures: disease-state and disease-activity signatures, reflecting disease establishment and exacerbation, respectively.After confirming the high replicability of both signatures in independent cohorts, we identified candidates of biological processes unique to each signature: e.g., upregulated E2F transcriptional activity in Th1, CD8+ memory T-lineage and NK cells, and dynamic increase of IL21 and CXCL13 in Th1 cells in an active phase of SLE. Pathway analysis highlighted the importance of immunometabolic process for SLE (e.g., oxidative phosphorylation) in cell-type-specific resolution.Moreover, we demonstrated cell-type-specific contributions to diverse organ involvement, e.g., Th1 for mucocutaneous, monocyte-lineage cells for musculoskeletal, neutrophil-lineage cells for renal activity, respectively.We also observed the strong associations of disease-activity signatures with treatment effect: (i) belimumab suppressed activity signatures from B-lineage cells, especially in good responders and (ii) mycophenolate mofetil substantially suppressed activity signatures from plasmablast, Th1, and central memory CD8 cells.However, through stratified LD score regression using large-scale SLE-GWASs, we revealed that disease-activity signatures were less enriched around SLE risk variants than disease-state signatures. Consistent with this result, the directions of SLE risk alleles’ expression quantitative trait locus (eQTL) effects were significantly concordant with the directions of disease-state signatures, but not with those of activity signatures. These findings suggested that the current genetic case-control studies may not well capture clinically vital biology linked to drug target discovery for SLE. Meanwhile, we also detected some examples of activity signatures that might contribute to the disease risk by modulating risk allele’s eQTL effects.Figure 1.ConclusionWe identified comprehensive gene signatures reflecting the establishment and exacerbation of SLE, which provide essential foundations for future genomic, genetic, and clinical studies.

Reprogramming of Hepatic Metabolism and Microenvironment in Nonalcoholic Steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD), a spectrum of metabolic liver disease associated with obesity, ranges from relatively benign hepatic steatosis to nonalcoholic steatohepatitis (NASH). The latter is characterized by persistent liver injury, inflammation, and liver fibrosis, which collectively increase the risk for end-stage liver diseases such as cirrhosis and hepatocellular carcinoma. Recent work has shed new light on the pathophysiology of NAFLD/NASH, particularly the role of genetic, epigenetic, and dietary factors and metabolic dysfunctions in other tissues in driving excess hepatic fat accumulation and liver injury. In parallel, single-cell RNA sequencing studies have revealed unprecedented details of the molecular nature of liver cell heterogeneity, intrahepatic cross talk, and disease-associated reprogramming of the liver immune and stromal vascular microenvironment. This review covers the recent advances in these areas, the emerging concepts of NASH pathogenesis, and potential new therapeutic opportunities.

Single-cell RNA-sequencing of mammalian brain development: insights and future directions.

Understanding human brain development is of fundamental interest but is also very challenging. Single-cell RNA-sequencing studies in mammals have revealed that brain development is a highly dynamic process with tremendous, previously concealed, cellular heterogeneity. This Spotlight discusses key insights from these studies and their implications for experimental models. We survey published single-cell RNA-sequencing studies of mouse and human brain development, organized by anatomical regions and developmental time points. We highlight remaining gaps in the field, predominantly concerning human brain development. We propose future directions to fill the remaining gaps, and necessary complementary techniques to create an atlas integrated in space and time of human brain development.

Single-Cell and Single-Nucleus RNAseq Analysis of Adult Neurogenesis.

The complexity of adult neurogenesis is becoming increasingly apparent as we learn more about cellular heterogeneity and diversity of the neurogenic lineages and stem cell niches within the adult brain. This complexity has been unraveled in part due to single-cell and single-nucleus RNA sequencing (sc-RNAseq and sn-RNAseq) studies that have focused on adult neurogenesis. This review summarizes 33 published studies in the field of adult neurogenesis that have used sc- or sn-RNAseq methods to answer questions about the three main regions that host adult neural stem cells (NSCs): the subventricular zone (SVZ), the dentate gyrus (DG) of the hippocampus, and the hypothalamus. The review explores the similarities and differences in methodology between these studies and provides an overview of how these studies have advanced the field and expanded possibilities for the future.

Toward Marrow Adipocytes: Adipogenic Trajectory of the Bone Marrow Stromal Cell Lineage.

Bone marrow contains precursor cells for osteoblasts and adipocytes in the stromal compartment. Bone marrow adipose tissue (BMAT) is an important constituent of the bone marrow that is particularly abundant in adults. BMAT is composed of the proximal “regulated” BMAT containing individual adipocytes interspersed within actively hematopoietic marrow, and the distal “constitutive” BMAT containing large adipocytes in the area of low hematopoiesis. Historically, bone marrow adipocytes were regarded as one of the terminal states of skeletal stem cells, which stand at the pinnacle of the lineage and possess trilineage differentiation potential into osteoblasts, chondrocytes and adipocytes. Recent single-cell RNA-sequencing studies uncover a discrete group of preadipocyte-like cells among bone marrow stromal cells (BMSCs), and recent mouse genetic lineage-tracing studies reveal that these adipocyte precursor cells possess diverse functions in homeostasis and regeneration. These adipogenic subsets of BMSCs are abundant in the central marrow space and can directly convert not only into lipid-laden adipocytes but also into skeletal stem cell-like cells and osteoblasts under regenerative conditions. It remains determined whether there are distinct adipocyte precursor cell types contributing to two types of BMATs. In this short review, we discuss the functions of the recently identified subsets of BMSCs and their trajectory toward marrow adipocytes, which is influenced by multiple modes of cell-autonomous and non-cell autonomous regulations.

Suppressed COP9 signalosome 5 promotes hemocyte proliferation through Cyclin E in the early G1 phase to defend against bacterial infection in crab.

Hemocytes are invertebrate immune cells that are similar to blood cells in vertebrates and play a crucial role in innate immunity. Previous work has found that mature circulating hemocytes lack the ability to proliferate. However, recent single-cell RNA sequencing and functional studies in invertebrate have challenged this view. Here, we report that bacteria induced hemocytes proliferation in the Chinese mitten crab, Eriocheir sinensis. Flow cytometry was used to collect non-proliferating and proliferating hemocytes populations, while the expression of EsCyclin E was highly expressed in proliferating hemocytes, but the expression of EsCsn5 was significantly suppressed in proliferating hemocytes. Subsequent studies have found EsCsn5 distributed in two fractions include holo-complex and monomeric form, whereas knockdown of EsCsn5has little impact on the amount of the holo-complex. EsCsn5 was widely expressed in different crab tissues, while its expression was significantly reduced upon bacterial infection. Crab hemocytes showed significantly enhanced proliferation when EsCsn5 was genetically knocked down, suggesting a critical role for CSN5 in the negative regulation of crab hemocyte proliferation. Moreover, EsCSN5 but not the EsCSN8 was demonstrated to negatively regulate the early G1 phase of the cell cycle by controlling the degradation of EsCyclin E through ubiquitination steps, rather than affecting its transcription. Furthermore, in the EsCyclin E-suppressed crab there was a significantly reduced survival rate and an up-regulated hemolymph bacterial concentration. Taken together, this study provides evidence demonstrating that invertebrate hemocytes down-regulate the expression of EsCsn5 upon bacterial challenge, thus promoting proliferation in an EsCyclin E-dependent manner in order to protect the crab from infection.

From the Editor’s Desk...

From the Editor’s Desk...

Hepatic stellate cells in physiology and pathology.

Hepatic stellate cells (HSCs) comprise a minor cell population in the liver but serve numerous critical functions in the normal liver and in response to injury. HSCs are primarily known for their activation upon liver injury and for producing the collagen-rich extracellular matrix in liver fibrosis. In the absence of liver injury, HSCs reside in a quiescent state, in which their main function appears to be the storage of retinoids or vitamin A-containing metabolites. Less appreciated functions of HSCs include amplifying the hepatic inflammatory response and expressing growth factors that are critical for liver development and both the initiation and termination of liver regeneration. Recent single-cell RNA sequencing studies have corroborated earlier studies indictaing that HSC activation involves a diverse array of phenotypic alterations and identified unique HSC populations. This review serves to highlight these many functions of HSCs, and to briefly describe the recent genetic tools that will help to thoroughly investigate the role of HSCs in hepatic physiology and pathology.

Abstract P3-09-16: Validating alternative splicing events between bulk and scRNA sequencing data: A bioinformatic approach

Abstract Background: As bulk and single-cell RNA (scRNA) sequencing studies and data continue to accrue to publicly accessible databases, bioinformatic tools continue being developed to analyze these datasets. The resulting pipelines often focus on either bulk or scRNA sequencing but rarely both. scRNA sequencing allows for in-depth characterization of transcription level events in distinct cell populations while bulk data offers a more global view, and for now, is more widely available in databases. Alternative splicing events in particular have been underexplored in a single-cell genomic architecture. The purpose of this study was to develop a bioinformatic pipeline to employ both bulk and single-cell mammary datasets to identify and validate alternative splicing events.Methods: Bulk fastq’s were utilized to identify AS events in luminal progenitor (LP) and mouse mammary stem cell (MSC) lineages in ovariectomized FVB mice that had been randomized into three treatment groups (SHAM [C], estradiol + progesterone [EP], EP + the selective progesterone receptor inhibitor telapristone acetate, TPA [EPT]). scRNA sequencing data from (Bach et al., 2017) was downloaded from the Gene Expression Omnibus (GEO) and utilized for validation of the AS events identified in the bulk data. To separate the data by progesterone level, the scRNA data was parsed into nulliparous, gestational, lactational, and post-involution groups. Further, to determine cell lineage level effects, the data was parsed into luminal and basal compartments using krt18 and krt5 expression. To accomplish the overall comparison between bulk and scRNA sequencing data, we developed a pipeline that would process and parse the scRNA data, identify the alternative splicing in bulk sequencing using rMATS and in the scRNA data using Outrigger (part of the Expedition suite). The resulting data from rMATS [bulk] LP/MSC: CvEP, CvEPT, & EPvEPT were filtered for events meeting statistical significance (p-value < 0.05, FDR < 0.01). After filtering, the events from each treatment comparison were then filtered so only events unique to LP or MSC CvEP, respectively, remain. The remaining significant and unique AS events are then compared to the Krt18-high and Krt5-high Outrigger results from each development stage based on genomic event coordinates (with a buffer +/- 20 base pairs).Results: 12 alternative splicing (AS) events were identified to be shared between the bulk and scRNA sequencing data in the context of increased progesterone exposure (EP and gestational, respectively) as well as cell lineage (LP and luminal respectively). Among the genes identified as having been alternatively spiced with an exact nucleotide match for the splice were Eif4a2, Pik3c2a, Brd4, Cdh1, and Enah. Conclusions: Alternative splicing events can be identified and validated between genomic sequencing methods, specifically bulk and single-cell RNA seq. We hypothesize that as scRNA-seq becomes more developed and quantification errors caused by short-read lengths fixed by full length scRNA seq methods currently in development, comparing data between single-cell and bulk may be made easier. However, we have shown that, when orthogonal methods for validation may not be feasible, events as specific as alternative splices can be validated using publicly available data and in-silico methods. Citation Format: Gannon Cottone, Benjamin T Spike, Elnaz Mirzaei Mehrabad, Seema A Khan, Susan Clare. Validating alternative splicing events between bulk and scRNA sequencing data: A bioinformatic approach [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-16.

Reversing T-cell Exhaustion in Cancer: Lessons Learned from PD-1/PD-L1 Immune Checkpoint Blockade.

Anti-PD-1/PD-L1 immune checkpoint blockade (ICB) therapy has revolutionized the treatment of many types of cancer over the past decade. The initial therapeutic hypothesis underlying the mechanism of anti-PD-1/PD-L1 ICB was built around the premise that it acts locally in the tumor, reversing the exhaustion of PD-1hiCD8+ T cells by "releasing the brakes." However, recent studies have provided unprecedented insight into the complexity within the CD8+ T-cell pool in the tumor microenvironment (TME). Single-cell RNA sequencing and epigenetic profiling studies have identified novel cell surface markers, revealing heterogeneity within CD8+ T-cell states classified as unique. Moreover, these studies highlighted that following ICB, CD8+ T-cell states within and outside the TME possess a differential capacity to respond, mobilize to the TME, and seed an effective antitumor immune response. In aggregate, these recent developments have led to a reevaluation of our understanding of both the underlying mechanisms and the sites of action of ICB therapy. Here, we discuss the evidence for the reversibility of CD8+ T-cell exhaustion after ICB treatment and its implication for the further development of cancer immunotherapy.

Development of an Inflammation-Related lncRNA-miRNA-mRNA Network Based on Competing Endogenous RNA in Breast Cancer at Single-Cell Resolution.

The role and mechanism of inflammation in breast cancer is unclear. This study aims to probe the relationship between inflammation and long non-coding RNAs (lncRNAs) and to stablish an inflammation-related competing endogenous RNA (ceRNA) network in breast cancer. Inflammation-related lncRNAs and target genes were screened based on the data from four single-cell RNA sequencing (scRNA-seq) studies and miRNAs were bioinformatically predicted according to ceRNA hypothesis. A series of in silico analyses were performed to construct an inflammation-related ceRNA network in breast cancer. Consequently, a total of seven inflammation-related lncRNAs were selected, after which LRRC75A-AS1 was identified as the most potential lncRNA in view of its expression and prognostic predictive value in breast cancer. Finally, an inflammation-related ceRNA network in breast cancer at the single cell level was established based on lncRNA LRRC75A-AS1, miR-3127-5p, miR-2114-3p, RPL36 and RPL27A mRNAs. Collectively, the lncRNA LRRC75A-AS1 and the LRRC75A-AS1-based on ceRNA network may exert crucial roles in modulating inflammation response during the initiation and progression of breast cancer.

FlexDotPlot: a universal and modular dot plot visualization tool for complex multifaceted data.

Dot plots are heatmap-like charts that provide a compact way to simultaneously display two quantitative information by means of dots of different sizes and colors. Despite the popularity of this visualization method, particularly in single-cell RNA-sequencing (scRNA-seq) studies, existing tools used to make dot plots are limited in terms of functionality and usability. We developed FlexDotPlot, an R package for generating dot plots from multifaceted data, including scRNA-seq data. It provides a universal and easy-to-use solution with a high versatility. An interactive R Shiny application is also available allowing non-R users to easily generate dot plots with several tunable parameters. Source code and detailed manual are available on CRAN (stable version) and at https://github.com/Simon-Leonard/FlexDotPlot (development version). Code to reproduce figures is available at https://github.com/Simon-Leonard/FlexDotPlot_paper. A Shiny app is available as a stand-alone application within the package. Supplementary data are available at Bioinformatics Advances online.