Marker Genes Research Articles

Tnfaip8 and Tipe2 Gene Deletion Ameliorates Immediate Proteoglycan Loss and Inflammatory Responses in the Injured Mouse Intervertebral Disc.

TNFAIP8 and TIPE2 belong to TNFa-induced protein 8 (TNFAIP8/TIPE) family. They control apoptosis and direct leukocyte migration. Nucleus pulposus cell loss is a hallmark of intervertebral disc degeneration in response to injury, and inflammation may cause pain. Here, we examined the effects of TNFAIP8/TIPE2 deficiency on the intervertebral discs in mice with these genes deleted. Tail intervertebral discs in Tnfaip8 or Tipe2 single and double knockout mice ( Tnfaip8 -/- , Tipe2 -/- , and Tnfaip8/Tipe2 dko) , and wild-type controls were injured. The spine motion segments were stained with safranin O to reveal proteoglycans. Macrophages were identified by immunostaining, and selected inflammatory marker and collagen gene expression was examined by Real Time PCR. The injured tail intervertebral discs of Tnfaip -/- , Tipe2 -/- , and Tnfaip8/Tipe2 dko mice all displayed higher levels of proteoglycans than wild-type controls. Fewer macrophages were found in the injured intervertebral discs of Tipe2 -/- and Tnfaip8/Tipe2 dko mice than wild type. Il6 , Adam8 , and Col1 gene expression was downregulated in the injured intervertebral discs of Tnfip8/Tipe2 dko mice. TNFAIP8 and TIPE2 loss of function ameliorated proteoglycan loss and inflammation in the injured intervertebral discs. They may serve as molecular targets to preserve disc structure and reduce inflammation.

Protective Effects of Hydroalcoholic Leaf Extract of Origanum Vulgare against Paraquat-induced Lung Toxicity in Rats: The Role of the Inflammation-related Markers and Oxidative Stress

Background:: The pesticide paraquat is hazardous to both humans and animals. Lung damage is a common complication of paraquat poisoning. Objective:: This research analyzes the preventive properties of Origanum vulgare leaf extract against paraquat-induced acute pulmonary toxicity and oxidative stress. Methods:: In this experiment, the rats were assigned to five distinct groups. Group 1 served as the control, group 2 got only 25 mg/kg bw paraquat, and groups 3, 4, and 5 received paraquat and Origanum vulgare leaf extract at dosages of 200, 400, and 800 mg/kg bw, respectively. In rats, inflammation- related indicators and oxidative stress were analyzed after therapy. Results:: Compared with the control group, the levels of the serum PC, MDA, and inflammatory markers such as TNF-α, IL-6, and IL-1β remarkably rose (p < 0.05) and the pulmonary contents of the SOD, CAT, and vitamin C greatly decreased (p < 0.05) in the paraquat-only group. Origanum vulgare leaf extract administration after paraquat exposure resulted in a substantial increase in lung SOD, CAT, and vitamin C levels as well as a significant reduction in MDA, PC, and lung inflammatory markers (TNF-α, IL-6, and IL-1β) gene expression relative to the paraquat-only group. Conclusion:: Origanum vulgare leaf extract might be supplied to lessen the toxicity of paraquat, based on the findings of this research, which indicated that Origanum vulgare leaf extract has antioxidant- enhancing properties and can improve the antioxidant defense system. other: Ethic number: IR. SKUMS. REC. 1395. 151.

Elongation factor 1A1 inhibition elicits changes in lipid droplet size, the bulk transcriptome, and cell type-associated gene expression in MASLD mouse liver.

Eukaryotic elongation factor 1A1 (EEF1A1), originally identified for its role in protein synthesis, has additional functions in diverse cellular processes. Of note, we previously discovered a role for EEF1A1 in hepatocyte lipotoxicity. We also demonstrated that a 2-wk intervention with the EEF1A1 inhibitor didemnin B (DB) (50 µg/kg) decreased liver steatosis in a mouse model of obesity and metabolic dysfunction-associated steatotic liver disease (MASLD) [129S6/SvEvTac mice fed Western diet (42% fat) for 26 wk]. Here, we further characterized the hepatic changes occurring in these mice by assessing lipid droplet (LD) size, bulk differential expression, and cell type-associated alterations in gene expression. Consistent with the previously demonstrated decrease in hepatic steatosis, we observed decreased median LD size in response to DB. Bulk RNA sequencing (RNA-Seq) followed by gene set enrichment analysis revealed alterations in pathways related to energy metabolism and proteostasis in DB-treated mouse livers. Deconvolution of bulk data identified decreased cell type association scores for cholangiocytes, mononuclear phagocytes, and mesenchymal cells in response to DB. Overrepresentation analyses of bulk data using cell type marker gene sets further identified hepatocytes and cholangiocytes as the primary contributors to bulk differential expression in response to DB. Thus, we show that chemical inhibition of EEF1A1 decreases hepatic LD size and decreases gene expression signatures associated with several liver cell types implicated in MASLD progression. Furthermore, changes in hepatic gene expression were primarily attributable to hepatocytes and cholangiocytes. This work demonstrates that EEF1A1 inhibition may be a viable strategy to target aspects of liver biology implicated in MASLD progression.NEW & NOTEWORTHY Chemical inhibition of EEF1A1 decreases hepatic lipid droplet size and decreases gene expression signatures associated with liver cell types that contribute to MASLD progression. Furthermore, changes in hepatic gene expression are primarily attributable to hepatocytes and cholangiocytes. This work highlights the therapeutic potential of targeting EEF1A1 in the setting of MASLD, and the utility of RNA-Seq deconvolution to reveal valuable information about tissue cell type composition and cell type-associated gene expression from bulk RNA-Seq data.

Single‐Cell RNA‐Sequencing of Soybean Reveals Transcriptional Changes and Antiviral Functions of GmGSTU23 and GmGSTU24 in Response to Soybean Mosaic Virus

ABSTRACTSoybean mosaic virus (SMV) stands as a prominent and widespread threat to soybean (Glycine max L. Merr.), the foremost legume crop globally. Attaining a thorough comprehension of the alterations in the transcriptional network of soybeans in response to SMV infection is imperative for a profound insight into the mechanisms of viral pathogenicity and host resistance. In this investigation, we isolated 50 294 protoplasts from the newly developed leaves of soybean plants subjected to both SMV infection and mock inoculation. Subsequently, we utilized single‐cell RNA sequencing (scRNA‐seq) to construct the transcriptional landscape at a single‐cell resolution. Nineteen distinct cell clusters were identified based on the transcriptomic profiles of scRNA‐seq. The annotation of three cell types—epidermal cells, mesophyll cells, and vascular cells—was established based on the expression of orthologs to reported marker genes in Arabidopsis thaliana. The differentially expressed genes between the SMV‐ and mock‐inoculated samples were analyzed for different cell types. Our investigation delved deeper into the tau class of glutathione S‐transferases (GSTUs), known for their significant contributions to plant responses against abiotic and biotic stress. A total of 57 GSTU genes were identified by a thorough genome‐wide investigation in the soybean genome G. max Wm82.a4.v1. Two specific candidates, GmGSTU23 and GmGSTU24, exhibited distinct upregulation in all three cell types in response to SMV infection, prompting their selection for further research. The transient overexpression of GmGSTU23 or GmGSTU24 in Nicotiana benthamiana resulted in the inhibition of SMV infection, indicating the antiviral function of soybean GSTU proteins.

The short-chain fatty acid receptors Gpr41/43 regulate bone mass by promoting adipogenic differentiation of mesenchymal stem cells

Bone is a dynamic tissue that is constantly remodeled throughout adult life. Recently, it has been shown that bone turnover decreases shortly after food consumption. This process has been linked to the fermentation of non-digestible food ingredients such as inulin by gut microbes, which results in the production of the short-chain fatty acids (SCFAs) acetate, propionate and butyrate. SCFAs exert various metabolic functions, which in part can be explained by activation of G protein-coupled receptors (Gpr) 41 and 43. However, the potential relevance of a SCFA-Gpr41/43 signaling axis for bone metabolism has not been established. The aim of our study is to investigate the role of Gpr41/43 in bone metabolism and osteogenic differentiation of mesenchymal stem cells. For this purpose, we analyzed the skeletal phenotype of wild type controls (WT) and Gpr41/43 double knockout (Gpr41/43 dKO) mice fed either a chow or an inulin-enriched diet. In addition, we isolated bone marrow derived mesenchymal stem cells from WT and Gpr41/43 dKO mice and differentiated them into osteoblasts in the absence or presence of acetate. MicroCT scanning of femoral bones of Gpr41/43 dKO mice revealed a significant increase of trabecular bone volume and trabecular compared to WT controls. Treatment of WT bone marrow-derived osteoblasts with acetate resulted in decreased mineralization and substantial downregulation of bone formation markers such as Phex, Ptgs2 and Col1a1. Notably, this effect was strongly attenuated in differentiated osteoblasts lacking Gpr41/43. Inversely, acetate supplementation resulted in higher levels of adipocyte marker genes including Pparg, Lpl and Adipoq in bone marrow-derived cells from WT mice, an effect blunted in differentiated cells isolated from Gpr41/43 dKO mice. Overall, these data indicate that acetate regulates bone architecture via SCFA-Gpr41/43 signaling by modulating the osteogenic versus adipogenic differentiation of mesenchymal stem cells.

Transcriptional Patterns in Stages of Alzheimer's Disease are Cell Type Specific and Partially Converge with the Effects of Alcohol Use Disorder in Humans

Advances in single cell technologies have led to the discovery and characterization of new brain cell-types, that in turn lead to a better understanding of the pathogenesis of Alzheimer’s disease (AD). Here, we present a detailed analysis of single nucleus (sn)RNA-seq data for three stages of AD from middle temporal gyrus (MTG) and compare it with snRNA-seq data from the prefrontal cortices from individuals with alcohol use disorder (AUD). We observed a significant decrease in both inhibitory and excitatory neurons, in general agreement with previous reports. We observed several cell-type specific gene expressions and pathways dysregulations that delineate AD stages. Endothelial and VLMCs (vascular leptomeningeal cells) showed the greatest degree of gene expression changes. Cell-type specific evidence of neurodegeneration was seen in multiple neuronal cell-types particularly in SST (somatostatin) and L5 ET (layer-5 extra-telencephalic) neurons, among others. Evidence of inflammatory responses were seen in non-neuronal cells, particularly in intermediate and advanced AD. We observed common perturbations in AD and AUD, particularly in pathways, like transcription, translation, apoptosis, autophagy, calcium signaling, neuroinflammation, and phosphorylation, that imply shared transcriptional pathogenic mechanisms and support a role for excessive alcohol intake in AD progression. Major AUD gene markers form and perturb a network of genes significantly associated with intermediate and advanced AD. Master regulator analysis from AUD gene markers revealed significant correlation with advanced AD of transcription factors that have implications in intellectual disability, neuroinflammation, and other neurodegenerative conditions, further suggesting a shared nexus of transcriptional changes between AD and AUD.Significance StatementThis study holds significant implications for understanding the intricate molecular landscape of Alzheimer's disease (AD) and its intersection with alcohol use disorder (AUD). By profiling transcriptional changes in the neocortex associated with AD progression and comparing them with those in AUD, we shed light on shared gene expression and pathway dysregulations between the two conditions. Our findings corroborate prior research on neuronal depletion and highlight novel insights into cell-type-specific gene expression patterns in AD stages. Moreover, the identification of common genetic signatures suggests a potential exacerbating effect of AUD on AD progression. This comprehensive analysis not only deepens our understanding of AD pathology but also underscores the importance of considering AUD as a potential risk factor for accelerating AD onset or severity.

Unraveling Morphological, Physiological, and Transcriptomic Alterations Underlying the Formation of Little Leaves in Phytoplasma-Infected Sweet Cherry Trees.

Phytoplasmas are minute phytopathogenic bacteria that induce excessive vegetative growth, known as witches'-broom (WB), in many infected plant species during the later stages of infection. The WB structure is characterized by densely clustered little (small) leaves, which are frequently accompanied by chlorosis (yellowing). The mechanisms behind the formation of little leaves within WB structures (LL-WB) are poorly understood. To address this gap, the LL-WB formation was extensively studied using sweet cherry virescence (SCV) phytoplasma-infected sweet cherry plants. Based on morphological examinations, signs of premature leaf senescence were observed in LL-WB samples, including reduced leaf size, chlorosis, and alterations in shape. Subsequent physiological analyses indicated decreased sucrose and glucose levels and changes in hormone concentrations in LL-WB samples. Additionally, the transcriptomic analysis revealed impaired ribosome biogenesis and DNA replication. As an essential process in protein production, the compromised ribosome biogenesis and the inhibited DNA replication led to cell cycle arrest, thus affecting leaf morphogenesis and further plant development. Moreover, the expression of marker genes involved in premature leaf senescence was significantly altered. These results indicate a complicated interplay between the development of leaves, premature leaf senescence, and the pathogen-induced stress responses in SCV phytoplasma-infected sweet cherry trees. The results of this study provide insight into understanding the underlying molecular mechanisms driving the formation of little leaves and interactions between plants and pathogens. The findings might help control phytoplasma diseases in sweet cherry cultivation.

Deconvolution from bulk gene expression by leveraging sample-wise and gene-wise similarities and single-cell RNA-Seq data

BackgroundThe widely adopted bulk RNA-seq measures the gene expression average of cells, masking cell type heterogeneity, which confounds downstream analyses. Therefore, identifying the cellular composition and cell type-specific gene expression profiles (GEPs) facilitates the study of the underlying mechanisms of various biological processes. Although single-cell RNA-seq focuses on cell type heterogeneity in gene expression, it requires specialized and expensive resources and currently is not practical for a large number of samples or a routine clinical setting. Recently, computational deconvolution methodologies have been developed, while many of them only estimate cell type composition or cell type-specific GEPs by requiring the other as input. The development of more accurate deconvolution methods to infer cell type abundance and cell type-specific GEPs is still essential.ResultsWe propose a new deconvolution algorithm, DSSC, which infers cell type-specific gene expression and cell type proportions of heterogeneous samples simultaneously by leveraging gene-gene and sample-sample similarities in bulk expression and single-cell RNA-seq data. Through comparisons with the other existing methods, we demonstrate that DSSC is effective in inferring both cell type proportions and cell type-specific GEPs across simulated pseudo-bulk data (including intra-dataset and inter-dataset simulations) and experimental bulk data (including mixture data and real experimental data). DSSC shows robustness to the change of marker gene number and sample size and also has cost and time efficiencies.ConclusionsDSSC provides a practical and promising alternative to the experimental techniques to characterize cellular composition and heterogeneity in the gene expression of heterogeneous samples.

3D printed gelatin methacryloyl hydrogels for perfusion culture of human trabecular meshwork cells and glaucoma studies.

Glaucoma, a progressive eye disease leading to irreversible blindness, currently affects over 70 million people globally. Elevated intraocular pressure (IOP) is implicated in its development. IOP is carefully regulated by the trabecular meshwork (TM). However, studying TM behavior has been limited to traditional tissue culture studies or costly ex vivo cultures of animal and donor eyes. Developing novel functional TM models could enhance cell/tissue behavior understanding and aid therapeutic development for glaucoma. In this study, we 3D printed a simplified and reproducible model of the human TM (hTM) and studied hTM cell behavior under static and dynamic cultures. Gelatin Methacryloyl bioinks proved suitable for printing with viable and proliferative hTM cells expressing crucial marker genes in response to glucocorticoid induction. This, to our knowledge, is the first functional 3D printed hTM model and aims to facilitate TM research. Moreover, this easily reproducible model could also be applicable in the study of numerous other cell types throughout the body.

PPARβ/δ Agonist GW0742 Modulates Microglial and Astroglial Gene Expression in a Rat Model of Temporal Lobe Epilepsy

The role of astroglial and microglial cells in the pathogenesis of epilepsy is currently under active investigation. It has been proposed that the activity of these cells may be regulated by the agonists of peroxisome proliferator-activated nuclear receptors (PPARs). This study investigated the effects of a seven-day treatment with the PPAR β/δ agonist GW0742 (Fitorine, 5 mg/kg/day) on the behavior and gene expression of the astroglial and microglial proteins involved in the regulation of epileptogenesis in the rat brain within a lithium–pilocarpine model of temporal lobe epilepsy (TLE). TLE resulted in decreased social and increased locomotor activity in the rats, increased expression of astro- and microglial activation marker genes (Gfap, Aif1), pro- and anti-inflammatory cytokine genes (Tnfa, Il1b, Il1rn), and altered expression of other microglial (Nlrp3, Arg1) and astroglial (Lcn2, S100a10) genes in the dorsal hippocampus and cerebral cortex. GW0742 attenuated, but did not completely block, some of these impairments. Specifically, the treatment affected Gfap gene expression in the dorsal hippocampus and Aif1 gene expression in the cortex. The GW0742 injections attenuated the TLE-specific enhancement of Nlrp3 and Il1rn gene expression in the cortex. These results suggest that GW0742 may affect the expression of some genes involved in the regulation of epileptogenesis.

Single-Cell Transcriptomics Applied in Plants

Single-cell RNA sequencing (scRNA-seq) is a high-tech method for characterizing the expression patterns of heterogeneous cells in the same tissue and has changed our evaluation of biological systems by increasing the number of individual cells analyzed. However, the full potential of scRNA-seq, particularly in plant science, has not yet been elucidated. To explore the utilization of scRNA-seq technology in plants, we firstly conducted a comprehensive review of significant scRNA-seq findings in the past few years. Secondly, we introduced the research and applications of scRNA-seq technology to plant tissues in recent years, primarily focusing on model plants, crops, and wood. We then offered five databases that could facilitate the identification of distinct expression marker genes for various cell types. Finally, we analyzed the potential problems, challenges, and directions for applying scRNA-seq in plants, with the aim of providing a theoretical foundation for the better use of this technique in future plant research.

Single-Neuron Labeling in Drosophila Using Multicolor FLP-Out.

Neurons exhibit some of the most striking examples of morphological diversity of any cell type. Thus, when studying neurons, the morphology of each neuron must be considered individually. However, neurons densely populate the central nervous system (CNS), making it difficult to ascertain fine morphological features due to a lack of spatial resolution. In Drosophila, this problem can be partially resolved by using driver lines that express the yeast transcription factor GAL4 in subsets of neurons. GAL4 can activate the expression of other introduced genetic elements such as genes for fluorescent proteins or other markers under the control of the GAL4 upstream activation sequences (UAS effectors). However, even highly specific GAL4 lines often label sets of potentially morphologically heterogeneous neurons. Here, we describe a protocol for using the multicolor flip-out (MCFO) technique in Drosophila melanogaster to stochastically label individual neurons within a GAL4 expression pattern. MCFO relies on the binary GAL4/UAS expression system in Drosophila but adds additional control for how densely the neurons within a GAL4 expression pattern are labeled via user-controlled heat shock. Specifically, three discrete UAS effector elements containing the sequences for unique epitope tags (FLAG, HA, and V5) linked to a gene for nonfluorescent GFP can be independently expressed under the control of GAL4 only when a transcriptional stop sequence in the UAS promoter sequence has been removed by heat shock-induced recombination. This effectively labels multiple individual neurons with either one or a combination of epitope tags that can be spectrally resolved with immunofluorescence. The MCFO technique is ideal for researchers who want to determine morphological features of CNS neurons in wild-type or mutant backgrounds.

Deciphering avian hematopoietic stem cells by surface marker screening and gene expression profiling

BackgroundAvian species have played a pivotal role in developmental hematopoiesis research, leading to numerous critical discoveries. Avian influenza, particularly the H5N1 strain, poses a significant threat to poultry and has zoonotic potential for humans. Infections often result in abnormal hematologic profiles, highlighting the complex interplay between avian diseases and hematopoiesis. Many avian diseases can suppress immune cells in the bone marrow (BM), impacting immune responses. Studying hematopoietic stem cells (HSCs) in avian BM is crucial for understanding these processes and developing effective vaccines and protection strategies for both avian and human health. MethodsThis study adapted methods from mouse studies to isolate avian HSCs as Lineage-negative (Lin-) cells. These isolated cells were further identified as Lin-Sca1+c-Kit+ (LSK) and were found to be more prevalent than in control groups. RT-PCR analyses were conducted, showing that genes like MEIS1 and TSC1 were upregulated, while SIRT1, FOXO1, and AHR were downregulated in these stem cells. Screening for LSK markers revealed ten unique surface antigens in the Sca1+c-Kit+ cell populations, including highly enriched antigens such as CD178, CD227, and CD184. Additionally, studies on quail HSCs demonstrated that similar labeling techniques were effective in quail BM. ResultsThe research demonstrated that the identification of avian HSC-specific surface antigens provides valuable insights into the pathogenesis of avian influenza and other diseases, enhancing our understanding of how these diseases suppress HSC function. Notably, the upregulation of MEIS1 and TSC1 genes in LSK cells underscores their critical roles in regulating hematopoietic processes. Conversely, the downregulation of SIRT1, FOXO1, and AHR genes provides important clues about their roles in differentiation and immune response mechanisms. DiscussionThe findings of this study deepen our understanding of the effects of avian diseases on the immune system by identifying surface markers specific to avian HSCs. The suppression of HSC function by pathogens such as influenza highlights the importance of understanding these cells in developing targeted vaccines. These results represent a significant step towards improving global health security by mitigating risks associated with avian pathogens.

Chromosomal-level reference genome assembly of muskox (Ovibos moschatus) from Banks Island in the Canadian Arctic, a resource for conservation genomics

The muskox (Ovibos moschatus), an integral component and iconic symbol of arctic biocultural diversity, is under threat by rapid environmental disruptions from climate change. We report a chromosomal-level haploid genome assembly of a muskox from Banks Island in the Canadian Arctic Archipelago. The assembly has a contig N50 of 44.7 Mbp, a scaffold N50 of 112.3 Mbp, a complete representation (100%) of the BUSCO v5.2.2 set of 9225 mammalian marker genes and is anchored to the 24 chromosomes of the muskox. Tabulation of heterozygous single nucleotide variants in our specimen revealed a very low level of genetic diversity, which is consistent with recent reports of the muskox having the lowest genome-wide heterozygosity among the ungulates. While muskox populations are currently showing no overt signs of inbreeding depression, environmental disruptions are expected to strain the genomic resilience of the species. One notable impact of rapid climate change in the Arctic is the spread of emerging infectious and parasitic diseases in the muskox, as exemplified by the range expansion of muskox lungworms, and the recent fatal outbreaks of Erysipelothrix rhusiopathiae, a pathogen normally associated with domestic swine and poultry. As a genomics resource for conservation management of the muskox against existing and emerging disease modalities, we annotated the genes of the major histocompatibility complex on chromosome 2 and performed an initial assessment of the genetic diversity of this complex. This resource is further supported by the annotation of the principal genes of the innate immunity system, genes that are rapidly evolving and under positive selection in the muskox, genes associated with environmental adaptations, and the genes associated with socioeconomic benefits for Arctic communities such as wool (qiviut) attributes. These annotations will benefit muskox management and conservation.

Histological differences related to autophagy in the minor salivary gland between primary and secondary types of Sjögren’s syndrome

Some forms of Sjögren’s syndrome (SS) follow a clinical course accompanied by systemic symptoms caused by lymphocyte infiltration and proliferation in the liver, kidneys, and other organs. To better understand the clinical outcomes of SS, here we used minor salivary gland tissues from patients and examine their molecular, biological, and pathological characteristics. A retrospective study was performed, combining clinical data and formalin-fixed paraffin-embedded (FFPE) samples from female patients over 60 years of age who underwent biopsies at Okayama University Hospital. We employed direct digital RNA counting with nCounter® and multiplex immunofluorescence analysis with a PhenoCycler™ on the labial gland biopsies. We compared FFPE samples from SS patients who presented with other connective tissue diseases (secondary SS) with those from stable SS patients with symptoms restricted to the exocrine glands (primary SS). Secondary SS tissues showed enhanced epithelial damage and lymphocytic infiltration accompanied by elevated expression of autophagy marker genes in the immune cells of the labial glands. The close intercellular distance between helper T cells and B cells positive for autophagy-associated molecules suggests accelerated autophagy in these lymphocytes and potential B cell activation by helper T cells. These findings indicate that examination of FFPE samples from labial gland biopsies can be an effective tool for evaluating molecular histological differences between secondary and primary SS through multiplexed analysis of gene expression and tissue imaging.

Abstract B052: Single-cell and spatial analysis of the immune landscape unveils a subset of potentially tumor-reactive T cells in patients with localized PDAC

Abstract Even in its localized stages, pancreatic ductal adenocarcinoma (PDAC) is virtually incurable, characterized by early dissemination and disease recurrence. The liver is the most common site of metastasis, and in most cases hepatic recurrences drive survival outcomes. This underscores the critical need to target and prevent liver metastases. We leveraged a novel biobanking strategy to perform paired single-cell RNA (scRNA-seq) and TCR sequencing (scTCR-seq) to profile multisite immunity in patients with localized PDAC across the tumor, peripheral blood, and premetastatic liver. This analysis of three patients included three tumor, three blood, and nine liver (three per patient from separate segments) samples, and generated 85,748 cells for downstream analysis. We evaluated populations of tumor-expanded T cells as potential indicators of antitumoral immunity, and utilized validated computational approaches to identify tumor-reactive T cells based on key markers of dysfunction and exhaustion. Gene set enrichment analysis was performed on tumor infiltrating lymphocytes (TILs) using signatures specific to tumor-reactive CD4 and CD8 T cells (“NeoTCR4” and “NeoTCR8,” respectively), and scored these using the AUCell package in R. Overall, 153 CD4 and 22 CD8 T cells fit candidate NeoTCR4 and NeoTCR8 signature states, representing 5.6% and 0.8% of the total TIL population respectively. These nominated T cells expressed key marker genes associated with tumor specificity such as CXCL13, TIGIT, PDCD1, ENTPD1, and TOX. Thirteen of these predicted tumor-reactive T cell clones identified within the tumor were also detected within our liver samples, suggestive of a potential antitumoral immune response in the premetastatic liver space. We next performed 10x Xenium in situ spatial transcriptomic analysis of primary tumor, normal adjacent pancreas, and premetastatic liver from two of these patients, using tissue microarrays to incorporate replicate 2mm core biopsies from each of these sites. A custom designed gene panel, guided by our scRNA-seq dataset, targeted tumor-specific mutations (e.g. KRAS G12V, G12D, G12R, and Q61) and immune related genes with a specific focus on markers of T cell activity and exhaustion. Leveraging these latter marker genes, which represent a subset of the NeoTCR4 and NeoTCR8 signatures, we identified a population of exhausted TILs within the tumor microenvironment, representing potentially tumor-reactive T cells whose interactions can now be explored at spatial resolution. Future directions are aimed at experimentally validating the tumor reactivity of the predicted neoantigen-specific TILs within our dataset and characterizing the immune neighborhoods that exist in both the tumor and liver of PDAC patients. This work establishes exciting opportunities for novel T-cell therapy in the treatment of PDAC, and we have already implemented these experimental findings in a forthcoming clinical trial investigating the applicability of this predictive signature score for the generation of TCR-engineered lymphocyte therapy in patients with PDAC. Citation Format: Elishama N Kanu, Ashley A Fletcher, Sri Krishna, Frank J Lowery, Haotian Zhuang, Ethan S Agritelley, Jiayin Bao, Austin M Eckhoff, Karrie Comatas, Tao Wang, Bin-Jin Hwang, Michael E Lidsky, Sabino Zani, Dan G Blazer III, Peter J Allen, Nicholas D Klemen, Zhicheng Ji, Daniel P Nussbaum, Erika J Crosby. Single-cell and spatial analysis of the immune landscape unveils a subset of potentially tumor-reactive T cells in patients with localized PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B052.

Abstract A081: Single-cell transcriptomic analysis of organoids derived from normal pancreata reveals epithelial cell heterogeneity and expression of PanIN-associated genes

Abstract Pancreatic intraepithelial neoplasia (PanIN) lesions are the microscopic pre-malignant precursor to pancreatic ductal adenocarcinoma (PDAC). Animal models have demonstrated that PanINs arise from acinar cells which have undergone acinar-to-ductal metaplasia (ADM), but the exact molecular mechanisms which govern this transformation remain poorly understood. Novel in vitro models of PanIN genesis are necessary to better characterize pancreatic carcinogenesis and identify clinically actionable biomarkers of early disease. Organoids cultured from normal human pancreata have generated promise in this regard, but the cellular identity and malignant potential of their component cells have not been comprehensively characterized. We performed single-cell RNA sequencing (10X Genomics) on 6 normal pancreatic organoid lines maintained in culture for 3 weeks as well as their parent pancreata obtained from donors with no history of PDAC or other exocrine pancreatic pathology. Integration of whole pancreatic and organoid data using the R package Seurat generated a UMAP with clearly defined “ductal” and “acinar” clusters based on marker gene expression. The majority of organoid cells (47.3-81.5%) colocalized to the “ductal” cluster, and expressed SOX9, KRT19, and PDX1. However, a subset of cells (1.0-33.3%) present in each organoid sample aligned with the “acinar” cluster and expressed PRSS1, PRSS3, REG3A, MUC1, and SPINK1. In accordance with prior reports, organoid cells did not meaningfully express the acinar/endocrine markers PTF1A, CPA1, CHGA, or INS. Further examination of the “acinar” organoid cells revealed heightened expression of CLDN18, TFF1, TFF2, CTSE, CA2, and LYZ, genes associated with PanIN in prior spatial RNA sequencing studies. Other genes significantly enriched in this “acinar” organoid cluster include MUC5AC, a known PanIN marker, OLFM4, a tumor suppressor implicated in intestinal carcinogenesis, and the chemokine CXCL17, which is expressed in cells undergoing ADM. Overall, this data suggests that organoids derived from normal pancreata represent a heterogeneous population of epithelial cells with both acinar and ductal features. Furthermore, these organoids may represent an in vitro model of ADM and/or early PanIN formation, though additional functional mechanistic studies are required for confirmation. Citation Format: Alexander W Bray, Eileen S Carpenter, Timothy L Frankel, Marina Pasca di Magliano. Single-cell transcriptomic analysis of organoids derived from normal pancreata reveals epithelial cell heterogeneity and expression of PanIN-associated genes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A081.

Single-cell profiling uncovers proliferative cells as key determinants of survival outcomes in lower-grade glioma patients

Lower-grade gliomas (LGGs), despite their generally indolent clinical course, are characterized by invasive growth patterns and genetic heterogeneity, which can lead to malignant transformation, underscoring the need for improved prognostic markers and therapeutic strategies. This study utilized single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq to identify a novel cell type, referred to as "Prol," characterized by increased proliferation and linked to a poor prognosis in patients with LGG, particularly under the context of immunotherapy interventions. A signature, termed the Prol signature, was constructed based on marker genes specific to the Prol cell type, utilizing an artificial intelligence (AI) network that integrates traditional regression, machine learning, and deep learning algorithms. This signature demonstrated enhanced predictive accuracy for LGG prognosis compared to existing models and showed pan-cancer prognostic potential. The mRNA expression of the key gene PTTG1 from the Prol signature was further validated through quantitative reverse transcription polymerase chain reaction (qRT-PCR). Our findings not only provide novel insights into the molecular and cellular mechanisms of LGG but also offer a promising avenue for the development of targeted biomarkers and therapeutic interventions.

PSV-6 Effect of maternal undernutrition on gene expression related to immune system in spleen of Japanese Black (Wagyu) fetuses

Abstract Underdevelopment in the fetal period increases the risk of disease in the neonatal period, and there are concerns about the effects on later growth and fattening. Previous studies have indicated that poor nutrition during gestation affects not only the development of skeletal muscle and liver, but also the formation of the thymus gland and spleen, which are involved in immunity. The purpose of this study was to investigate the effects of maternal undernutrition on gene expression related to the immunity of the spleen. Multiparous Wagyu cattle (n = 12) were randomly assigned to two nutrition groups: the control group (CON: 120% of Japanese feeding standard for beef cattle; n = 6) and the maternal undernutrition group (MUN: 60% of Japanese feeding standard for beef cattle; n = 6) from conception to d 260 of gestation. Fetuses (n = 12) were harvested from the maternal cows and fetal spleens were collected. Total RNA was extracted from each spleen to synthesize cDNA. The quantitative RT-PCR (qRT-PCR) analysis was carried out to investigate the expression of 17 target genes [CD4, CD8a, CD8b, CD19, CD21, CD79a, CD205, interferon (IFN) -γ, NFk-β, toll-like receptor (TLR) 4, interleukin (IL) 2, IL4, IL6, IL7, IL10, IL18, and IFI16) related to the immune system. Ribosomal protein S18 was used as an internal control. The specificity of PCR products was confirmed by analyzing the dissociation curves. Data were analyzed by student t-test or Welch’s t-test after the Smirnov-Grubbs test and F-test to remove outliers and compare variances. The expression of CD8a (P < 0.1) and CD8b (P < 0.1) related to Cytotoxic T cell tended to be less in MUN than in CON (Figure 1). Other immunological cell marker genes expression (CD4, CD19, CD21, CD79a, CD205) did not show any significant differences between CON and MUN (P > 0.11). In genes related to immunological mechanisms (IFN-γ, NFk-β, TLR4, IL2, IL4, IL6, IL7, IL10, IL18, and IFI16), the expression of NFk-β was lless in MUN than in CON (P < 0.05). NFk-β was reported to regulate more than 400 genes including immune function. In addition, the expression of IL10 was less in MUN than in CON (P < 0.05). The cytokine of IL-10 has functions to suppress over-activated cytotoxic immunity by CD8+ T cell. These results might suggest that maternal undernutrition induces cytotoxic immune deactivation through NFk-β expression decline in the fetal spleen.

StarTracer is an accelerated approach for precise marker gene identification in single-cell RNA-Seq analysis

Revealing the heterogeneity among tissues is the greatest advantage of single-cell-sequencing. Marker genes not only act as the key to correctly identify cell types, but also the bio-markers for cell-status under certain experimental imputations. Current analysis methods such as Seurat and Monocle employ algorithms which compares one cluster to all the rest and select markers according to statistical tests. This pattern brings redundant calculations and thus, results in low calculation efficiency, specificity and accuracy. To address these issues, we introduce starTracer, a novel algorithm designed to enhance the efficiency, specificity and accuracy of marker gene identification in single-cell RNA-seq data analysis. starTracer operates as an independent pipeline, which exhibits great flexibility by accepting multiple input file types. The primary output is a marker matrix, where genes are sorted by the potential to function as markers, with those exhibiting the greatest potential positioned at the top. The speed improvement ranges by 2 ~ 3 orders of magnitude compared to Seurat, as observed across three independent datasets with lower false positive rate as observed in a simulated testing dataset with ground-truth. It’s worth noting that starTracer exhibits increasing speed improvement with larger data volumes. It also excels in identifying markers in smaller clusters. These advantages solidify starTracer as an important tool for single-cell RNA-seq data, merging robust accuracy with exceptional speed.