Differences In Cellular Composition Research Articles

Abstract B010: Improving pancreatic cancer image classification with transfer learning: CD45 vs. PANCK

Abstract Pancreatic ductal adenocarcinoma (PDAC) is known for its high aggressiveness and is the fourth most common cause of death of cancer-related death in the United States. It is highly malignant and often goes undiagnosed until advanced stages due to its late-onset symptoms, resulting in poor prognosis. Using quantitative analyses of histological features, such as the expression of prognostic biomarker, can provide valuable insight to guide treatment decisions, monitor disease progression and predict outcomes. Previous researchers have found that CD45 and PANCK play a huge role in characterizing the tumor microenvironment of PDAC. One study using single-cell RNA sequencing compared the microenvironments of healthy pancreata and pancreatic tumors, revealing differences in cellular composition. Tumor samples showed a higher proportion of immune cells, including CD45+ cells, compared to heathy samples and utilized PANCK to differentiate between epithelial and non-epithelial cells. This study aims to enhance the accuracy of classifying pancreatic cancer pathological images based on prognostic markers using transfer learning techniques. We curated and pre-processed a dataset of pancreatic cancer pathological images with different prognostic markers. Using the pre-trained convolutional neural network (CNN) model VGG16, we fine-tuned it with our dataset to better differentiate between two prognostic markers, CD45 and PANCK. The model's performance was evaluated using a similarity matrix to illustrate distinctions among the two prognostic markers. Our results demonstrate that the transfer learning approach significantly improves classification accuracy, achieving confusion matrix validation results of 90% between CD45 and PANCK prognostic markers. SSIM values were relatively low, highlighting the ability of our model to differentiate between the two prognostic markers. The low SSIM value also shows variability within the two prognostic markers, indicating heterogeneity in structural changes due to pancreatic cancer. These findings indicate that transfer learning can be a powerful tool for improving the classification of pathological images and that there is notable variability in the structural features associated with different prognostic markers in pancreatic cancer. Citation Format: Miracle Thomas. Improving pancreatic cancer image classification with transfer learning: CD45 vs. PANCK [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B010.

Single-cell transcriptomics link gene expression signatures to clinicopathological features of gonadotroph and lactotroph PitNET.

Pituitary neuroendocrine tumors (PitNET) are among the most common intracranial tumors. Despite a frequent benign course, aggressive behavior can occur. Tumor behavior is known to be under the influence of the tumor microenvironment (TME). However, the relationship between TME cells and aggressive tumor behavior has not been adequately explored in PitNET. We performed differential expression analysis as well as gene expression program identification based on single-cell RNA sequencing to comparatively characterize the transcriptome of seven gonadotroph and three lactotroph PitNET and correlate it with clinical features using bulk RNA-seq data from an independent cohort of 134 PitNET. Tumor immune infiltration was quantified via immunostaining on tissue sections of gonadotroph and lactotroph PitNET. In lactotroph PitNET, we detect a highly proliferative gene profile with significantly increased expression levels in aggressively growing tumors within bulk RNA-seq data of an independent cohort of 134 PitNET samples. We also report high intratumoral heterogeneity in gonadotroph PitNET (GoPN) and lactotroph PitNET (LaPN) and identify signatures of epithelial, endocrine, and immunological gene networks in both subtypes. A comparison of their TME composition shows enrichment of SPP1+ macrophages and CD4+ T cells in GoPN, as well as enrichment of CD4/CD8 double-negative T cells (DN) and natural killer cells (NK) in LaPN. Also notable is the presence of proliferative lymphocytes, the occurrence of which positively correlates with more aggressive tumor behavior in the bulk RNA-seq cohort. However, increased CD8+ T and NK cell abundances correlate significantly with reduced aggressiveness indicating potential anti-tumoral effects. Our study expands the knowledge of the differences in cellular composition of gonadotroph and lactotroph PitNET subtypes. It lays the foundation for further studies on the influence of lymphoid cells on the variable aggressive behavior of PitNET. Regarding the treatment of drug-resistant lactotroph PitNET, proliferative lymphocytes, CD8+ T, and NK cells could represent potentially valuable targets for developing new cancer immunotherapies.

Abstract 4137296: Single-cell Atlas of Human Epicardial Adipose Tissue Reveals Aging IL4R + Naive B cell Contributing To Atrial Fibrillation

Background: Numerous recent evidence suggested a role of epicardial adipose tissue (EAT), the visceral fat depot of the heart, in the development of Atrial Fibrillation (AF). However, the cellular and molecular mechanisms underlying the association remain elusive. Methods: We performed single-nucleus RNA sequencing on eleven EAT samples collected from 2 groups of subjects: patients with nonvalvular AF undergoing coronary bypass surgery for severe CAD (n=7), and a control group of patients without concomitant AF (n=4). Comparative analyses were performed between the groups, including cellular compositional analysis, cell type–resolved transcriptomic changes, and functional analysis. Results: Unsupervised clustering of 92734 nuclei identified 11 clusters, encompassing all known cell types in the adipose tissue. Pseudobulk differential expression analysis between the AF and control groups revealed pronounced differences in a select subset of genes located on the X and Y chromosomes, including XIST and TSIX. Subsequent pathway analysis identified that the most significantly impacted pathways were those associated with immune cell functions. Meanwhile, differences in cellular composition and transcriptomic profiles, particularly concerning B cells, were observed between normal and AF conditions, highlighting distinct immune cell dynamics. Comparison of pathways across different B cell states revealed the enrichment of distinct pathways within each state, including those involved in cellular senescence, antigen processing and presentation, and programmed cell death signatures. Notably, aging IL4R + naive B cell and inflammatory ITGB1 + memory B cell were identified as important regulators potentially involved in functional changes of EAT and AF pathogenesis. Conclusions: We built a complete single-nucleus transcriptomic atlas of human EAT in normal and AF conditions. Our study lays the foundation for developing novel therapeutic strategies for treating AF by targeting and modifying B cell functions within EAT.

Focal adhesion kinase regulates tendon cell mechanoresponse and physiological tendon development.

Tendons enable locomotion by transmitting high tensile mechanical forces between muscle and bone via their dense extracellular matrix (ECM). The application of extrinsic mechanical stimuli via muscle contraction is necessary to regulate healthy tendon function. Specifically, applied physiological levels of mechanical loading elicit an anabolic tendon cell response, while decreased mechanical loading evokes a degradative tendon state. Although the tendon response to mechanical stimuli has implications in disease pathogenesis and clinical treatment strategies, the cell signaling mechanisms by which tendon cells sense and respond to mechanical stimuli within the native tendon ECM remain largely unknown. Therefore, we explored the role of cell-ECM adhesions in regulating tendon cell mechanotransduction by perturbing the genetic expression and signaling activity of focal adhesion kinase (FAK) through both invitro and invivo approaches. We determined that FAK regulates tendon cell spreading behavior and focal adhesion morphology, nuclear deformation in response to applied mechanical strain, and mechanosensitive gene expression. In addition, our data reveal that FAK signaling plays an essential role in invivo tendon development and postnatal growth, as FAK-knockout mouse tendons demonstrated reduced tendon size, altered mechanical properties, differences in cellular composition, and reduced maturity of the deposited ECM. These data provide a foundational understanding of the role of FAK signaling as a critical regulator of insitu tendon cell mechanotransduction. Importantly, an increased understanding of tendon cell mechanotransductive mechanisms may inform clinical practice as well as lead to the discovery of diagnostic and/or therapeutic molecular targets.

Self-organizing ovarian somatic organoids preserve cellular heterogeneity and reveal cellular contributions to ovarian aging.

Ovarian somatic cells are essential for reproductive function, but no existing ex vivo models recapitulate the cellular heterogeneity or interactions within this compartment. We engineered a novel ovarian somatic organoid model by culturing a stroma-enriched fraction of mouse ovaries in scaffold-free agarose micromolds. Ovarian somatic organoids self-organized, maintained diverse cell populations, produced extracellular matrix, and secreted hormones. Organoids generated from reproductively old mice exhibited reduced aggregation and growth compared to young counterparts, as well as differences in cellular composition. Interestingly, matrix fibroblasts from old mice demonstrated upregulation of pathways associated with the actin cytoskeleton and downregulation of cell adhesion pathways, indicative of increased cellular stiffness which may impair organoid aggregation. Cellular morphology, which is regulated by the cytoskeleton, significantly changed with age and in response to actin depolymerization. Moreover, actin depolymerization rescued age-associated organoid aggregation deficiency. Overall, ovarian somatic organoids have advanced fundamental knowledge of cellular contributions to ovarian aging.

Electrophysiological activity pattern of mouse hippocampal CA1 and dentate gyrus under isoflurane anesthesia.

General anesthesia can impact a patient's memory and cognition by influencing hippocampal function. The CA1 and dentate gyrus (DG), serving as the primary efferent and gateway of the hippocampal trisynaptic circuit facilitating cognitive learning and memory functions, exhibit significant differences in cellular composition, molecular makeup, and responses to various stimuli. However, the effects of isoflurane-induced general anesthesia on CA1 and DG neuronal activity in mice are not well understood. In this study, utilizing electrophysiological recordings, we examined neuronal population dynamics and single-unit activity (SUA) of CA1 and DG in freely behaving mice during natural sleep and general anesthesia. Our findings reveal that isoflurane anesthesia shifts local field potential (LFP) to delta frequency and reduces the firing rate of SUA in both CA1 and DG, compared to wakefulness. Additionally, the firing rates of DG neurons are significantly lower than CA1 neurons during isoflurane anesthesia, and the recovery of theta power is slower in DG than in CA1 during the transition from anesthesia to wakefulness, indicating a stronger and more prolonged impact of isoflurane anesthesia on DG. This work presents a suitable approach for studying brain activities during general anesthesia and provides evidence for distinct effects of isoflurane anesthesia on hippocampal subregions.

Bacterial Biohybrids for Invasion of Tumor Cells Promote Antigen Cross-Presentation Through Gap Junction.

Due to inherent differences in cellular composition and metabolic behavior with host cells, tumor-harbored bacteria can discriminatorily affect tumor immune landscape. However, the mechanisms by which intracellular bacteria affect antigen presentation process between tumor cells and antigen-presenting cells (APCs) are largely unknown. The invasion behavior of attenuated Salmonella VNP20009 (VNP) into tumor cells is investigated and an attempt is made to modulate this behavior by modifying positively charged polymers on the surface of VNP. It is found that non-toxic chitosan oligosaccharide (COS) modified VNP (VNP@COS) bolsters the formation of gap junction between tumor cells and APCs by enhancing the ability of VNP to infect tumor cells. On this basis, a bacterial biohybrid is designed to promote in situ antigen cross-presentation through intracellular bacteria induced gap junction. This bacterial biohybrid also enhances the expression of major histocompatibility complex class I molecules on the surface of tumor cells through the incorporation of Mdivi-1 coupled with VNP@COS. This strategic integration serves to heighten the immunogenic exposure of tumor antigens; while, preserving the cytotoxic potency of T cells. A strategy is proposed to precisely controlling the function and local effects of microorganisms within tumors.

Abstract LB331: Integrating multiple spatial transcriptomic and proteomic technologies to generate a cancer atlas to understand the tumor microenvironment

Abstract Deep single-cell genomics has provided a monumental leap in our understanding and categorical analysis of cancer cells and their unique molecular properties. With spatial platforms that preserve the tissue architecture, an emerging understanding of complex cell interactions and local environment influences is providing new perspectives for targeted therapies. Here we describe a comprehensive approach at profiling the tumor immune microenvironment across multiple solid malignancies to provide an atlas of tumor biology. To generate this atlas, we created a MERFISH panel of 500 genes representing over 40 cell phenotyping pathways and dozens of signaling pathways including chemokines, cytokines, interferon, and ligand-receptor interactions. This panel was used across 15 patients representing multiple solid malignancies. We also utilized the Immuno-Oncology focused Xenium panel to profile 16 different patients across the same indications. With the Xenium data we simultaneously assessed proteomic expression using a 16 plex panel focused on immuno-oncology targets and subsequently did H&E staining, allowing multiple -omic data collections within a single slide. After initial filtering and quality control this projects produced a total dataset with over 15 million cells and billions of transcripts. The 500 gene MERFISH panel revealed complex relationships between diverse cell types within the tumor microenvironment that was dependent on cellular location and surrounding area. While the presence of major immune cell types was observed in all cancer tissues, the ratio and spatial relationship to cancer cells differed between each tumor. Accordingly, we observed that differences in cellular composition of the microenvironment corresponded with associated changes in cellular signaling. Similarly, with Xenium we identified new spatial relationships between cancer and immune cells with their environment while confirming part of these relationships with subsequent immunofluorescence data. The H&E data provided an opportunity to overlay this data with standard pathological annotation. We further investigated how each cellular microneighborhood varied throughout the tumor and varied from patient. This comprehensive map of the tumor immune microenvironment is a deep cellular dive into cellular architecture. As this Cancer Atlas evolves this will drive the discovery of several new potential biological targets and cell types of interest. This will also be critical for the assessment of current cellular and drug therapies in preclinical and clinical models. This effort will continue with more patient samples across a greater number of disease indications and will include more technologies. We will also expand the development and implementation of analytical tools to maximum the knowledge and insights gained from this Atlas. Together, this will be a critical tool in the continuing fight against cancer. Citation Format: Colles Price, Kazuho Nishimura, Som Bandyopadhyay, Darrell Borger, Russell Weiner. Integrating multiple spatial transcriptomic and proteomic technologies to generate a cancer atlas to understand the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB331.

Abstract B039: Identification of sex-specific molecular features in glioma transcriptome

Abstract Glioma is a lethal disease with limited treatment options available. Sex differences in glioma are well-documented in clinical settings, with males having twice the risk of developing the disease and increased mortality compared to female counterparts. To investigate the molecular basis of sex differences in glioma, we analyzed transcriptomic sex differences in a collection of three independent glioma datasets and validated them in additional independent studies. The impact of differentially expressed genes were assessed through Gene Set Enrichment Analysis (GSEA) on approximately 5000 gene sets to interpret their biological relevance. Given the well-recognized heterogeneity of glioma, we also integrated three single-cell RNA (scRNA) sequencing datasets to decipher the sex differences in cellular composition and cell-type specific differential gene expression between male and female tumors. The effects of sex on gene expression are modest and primarily driven by the X chromosome (14 out of 16 differentially expressed genes were on the X chromosome). These genes are associated with escape from X-chromosome inactivation (e.g., KDM5C, KDM6A) and are also sex-differentially expressed (DE) in the healthy human brain according to Genotype-Tissue Expression (GTEx) data. GSEA results suggest increased activity in cell proliferation and neuronal signaling in male GBM samples, whereas females show enrichment in glycoprotein/lipid metabolism. By deconvoluting the cell-type composition, we identified a higher percentage of pericytes in the male glioma tumor microenvironment compared to those in females, which is consistent with recent spatial-omic discoveries in glioma. By integrating scRNA sequencing data, we found that both differentially expressed genes and pathways show cell-type-specific enrichment. For example, the female-biased gene GBGT1 is exclusively expressed in the myeloid lineage, while male-biased neuronal pathways are enriched in cancer stem cells. Integrating with the inference of cell trajectories, we observed that male tumors harbor more unconnected, invasive malignant cells than females, which might explain the poor prognosis in male glioma patients. In summary, our results provide a comprehensive multi-level characterization of sex differences in glioma tumors, and these findings will provide novel insights into glioma disease progression and development of new therapy. Citation Format: Yingbo Huang, Yuting Shan, Weijie Zhang, Christina Printzis, Lorenzo Pesce, Barbara Elaine Stranger, R. Stephanie Huang. Identification of sex-specific molecular features in glioma transcriptome [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B039.

Single cell analysis unveils the commonality and heterogeneity between nasopharyngeal and oropharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) and oropharyngeal carcinoma (OPC) are subtypes of head and neck cancer with different treatment effects due to the heterogeneity of tumor microenvironments. This study was to investigate the distinctive tumor microenvironments of NPC and OPC. Analyzing single-cell data from 10 cases of each subtype, we reveal significant differences in cellular composition, with NPC microenvironment dominated by T/NK and B cells, and OPC characterized by prevalent epithelial cells and fibroblasts. Dynamic transitions of CD8 T cells are observed in both tumor types, involving shifts from naivety to cytotoxicity, proliferation, and eventual exhaustion/exhausted states. Additionally, Tregs exhibit heightened proliferative abilities in later developmental stages, concomitant with exhaustion. These highly proliferative T cells and Tregs manifest elevated glycolysis and lactate metabolism activities. Furthermore, we explore intercellular communication between glycolytic malignant epithelial cells and these proliferative T cells. These findings offer comprehensive insights into the heterogeneity of tumor microenvironments and provide a solid foundation for future therapeutic strategies and targeted interventions.

Abstract WMP118: Utilizing Patient-Derived Induced Pluripotent Stem Cells as a Cellular Model for Studying Moyamoya Disease

Introduction: Moyamoya disease (MMD) is a rare cerebrovascular disease that can lead to stroke. A histopathological characteristic in MMD is the thickening of the intimal layer that causes narrowing of the vessel, suggesting dysfunctions in the vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). The mechanism of MMD is largely unknown, due to a lack of established MMD-specific cellular or animal models. In this study, we established patient-derived cellular co-culture models using MMD iPSC-derived ECs and VSMCs. We also generated a 3D vascular organoid model to more closely mimic the MMD vasculature and function. Methods: Peripheral blood mononuclear cells (PBMCs) from MMD patients or healthy controls were used to generate iPSCs (n=4 per group). Differentiated ECs and VSMCs were validated and their functional characteristics were investigated in single and co-cultures. For co-cultures, ECs and VSMCs were labeled with Vibrant cell labeling dyes (DiO and DiD), followed by either transwell migration assay (ECs on bottom of cell culture plate and VSMCs on top Transwell insert) or in vitro angiogenic tube formation assay. Control and MMD iPSCs were used to generate 3D cellular organoids, and their cellular characteristics were examined using immunohistochemistry with antibodies labeling ECs and VSMCs. Results: While single cultures of ECs displayed similar tube formation patterns between control and MMD, co-cultures of ECs with VSMCs showed that MMD ECs failed to stabilize in vitro angiogenic tubes and formed aggregates at 24hr, when compared to control ECs. Transwell migration assays showed MMD VSMCs migrated faster than control VSMCs. Immunostaining of organoids with EC and VSMC markers showed notable differences in cellular composition, with MMD organoid showing compromised integrity of vascular networks, when compared with controls. Conclusions: We have established 3D organoid cellular models and in vitro co-cultures using MMD patient-derived iPSCs. Our co-culture studies revealed functional impairment in MMD EC/VSMC and organoid models revealed distinct characteristics in their vascular networks, possibly contributing to MMD pathology. These results shed useful insight into the cellular and molecular mechanisms driving MMD.

Длинная некодирующая РНК NEAT1_1 снижает выживаемость первичных нейронных клеток при ЭПР-стрессе

NEAT1 long non-coding RNAs play an important role in the central nervous system (CNS) and are associated with a number of pathological conditions. Increased levels of NEAT1 in the brain have been observed in neurodegenerative and psychiatric diseases — the significance of such an increase is still poorly understood. Functionally, NEAT1 is associated with cellular stress pathways in the nervous system. The aim of the current study was to evaluate the effect of increased levels of the short isoform NEAT1_1 on survival of mice primary hippocampal cultures under ER-stress induced by MG132 proteasome inhibitor. Primary cultures were obtained from transgenic animals expressing human NEAT1_1. Cellular composition and apoptosis were assessed using immunocytochemical staining. The expression of apoptosis signaling pathway genes was analyzed by quantitative PCR with reverse transcription. No differences in cellular composition and morphological characteristics of neurons were observed in primary neuronal cultures obtained from transgenic animals as compared to wild type cultures. Induction of ER-stress resulted in a more significant increase in apoptotic death of cells including neurons in NEAT1_1 expressing cultures in comparison with the wild type cultures. ER-stress signaling pathway genes Atf4 and Ddit3 were less expressed in transgenic cultures under stress. Expression of Bcl2l2 and Mcl1 anti-apoptotic genes was reduced as well. Thus, high levels of NEAT1_1 in primary neuronal cultures increased apoptotic cell death under ER-stress.

The expression of immune related genes and potential regulatory mechanisms in schizophrenia

ObjectiveThis study aimed to investigate the role of immune dysfunction in the pathogenesis of schizophrenia through single-cell transcriptome and bulk RNA data analyses. MethodsThe single-cell RNA sequencing (scRNA-seq) was selected to assess the cellular composition and gene expression profiles of the brain tissue. Further, bulk RNA sequencing data was utilized to corroborate findings from the single-cell analyses and provide additional insights into the molecular changes associated with the disease. Gene-drug interaction data was also included to identify potential therapeutic drugs targeting these dysregulated immune-related genes in schizophrenia. ResultsWe discovered significant differences in cellular composition within schizophrenia tissue, including increased infiltration of fibroblasts, horizontal basal cells, monocytes, mesenchymal cells, and smooth muscle cells. The investigation of immune-related genes revealed significantly different expression of genes such as S100A2, CCL14, IGHA1, BPIFA1, GDF15, IL32, BPIFB2, HLA-DRA, S100A8, PTX3, TPM2, TNFRSF12A, GREM1 and others. These genes possibly contribute to the progression of schizophrenia through various pathways such as humoral immune response, IL-17 signaling pathway, adaptive immune response, antigen processing and presentation, and gut IgA production. Our findings also suggest possible transcriptional regulation in schizophrenia's immune dysfunction by transcription factors in monocytes, neutrophils, endothelial cells, and epithelial cells. Lastly, potential therapeutic drugs were identified through gene-drug interaction data, such as those targeting HLA-A and HLAB. ConclusionThe cellular heterogeneity and immune-related gene dysregulation play important roles in schizophrenia, which provides a foundation for understanding the pathogenesis and developing new treatment methods.

Construction of a prognostic 6-gene signature for breast cancer based on multi-omics and single-cell data

BackgroundBreast cancer (BC) is one of the females’ most common malignant tumors there are large individual differences in its prognosis. We intended to uncover novel useful genetic biomarkers and a risk signature for BC to aid determining clinical strategies.MethodsA combined significance (pcombined) was calculated for each gene by Fisher’s method based on the RNA-seq, CNV, and DNA methylation data from TCGA-BRCA. Genes with a pcombined< 0.01 were subjected to univariate cox and Lasso regression, whereby an RS signature was established. The predicted performance of the RS signature would be assessed in GSE7390 and GSE20685, and emphatically analyzed in triple-negative breast cancer (TNBC) patients, while the expression of immune checkpoints and drug sensitivity were also examined. GSE176078, a single-cell dataset, was used to validate the differences in cellular composition in tumors between TNBC patients with different RS.ResultsThe RS signature consisted of C15orf52, C1orf228, CEL, FUZ, PAK6, and SIRPG showed good performance. It could distinguish the prognosis of patients well, even stratified by disease stages or subtypes and also showed a stronger predictive ability than traditional clinical indicators. The down-regulated expressions of many immune checkpoints, while the decreased sensitivity of many antitumor drugs was observed in TNBC patients with higher RS. The overall cells and lymphocytes composition differed between patients with different RS, which could facilitate a more personalized treatment.ConclusionThe six genes RS signature established based on multi-omics data exhibited well performance in predicting the prognosis of BC patients, regardless of disease stages or subtypes. Contributing to a more personalized treatment, our signature might benefit the outcome of BC patients.

Resilience of Emiliania huxleyi to future changes in subantarctic waters.

Lower pH and elevated temperature alter phytoplankton growth and biomass in short-term incubations, but longer-term responses and adaptation potential are less well-studied. To determine the future of the coccolithophore Emiliania huxleyi, a mixed genotype culture from subantarctic water was incubated for 720 days under present-day temperature and pH, and also projected future conditions by the year 2100. The future population exhibited a higher growth rate relative to present-day cells transferred to future conditions after 309 days, indicating adaptation or genotype selection; this was reflected by an increase in optimum growth temperature of ~2.5°C by the end of the experiment. Following transfer to opposing conditions in short-term cross-over incubations, cell volume responded rapidly, within eight generations, confirming trait plasticity. The changes in growth rate and cell volume were larger than reported in previous single stressor relationships and incubations, suggesting synergistic or additive effects of combined elevated temperature and lower pH and highlighting the importance of long-term multiple stressor experiments. At the end of the incubation there were no significant differences in cellular composition (particulate organic content and chlorophyll a), or primary production between present-day and future populations. Conversely, two independent methods showed a 50% decrease in both particulate inorganic carbon and calcification rate, consistent with the decrease in cell volume, in the future population. The observed plasticity and adaptive capacity of E. huxleyi indicate resilience to future conditions in subantarctic waters, although changes in cell volume and carbonate may alter grazing loss and cell ballast, so influencing carbon export to the deep ocean.

Abstract 15: Single-cell Immune Landscape Of Human Clot Retrieved At Mechanical Thrombectomy. Association With Stroke Origin.

Background: Mechanical thrombectomy (MT) has significantly improved clinical outcomes in acute ischemic stroke. We used single-cell RNA sequencing (scRNA-seq) to describe the landscape of human clots at single cell resolution seeking to provide greater mechanistic insight into stroke pathophysiology and to uncover an association with stroke origin. Methods: scRNA-seq was performed on 14 clots retrieved from 14 patients undergoing MT between October 2020-July 2022. We followed standard molecular biology and bioinformatics workflows for sample processing and data to characterize differences in cellular composition between clots of different stroke origin (atrial fibrillation (AF) and carotid atherosclerosis (CA)). Results: Clots revealed a significant heterogeneity of immune cells with major constituents including cells of the mononuclear phagocyte system (MPS) and neutrophils. The cellular populations most enriched in CA clots were B-cells and CD4+ T-cells. The cellular population most enriched in AF clots was red blood cells. When looking at cellular subpopulations of the MPS component, we found that CD16+ monocytes (intermediate and non-classical) were significantly more abundant than CD14+CD16- (classical) monocytes in all clots. Moreover, the proportions of CD16+ monocytes varied with stroke origin: the proportion of non-classical (CD14-CD16+) monocytes was higher in CA clots (p=0.05) whereas the proportion of intermediate monocytes (CD14+CD16+) was higher in FA clots (p=0.03). AF clots had upregulated biomarkers and pathways involved with AF or contractile dysfunction (IFI30, FABP4, PPAR, HOTAIR, ILK, Integrin). CA clots had upregulated biomarkers and pathways associated with cardiovascular diseases and atherogenesis (RNSE1, POLR2J3.1, PLTP, Sirtuin, FAK). (Figure) Conclusions: These results reveal a significant heterogeneity of immune cells and point to important differences in cellular composition associated with stroke etiology.

Impact of Changes in Human Airway Epithelial Cellular Composition and Differentiation on SARS-CoV-2 Infection Biology

The consequences of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can range from asymptomatic to fatal disease. Variations in epithelial susceptibility to SARS-CoV-2 infection depend on the anatomical location from the proximal to distal respiratory tract. However, the cellular biology underlying these variations is not completely understood. Thus, air-liquid interface cultures of well-differentiated primary human tracheal and bronchial epithelial cells were employed to study the impact of epithelial cellular composition and differentiation on SARS-CoV-2 infection by transcriptional (RNA sequencing) and immunofluorescent analyses. Changes of cellular composition were investigated by varying time of differentiation or by using specific compounds. We found that SARS-CoV-2 primarily infected not only ciliated cells but also goblet cells and transient secretory cells. Viral replication was impacted by differences in cellular composition, which depended on culturing time and anatomical origin. A higher percentage of ciliated cells correlated with a higher viral load. However, DAPT treatment, which increased the number of ciliated cells and reduced goblet cells, decreased viral load, indicating the contribution of goblet cells to infection. Cell entry factors, especially cathepsin L and transmembrane protease serine 2, were also affected by differentiation time. In conclusion, our study demonstrates that viral replication is affected by changes in cellular composition, especially in cells related to the mucociliary system. This could explain in part the variable susceptibility to SARS-CoV-2 infection between individuals and between anatomical locations in the respiratory tract.

Divergent Cellular Energetics, Glutamate Metabolism, and Mitochondrial Function Between Human and Mouse Cerebral Cortex.

Disruptions of brain energy and neurotransmitter metabolism are associated with several pathological conditions including neurodegenerative diseases such as Alzheimer's disease. Transgenic rodent models, and in vitro preparations hereof, are often applied for studying pathological aspects of brain metabolism. However, despite the conserved cerebral development across mammalian species, distinct differences in cellular composition and structure may influence metabolism of the rodent and human brain. To address this, we investigated the metabolic function of acutely isolated brain slices and non-synaptic mitochondria obtained from the cerebral cortex of mice and neurosurgically resected neocortical tissue of humans. Utilizing dynamic isotope labeling with 13C-enriched metabolic substrates, we show that metabolism of glucose, acetate, β-hydroxybutyrate, and glutamine operates at lower rates in human cerebral cortical slices when compared to mouse slices. In contrast, human cerebral cortical slices display a higher capacity for converting exogenous glutamate into glutamine, which subsequently supports neuronal GABA synthesis, whereas mouse slices primarily convert glutamate into aspartate. In line with the reduced metabolic rate of the human brain slices, isolated non-synaptic mitochondria of the human cerebral cortex have a lower oxygen consumption rate when provided succinate as substrate. However, when provided pyruvate and malate, human mitochondria display a higher coupled respiration and lower proton leak, signifying a more efficient mitochondrial coupling compared to mouse mitochondria. This study reveals key differences between mouse and human brain metabolism concerning both neurons and astrocytes, which must be taken into account when applying in vitro rodent preparations as a model system of the human brain.

Abstract P3006: A Human Arterial Cell Atlas

Background: Human vascular diseases are the worldwide leading causes of morbidity and mortality. Nearly all human vascular diseases have arterial segment-specific tropisms despite identical exposures to genetic and environmental risk factors. Understanding the cellular and transcriptomic determinants of arterial identities may hold the key to identifying novel pathophysiology and potential therapies. Methods: To specifically determine arterial site-specific differences independent of inter-individual variation, we have generated a human arterial cellular atlas by simultaneously collecting and analyzing up to 8 arterial sites from multiple healthy transplant donors. We performed single cell transcriptomic analysis on arterial segments to determine the differences in cellular composition and transcriptomic programs. We subsequently integrated human genetic data with cell-type specific transcriptomic differences across vascular beds to identify probable causal cells and causal genes associated with human vascular phenotypes. Results/Conclusions: Single cell transcriptomic analysis of >150,000 cells sequenced at >50,000 reads per cell revealed that the dominant cellular drivers of transcriptomic differences between distinct arterial segments, i.e. determinants of arterial identity, are fibroblasts and smooth muscle cells, not endothelial cells or macrophages. Adult vascular cells transcriptomes from different segments are most influenced by their embryonic origins but not by anatomical proximity. Differentially regulated genes in fibroblast across different vascular beds were particularly enriched for vascular disease associated genetic signals, suggesting a prominent role for these cells in human disease. While the majority of endothelial cells were transcriptionally similar across vascular beds, a rare, previously undescribed, cluster of endothelial cells were identified who expressed segment-specific transcriptomic signatures. Differentially expressed genes in these cells were enriched for vascular disease signals, suggesting a possible role of these rare cells in human disease.

Single-cell immune mapping of adenoid cystic carcinoma (ACC) reveals potential therapeutic targets for the aggressive solid subtype.

6090 Background: ACC is a common salivary gland malignancy for which there is no FDA approved therapies. Despite a quiet genome, ACC has significant tumoral heterogeneity, which may facilitate the metastatic relapse. Studies focusing on the deep profile of ACC tumor microenvironment (TME) are lacking. Here we explored the TME of ACC using imaging mass cytometry (IMC) and assessed TME attributes and its association with histology and clinical outcomes. Methods: Two tissue microarrays from 62 ACC patients (pts) were built and stained with 37 metal-tagged markers. IMC was performed with the Fluidigm Helios CyTOF instrument utilizing the Hyperion Imaging System Laser ablation. Tissue and cell segmentation and multiplex imaging analysis were performed with Visiopharm software using pre-trained artificial intelligence algorithms. Comparison of cell types and markers densities among histology sub-groups were analyzed with Wilcoxon signed-rank test and its association with overall survival (OS) with log-rank and Cox Proportional Hazards. Results: Of 62 pts, 37% (23/62) had solid histology, 19% (12/62) were metastatic at diagnosis, and 55% (30/54) had disease recurrence during follow-up. With a median follow-up of 7.9 y, the median OS was 9.3y (CI 95%, 7.8-NR). Pts with solid subtype had poorer OS than non-solid histology (5.2 vs 14.6 y, p = 0.004). The IMC final dataset comprised 507,524 single-cells. No significant differences were found in cell subpopulations density between solid and non-solid histology. The most represented cell population in the stroma were macrophages, followed by CD8+ T cells, and fibroblasts. A higher percentage of M2-macrophage was associated with poor survival (p = 0.001), whereas, a higher percentage of M1-macrophage (M1:M2 ratio) was associated with better prognosis. No other immune cell type, fibroblasts or immune cell functional markers (TIGIT, TIM3, granzyme B, and PD-L1) correlated with survival. Regarding tumor markers, a higher expression of BCL-2, B7-H4, and Ki67 in the tumor cells were associated with worst survival; and remained statistically associated after adjustment for histology and staging (all p < 0.001). Solid histology had a significantly higher density of tumor cells expressing B7-H4, BCL-2, and Ki67 compared to cribriform and tubular histology. A higher expression of myoepithelial marker (p63+) were associated with a better survival when compared with tumors with low p63 expression. Conclusions: ACC’s TME is composed mainly of macrophages. Despite having no significant differences in cellular composition, a higher density of tumor cells expressing BCL-2, B7-H4 and Ki67 were found in the solid histology and these markers were independent predictors of poor prognosis. The overexpression of BCL-2 and B7-H4 in the solid histology provides a scientific rationale for BCL-2 and/or B7-H4 targeting for the most aggressive ACC.