Markers For Subpopulations Research Articles

A Reproducible Protocol for the Isolation of Malaria-Derived Extracellular Vesicles by Differential Centrifugation.

Over the last few decades, malaria-derived extracellular vesicles (EVs) have gained increasing interest due to their role in disease pathophysiology and parasite biology. Unlike other EV research fields, the isolation of malaria EVs is not standardized, hampering inter-study comparisons. Most malaria EV studies isolate vesicles by the "gold-standard" technique of differential (ultra)centrifugation (DC). Here, we describe in detail an optimized and reproducible protocol for the isolation of malaria-derived EVs by DC. The protocol begins with a description of cultivating high-parasitemia, synchronous P. falciparum cultures that are the source of EV-containing conditioned culture media. The isolation protocol generates two EV subtypes, and we provide details of characterizing these distinct subtypes by analyzing human and parasite proteins by Western blot analysis. We identify some of these proteins as suitable markers for malaria EV subpopulations and subtypes.

Dissecting the Single-Cell Diversity and Heterogeneity Underlying Cervical Precancerous Lesions and Cancer Tissues.

The underlying cellular diversity and heterogeneity from cervix precancerous lesions to cervical squamous cell carcinoma (CSCC) is investigated. Four single-cell datasets including normal tissues, normal adjacent tissues, precancerous lesions, and cervical tumors were integrated to perform disease stage analysis. Single-cell compositional data analysis (scCODA) was utilized to reveal the compositional changes of each cell type. Differentially expressed genes (DEGs) among cell types were annotated using BioCarta. An assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis was performed to correlate epigenetic alterations with gene expression profiles. Lastly, a logistic regression model was used to assess the similarity between the original and new cohort data (HRA001742). After global annotation, seven distinct cell types were categorized. Eight consensus-upregulated DEGs were identified in B cells among different disease statuses, which could be utilized to predict the overall survival of CSCC patients. Inferred copy number variation (CNV) analysis of epithelial cells guided disease progression classification. Trajectory and ATAC-seq integration analysis identified 95 key transcription factors (TF) and one immunohistochemistry (IHC) testified key-node TF (YY1) involved in epithelial cells from CSCC initiation to progression. The consistency of epithelial cell subpopulation markers was revealed with single-cell sequencing, bulk sequencing, and RT-qPCR detection. KRT8 and KRT15, markers of Epi6, showed progressively higher expression with disease progression as revealed by IHC detection. The logistic regression model testified the robustness of the resemblance of clusters among the various datasets utilized in this study. Valuable insights into CSCC cellular diversity and heterogeneity provide a foundation for future targeted therapy.

NK Cell Degranulation Triggered by Rituximab Identifies Potential Markers of Subpopulations with Enhanced Cytotoxicity toward Malignant B Cells.

A promising strategy in cancer immunotherapy is to restore or enhance the cytotoxicity of NK cells, among others, by activating the mechanism of antibody-dependent cellular cytotoxicity (ADCC). Monoclonal antibodies targeting tumor antigens, such as rituximab (targeting CD20), induce NK cell-mediated ADCC and have been used to treat B cell malignancies, such as non-Hodgkin lymphoma, but not always successfully. The aim of this study was to analyze the gene expression profile of the NK cells involved in the cytolytic response stimulated by rituximab. NK cells were co-cultured with rituximab-opsonized Raji cells. Sorting into responder and non-responder groups was based on the presence of CD107a, which is a degranulation marker. RNA-seq results showed that the KIT and TNFSF4 genes were strongly down-regulated in the degranulating population of NK cells (responders); this was further confirmed by qRT-PCR. Both genes encode surface proteins with cellular signaling abilities, namely c-KIT and the OX40 ligand. Consistent with our findings, c-KIT was previously reported to correlate inversely with cytokine production by activated NK cells. The significance of these findings for cancer immunotherapy seems essential, as the pharmacological inhibition of c-KIT and OX40L, or gene ablation, could be further tested for the enhancement of the anti-tumor activity of NK cells in response to rituximab.

Assessment of non-classical lymphocyte populations in patients with advanced lung cancer treated with Biomodulina T following platinum-based chemotherapy

Aim: Currently, malignant diseases represent a health issue worldwide. Among these, lung cancer is of growing importance, due to its high incidence and mortality. Chemotherapy, one of the most frequently used treatments, has shown its ability to induce accelerated immunosenescence in classic and as well non-classic lymphocyte compartments, being less described in the latter. The immune restoration strategies have demonstrated their ability to reverse immunosenescence and exhaustion markers in conventional lymphocyte subpopulations after chemotherapy. However, the possible immunorestorative effect on non-classical lymphocytes has not been widely reported. The aim of this study was to evaluate the effect of chemotherapy and the administration of a thymic polypeptide factor on non-classical lymphocyte populations in patients with advanced lung cancer. Methods: Eighteen patients with advanced lung cancer, were evaluated at baseline before and after platinum-based chemotherapy (4–6 cycles). All patients could complete treatment with a thymic polypeptide factor [Biomodulina T (BT)] at the end of chemotherapy. Blood from patients was collected by venipuncture in heparinized tubes before and after chemotherapy and at the end of BT treatment to analyze the frequencies of non-classical immune subpopulations by flow cytometry. Results: Natural killer (NK), natural killer T cells (NKT), and double-positive T lymphocyte (DPT) proportions reached normal values in patients diagnosed with advanced lung cancer before receiving cytotoxic treatment. Chemotherapy did not induce modifications in the total percent of NK, NKT, and DPT populations in these patients. However, the administration of BT decreased DPTs and NK cells expressing the cluster of differentiation (CD)57 molecule, which is considered a marker of immunosenescence. Conclusions: These results suggest a lower influence of platinum-based chemotherapy on non-classical lymphocytes and the potential to generate a reconstitution of lymphocyte subpopulations in patients with advanced lung cancer by using the thymic factor BT, which reveals a new possibility for improving the response to cancer immunotherapies [Cuban Public Registry of Clinical Trial (RPCEC, https://rpcec.sld.cu/en/trials/RPCEC00000358-En) identifier: RPCEC00000358].

Markers of Dermal Fibroblast Subpopulations for Viable Cell Isolation via Cell Sorting: A Comprehensive Review.

Fibroblasts are among the most abundant cell types in the human body, playing crucial roles in numerous physiological processes, including the structural maintenance of the dermis, production of extracellular matrix components, and mediation of inflammatory responses. Despite their importance, fibroblasts remain one of the least characterized cell populations. The advent of single-cell analysis techniques, particularly single-cell RNA sequencing (scRNA-seq) and fluorescence-activated cell sorting (FACS), has enabled detailed investigations into fibroblast biology. In this study, we present an extensive analysis of fibroblast surface markers suitable for cell sorting and subsequent functional studies. We reviewed over three thousand research articles describing fibroblast populations and their markers, characterizing and comparing subtypes based on their surface markers, as well as their intra- and extracellular proteins. Our detailed analysis identified a variety of distinct fibroblast subpopulations, each with unique markers, characteristics dependent on their location, and the physiological or pathophysiological environment. These findings underscore the diversity of fibroblasts as a cellular population and could lead to the development of novel diagnostic and therapeutic tools.

Predictors of Clinical Outcomes among People with HIV and Tuberculosis Symptoms after Rapid Treatment Initiation in Haiti.

Few studies have evaluated baseline predictors of clinical outcomes among people with HIV starting antiretroviral therapy (ART) in the modern era of rapid ART initiation. We conducted a secondary analysis of a randomized controlled trial of two rapid treatment initiation strategies for people with treatment-naïve HIV and tuberculosis symptoms at an urban clinic in Haiti. We used logistic regression models to assess associations between baseline characteristics and (1) retention in care at 48 weeks, (2) HIV viral load suppression at 48 weeks (among participants who underwent viral load testing), and (3) all-cause mortality. 500 participants were enrolled in the study 11/2017-1/2020. Eighty-eight (18%) participants were diagnosed with tuberculosis, and ART was started in 494 (99%). After adjustment, less than secondary education (adjusted odds ratio [AOR] 0.21, 95% CI 0.10-0.46), dolutegravir initiation (AOR 2.57, 95% CI 1.22-5.43), age (AOR 1.42 per 10-year increase, 95% CI 1.01-1.99), and tuberculosis diagnosis (AOR 3.92, 95% CI 1.36-11.28) were significantly associated with retention. Age (AOR 1.36, 95% CI 1.05-1.75), dolutegravir initiation (AOR 1.75, 95% CI 1.07-2.85), and tuberculosis diagnosis (AOR 0.50, 95% CI 0.28-0.89) were associated with viral suppression. Higher CD4 cell count at enrollment (unadjusted odds ratio [OR] 0.69, 95% CI 0.55-0.87) and anemia (OR 4.86, 95% CI 1.71-13.81) were associated with mortality. We identified sociodemographic, treatment-related, clinical, and laboratory-based predictors of clinical outcomes. These characteristics may serve as markers of sub-populations that could benefit from additional interventions to support treatment success after rapid treatment initiation.

Left atrial strain analysis in the realm of pediatric cardiology: Advantages and implications.

Left atrial (LA) strain analysis has emerged as a noninvasive technique for assessing LA function and early detection of myocardial deformation. Recently, its application has also shown promise in the pediatric population, spanning diverse cardiac conditions that demand accurate and sensitive diagnostic measures. This research article endeavors to explore the role of LA strain parameters and contribute to the growing body of knowledge in pediatric cardiology, paving the way for more effective and tailored approaches to patient care. A comprehensive literature review was conducted to gather evidence from studies using echocardiographic strain imaging techniques across pediatric populations. LA strain parameters exhibited greater sensitivity than conventional atrial function indicators, with early detection of diastolic dysfunction and LA remodeling in pediatric cardiomyopathy, children with multisystem inflammatory syndrome, rheumatic heart disease, as well as childhood renal insufficiency and obesity offering prognostic relevance as potential markers in these pediatric subpopulations. However, there remains a paucity of evidence concerning pediatric mitral valve pathology, justifying further exploration. LA strain analysis carries crucial clinical and prognostic implications in pediatric cardiac conditions, with reliable accuracy and sensitivity to early functional changes.

Application of Deep Learning Models on Single-Cell RNA Sequencing Analysis Uncovers Novel Markers of Double Negative T Cells

Abstract Double negative T (DNT) cells, a unique subset of CD3+TCRαβ+ T lymphocytes lacking CD4, CD8, or NK1.1 expression, constitute 3-5% of the total T cell population in C57BL/6 mice. Traditional machine learning models such as principal component analysis in single-cell RNA sequencing (scRNA-seq) analysis have been utilized to characterize this subset, but only deep learning models such as Single Cell Variational Inference (SCVI) can capture nonlinear gene expression in the sequencing data. In this study, using the deep learning approach, we have uncovered novel markers of splenic DNT cells in C57BL/6 mice. We classified DNT cells into two subgroups, naïve DNT (nDNT) cells and activated DNT (aDNT) cells, which could be differentiated by the expression of Ly6C and MHC-II, respectively. In addition, our data identified CD153 and CD153 as unique markers for aDNT cells. SCVI analysis suggested, and flow cytometry analysis confirmed, that Ly49G2 and CCR2 are respective markers for two distinct subsets within nDNT and aDNT populations. In addition to cell surface markers, we further discovered that nDNT cells expressed higher Rgs2 that is required for T cell activation and that aDNT cells expressed higher Rbpj involved in Notch signaling. Together, our comprehensive analysis has uncovered and validated novel markers for different subpopulations of DNT cells that can be used in the phenotypic and/or functional characterization of these relatively rare cells in health and disease.

Abstract 6883: Investigation of tumor-immune interactions in pediatric sarcomas using single-cell multi-omics

Abstract Background: Pediatric sarcoma represents the second most common solid tumor in children. Despite advances in multimodal therapies, one-third of sarcoma patients do not survive the disease. Over the past few decades, the development of novel therapeutic strategies for sarcoma has been impeded by an insufficient understanding of the tumor microenvironment (TME). In particular, the potential of immunotherapy remains constrained by the gap of knowledge about the molecular and functional phenotypes of the immune cells in the sarcoma TME. Methods: To address this knowledge gap, we collected treatment-naïve tumor samples from 32 patients with five pediatric sarcoma subtypes. We employed a single-nuclei RNA+ATAC-Seq multi-omics assay to comprehensively characterize the transcriptomic and epigenomic heterogeneity of tumor and immune cells within the sarcoma TME. Results: Our analysis revealed that tumor-associated macrophages (TAMs) were the most abundant immune cell type in the sarcoma TME. We identified seven distinct TAM subpopulations, each exhibiting unique gene expression and chromatin accessibility patterns. Among these, TAM subsets with higher expression of SPP1 and C1QC genes displayed the strongest immunosuppressive potential. Transcriptional regulatory network (TRN) analysis identified transcription factors that were most likely to drive the phenotypes of SPP1+ and C1QC+ TAMs, including ELF2, JUND and RUNX1. Through cell-cell communication analysis, we discovered that SPP1+ TAMs interact with T cells via SPP1-CD44 signaling, while C1QC+ TAMs interact with NK cells through HLA-E-KLRC1 signaling, suggesting the potential involvement of these signaling pathways in TAM-mediated suppression of these cytotoxic lymphocytes. We prioritized ligand-receptor gene pairs, such as SEMA3D-NRP1 and LTBP3-ITGB5, which likely mediate the interaction between malignant sarcoma cells and SPP1+/C1QC+ TAMs based on their dominance across sarcoma subtypes and association with poorer clinical outcomes. On the other hand, our analysis revealed that sarcoma subtype-specific interactions between TAMs and malignant cells are common, highlighting the heterogeneity of signaling events across sarcoma subtypes. Conclusion and Significance: Our study presents a comprehensive single-cell multi-omics characterization of pediatric sarcomas and sheds light on its tumor microenvironment. By identifying unique markers for immunosuppressive TAM subpopulations and elucidating intercellular interactions promoting tumor development, this study proposes potential therapeutic targets for sarcoma treatment based on the role of TAMs in sarcoma TME. Citation Format: Zhan Zhang, Kyung Jin Ahn, Changya Chen, Rumeysa Biyik Sit, Hesham Mohei, Brian Lockhart, Malay Haldar, Kathrin Bernt, Vinodh Pillai, Kai Tan. Investigation of tumor-immune interactions in pediatric sarcomas using single-cell multi-omics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6883.

Spherical harmonics analysis reveals cell shape-fate relationships in zebrafish lateral line neuromasts.

Cell shape is a powerful readout of cell state, fate and function. We describe a custom workflow to perform semi-automated, 3D cell and nucleus segmentation, and spherical harmonics and principal components analysis to distill cell and nuclear shape variation into discrete biologically meaningful parameters. We apply these methods to analyze shape in the neuromast cells of the zebrafish lateral line system, finding that shapes vary with cell location and identity. The distinction between hair cells and support cells accounted for much of the variation, which allowed us to train classifiers to predict cell identity from shape features. Using transgenic markers for support cell subpopulations, we found that subtypes had different shapes from each other. To investigate how loss of a neuromast cell type altered cell shape distributions, we examined atoh1a mutants that lack hair cells. We found that mutant neuromasts lacked the cell shape phenotype associated with hair cells, but did not exhibit a mutant-specific cell shape. Our results demonstrate the utility of using 3D cell shape features to characterize, compare and classify cells in a living developing organism.

Expression of CD40 and CD192 in Classical Monocytes in Multiple Sclerosis Patients Assessed with Transcranial Magnetic Stimulation.

Expression of CD40 and CD192 markers in different monocyte subpopulations has been reported to be altered in people with MS (pwMS). Also, functional connectivity of the corticospinal motor system pathway alterations has been proved by transcranial magnetic stimulation (TMS). The study objective was to investigate the expression of CD40 and CD192 in classical (CD14++CD16-), intermediate CD14++CD16+ and non-classical (CD14+CD16++) blood monocyte subpopulations in pwMS, undergoing neurophysiological TMS assessment of the corticospinal tract integrity by recording motor-evoked potentials (MEPs). Radiological examination on lesion detection with MRI was performed for 23 patients with relapsing-remitting MS treated with teriflunomide. Then, immunological analysis was conducted on peripheral blood samples collected from the patients and 10 healthy controls (HC). The blood samples were incubated with anti-human CD14, CD16, CD40 and CD192 antibodies. Next, pwMS underwent neurological testing of functional disability (EDSS) and TMS assessment with recording MEPs from upper and lower extremity muscles. The results show that in comparison to HC subjects, both pwMS with normal and altered MEP findings (prolonged MEP latency or absent MEP response) had significantly decreased surface receptor expression measured (MFIs) of CD192 and increased CD40 MFI in classical monocytes, and significantly increased percentages of classical and total monocytes positive for CD40. Knowing CD40's pro-inflammatory action, and CD192 as a molecule that enables the passing of monocytes into the brain, decreased CD192 in classical monocytes could represent a beneficial anti-inflammatory parameter.

Integrated single-cell and bulk RNA sequencing analysis identified pyroptosis-related signature for diagnosis and prognosis in osteoarthritis

Osteoarthritis (OA), a degenerative disease of the joints, has one of the highest disability rates worldwide. This study investigates the role of pyroptosis-related genes in osteoarthritis and their expression in different chondrocyte subtypes at the individual cell level. Using OA-related datasets for single-cell RNA sequencing and RNA-seq, the study identified PRDEGs and DEGs and conducted Cox regression analysis to identify independent prognostic factors for OA. CASP6, NOD1, and PYCARD were found to be prognostic factors. Combined Weighted Gene Correlation Network Analysis with PPI network, a total of 15 hub genes related to pyroptosis were involved in the notch and oxidative phosphorylation pathways, which could serve as biomarkers for the diagnosis and prognosis of OA patients. The study also explored the heterogeneity of chondrocytes between OA and normal samples, identifying 19 single-cell subpopulation marker genes that were significantly different among 7 chondrocyte cell clusters. AGT, CTSD, CYBC, and THYS1 were expressed differentially among different cell subpopulations, which were associated with cartilage development and metabolism. These findings provide valuable insights into the molecular mechanisms underlying OA and could facilitate the development of new therapeutic strategies for this debilitating disease.

Localization of Calretinin, Parvalbumin, and S100 Protein in Nothobranchius guentheri Retina: A Suitable Model for the Retina Aging.

Calcium-binding proteins (CaBPs) are members of a heterogeneous family of proteins able to buffer intracellular Ca2+ ion concentration. CaBPs are expressed in the central and peripheral nervous system, including a subpopulation of retinal neurons. Since neurons expressing different CaBPs show different susceptibility to degeneration, it could be hypothesized that they are not just markers of different neuronal subpopulations, but that they might be crucial in survival. CaBPs' ability to buffer Ca2+ cytoplasmatic concentration makes them able to defend against a toxic increase in intracellular calcium that can lead to neurodegenerative processes, including those related to aging. An emergent model for aging studies is the annual killifish belonging to the Nothobranchius genus, thanks to its short lifespan. Members of this genus, such as Nothobranchius guentheri, show a retinal stratigraphy similar to that of other actinopterygian fishes and humans. However, according to our knowledge, CaBPs' occurrence and distribution in the retina of N. guentheri have never been investigated before. Therefore, the present study aimed to localize Calretinin N-18, Parvalbumin, and S100 protein (S100p) in the N. guentheri retina with immunohistochemistry methods. The results of the present investigation demonstrate for the first time the occurrence of Calretinin N-18, Parvalbumin, and S100p in N. guentheri retina and, consequently, the potential key role of these CaBPs in the biology of the retinal cells. Hence, the suitability of N. guentheri as a model to study the changes in CaBPs' expression patterns during neurodegenerative processes affecting the retina related both to disease and aging can be assumed.

The Role of Toll-like Receptor-4 in Macrophage Imbalance in Lethal COVID-19 Lung Disease, and Its Correlation with Galectin-3.

To the current data, there have been 6,955,141 COVID-19-related deaths worldwide, reported to WHO. Toll-like receptors (TLRs) implicated in bacterial and virus sensing could be a crosstalk between activation of persistent innate-immune inflammation, and macrophage's sub-population alterations, implicated in cytokine storm, macrophage over-activation syndrome, unresolved Acute Respiratory Disease Syndrome (ARDS), and death. The aim of this study is to demonstrate the association between Toll-like-receptor-4 (TLR-4)-induced inflammation and macrophage imbalance in the lung inflammatory infiltrate of lethal COVID-19 disease. Twenty-five cases of autopsy lung tissues were studied by digital pathology-based immunohistochemistry to evaluate expression levels of TLR-4 (CD 284), pan-macrophage marker CD68 (clone KP1), sub-population marker related to alveolar macrophage Galectin-3 (GAL-3) (clone 9C4), and myeloid derived CD163 (clone MRQ-26), respectively. SARS-CoV-2 viral persistence has been evaluated by in situ hybridation (ISH) method. This study showed TLR-4 up-regulation in a subgroup of patients, increased macrophage infiltration in both Spike-1(+) and Spike-1(-) lungs (p < 0.0001), and a macrophage shift with important down-regulation of GAL-3(+) alveolar macrophages associated with Spike-1 persistence (p < 0.05), in favor of CD163(+) myeloid derived monocyte-macrophages. Data show that TLR-4 expression induces a persistent activation of the inflammation, with inefficient resolution, and pathological macrophage shift, thus explaining one of the mechanisms of lethal COVID-19.

Developmental Expression of the Cell Cycle Regulator p16INK4a in Retinal Glial Cells: A Novel Marker for Immature Ocular Astrocytes?

Retinal astrocytes are vital for neuronal homeostasis in the retina. Together with Müller glia, they provide retinal cells with neurotrophic factors, antioxidative support, and defense mechanisms such as the formation of the blood-retinal barrier. Substantial heterogeneity of astrocyte morphology and function represents a challenge for identification of distinct subtypes which may be potential targets for therapeutic purposes. Hence, identification of novel markers of astrocyte subpopulations is highly relevant to better understand the molecular mechanisms involved in retinal development, homeostasis, and pathology. In this study, we observed that the cell cycle regulator, p16INK4a, is expressed in immature astrocytes in the mouse retina. Immunohistochemical analysis showed p16INK4a expression in the optic nerve of wild-type mice from 3 days to 3 months of age and in the nerve fiber layer of the adult mouse retina. Colocalization of p16INK4a expression and glial fibrillary acidic protein (immature/mature astrocyte marker) tends to decrease with age. However, colocalization of p16INK4a expression and vimentin (immature astrocyte marker) remains high in the optic nerve from the early postnatal period to adulthood. The observations from this study provide a valuable tool for further investigations of ocular astrocytes in the developing retina as well as in degenerative retinopathies.

Small RNA transcriptome analysis using parallel single-cell small RNA sequencing

miRNA and other forms of small RNAs are known to regulate many biological processes. Single-cell small RNA sequencing can be used to profile small RNAs of individual cells; however, limitations of efficiency and scale prevent its widespread application. Here, we developed parallel single-cell small RNA sequencing (PSCSR-seq), which can overcome the limitations of existing methods and enable high-throughput small RNA expression profiling of individual cells. Analysis of PSCSR-seq data indicated that diverse cell types could be identified based on patterns of miRNA expression, and showed that miRNA content in nuclei is informative (for example, cell type marker miRNAs can be detected in isolated nuclei). PSCSR-seq is very sensitive: analysis of only 732 peripheral blood mononuclear cells (PBMCs) detected 774 miRNAs, whereas bulk small RNA analysis would require input RNA from approximately 106 cells to detect as many miRNAs. We identified 42 miRNAs as markers for PBMC subpopulations. Moreover, we analyzed the miRNA profiles of 9,533 cells from lung cancer biopsies, and by dissecting cell subpopulations, we identified potentially diagnostic and therapeutic miRNAs for lung cancers. Our study demonstrates that PSCSR-seq is highly sensitive and reproducible, thus making it an advanced tool for miRNA analysis in cancer and life science research.

Heterogeneity of glycan biomarker clusters as an indicator of recurrence in pancreatic cancer.

Outcomes following tumor resection vary dramatically among patients with pancreatic ductal adenocarcinoma (PDAC). A challenge in defining predictive biomarkers is to discern within the complex tumor tissue the specific subpopulations and relationships that drive recurrence. Multiplexed immunofluorescence is valuable for such studies when supplied with markers of relevant subpopulations and analysis methods to sort out the intra-tumor relationships that are informative of tumor behavior. We hypothesized that the glycan biomarkers CA19-9 and STRA, which detect separate subpopulations of cancer cells, define intra-tumoral features associated with recurrence. We probed this question using automated signal thresholding and spatial cluster analysis applied to the immunofluorescence images of the STRA and CA19-9 glycan biomarkers in whole-block sections of PDAC tumors collected from curative resections. The tumors (N = 22) displayed extreme diversity between them in the amounts of the glycans and in the levels of spatial clustering, but neither the amounts nor the clusters of the individual and combined glycans associated with recurrence. The combined glycans, however, marked divergent types of spatial clusters, alternatively only STRA, only CA19-9, or both. The co-occurrence of more than one cluster type within a tumor associated significantly with disease recurrence, in contrast to the independent occurrence of each type of cluster. In addition, intra-tumoral regions with heterogeneity in biomarker clusters spatially aligned with pathology-confirmed cancer cells, whereas regions with homogeneous biomarker clusters aligned with various non-cancer cells. Thus, the STRA and CA19-9 glycans are markers of distinct and co-occurring subpopulations of cancer cells that in combination are associated with recurrence. Furthermore, automated signal thresholding and spatial clustering provides a tool for quantifying intra-tumoral subpopulations that are informative of outcome.

Abstract 4159: The COX-2/PGE2 pathway as a mediator of resistance to anti-PD-1 therapy

Abstract Background: We previously found upregulation of the cyclooxygenase-2/prostaglandin E2 (COX-2/PGE2) pathway in the tumor microenvironment (TME) of cancer types that respond poorly to anti-PD-1 therapy, and showed that TME-resident cytokines such as IL-1B induce COX-2 expression in human tumor and myeloid cells. To investigate a potential role for COX-2/PGE2 in anti-PD-1 resistance, we studied PGE2-mediated effects on human myeloid and T cells and their dependence on prostaglandin (EP) receptor signaling. Methods: Monocytes (Monos) and T cells were enriched from normal donor peripheral blood by negative selection. PGE2 effects were tested on Monos activated with IFN-g (PD-L1 expression; FACS) or LPS (cytokine secretion; ELISA). PGE2 effects on monocytic dendritic cells (DCs) generated with GM-CSF+IL-4 and matured with CD40L were assessed by phenotyping for costimulatory molecules (CD80, CD86) and subpopulation markers (CD14, CD16, CD83). T cells activated with anti-CD3/CD28 +/- PGE2 for 2-6 days were assessed for expression of CD25, CD69, CD107a, CD137, OX40, GITR, LAG-3, PD-1, PD-1, PD-L1 and TIM3 (FACS); proliferation markers (Ki67, CFSE; FACS); cytokine secretion (IFN-g, IL-2, IL-10, TGF-b; ELISA); and metabolic function (Seahorse assay). In some experiments, cells were pre-incubated with inhibitors of EP2 (PF-04418948), EP4 (ONO-AE3-208), or the dual inhibitor TPST-1495 (Tempest Therapeutics). Results: Myeloid cell functions were suppressed by PGE2, which reduced IFNg-induced PD-L1 expression and modulated LPS-induced cytokine secretion. Furthermore, PGE2 inhibited DC maturation, evidenced by persistent CD14 and CD16 expression and a decreased CD83+ subset. In CD4 and CD8 T cells activated with anti-CD3/CD28, PGE2 reduced expression of CD25 and CD69 (early activation markers), CD107a (cytolytic potential), Ki67 (cell proliferation), co-stimulatory molecules (CD137, OX40, GITR) and checkpoint molecules (LAG-3, PD-1, PD-L1, TIM3) in a dose-dependent manner. PGE2 also reduced the CFSE-low (proliferating) population in activated CD4, CD8 and Treg cells. Secretion of proinflammatory cytokines IFN-g and IL-2, and inhibitory cytokines IL-10 and TGF-b from activated T cells was also suppressed by PGE2. These effects were specifically reversed by chemical inhibitors of EP2 and/or EP4. PGE2 exposure at the time of T cell activation was associated with depressed mitochondrial oxidation and cellular glycolysis, suggesting global effects on T cell metabolism. Conclusion: Understanding and preventing anti-PD-1 resistance is a critical goal in oncology. Our results suggest that the COX-2/PGE2 pathway exerts immunosuppressive effects on myeloid and T cells via the EP2 and EP4 receptors. COX-2 and PD-L1 are distinct immune checkpoints, providing a rationale to test anti-PD-1 in conjunction with COX-2 pathway inhibitors such as IL-1B antagonists and highly specific EP2 and EP4 inhibitors in patients with cancer. Citation Format: Shuming Chen, Seoho Lee, Tracee L. McMiller, Isaac Morales, Preethi Sankaran, Brian Francica, Robert D. Leone, Tom W. Dubensky, Suzanne L. Topalian. The COX-2/PGE2 pathway as a mediator of resistance to anti-PD-1 therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4159.

Temporal transcriptome profiling of floating apical out chicken enteroids suggest stability and reproducibility

Enteroids are miniature self-organising three-dimensional (3D) tissue cultures which replicate much of the complexity of the intestinal epithelium. We recently developed an apical-out leukocyte-containing chicken enteroid model providing a novel physiologically relevant in vitro tool to explore host–pathogen interactions in the avian gut. However, the replicate consistency and culture stability have not yet been fully explored at the transcript level. In addition, causes for the inability to passage apical-out enteroids were not determined. Here we report the transcriptional profiling of chicken embryonic intestinal villi and chicken enteroid cultures using bulk RNA-seq. Comparison of the transcriptomes of biological and technical replicate enteroid cultures confirmed their high level of reproducibility. Detailed analysis of cell subpopulation and function markers revealed that the mature enteroids differentiate from late embryonic intestinal villi to recapitulate many digestive, immune and gut-barrier functions present in the avian intestine. These transcriptomic results demonstrate that the chicken enteroid cultures are highly reproducible, and within the first week of culture they morphologically mature to appear similar to the in vivo intestine, therefore representing a physiologically-relevant in vitro model of the chicken intestine.

Abstract P075: Comparison of the tumor and lymph node immune microenvironment in early non-small cell lung cancer through multimodal single cell sequencing

Abstract Background/Purpose: Previous studies have analyzed the tumor and local immune microenvironments in lung cancers and suggest immune modulation is associated with worse clinical outcome. However, the tumor-immune microenvironment in early-stage lung tumors and lymph nodes (LNs) have not been fully described. We aim to compare cell states in the immune microenvironments between lung tumors and LNs through multi-modal profiling of the transcriptome and surface proteins. Methods: Needle biopsy samples were taken from 10 treatment-naive early-stage lung cancer patients undergoing lung cancer resections. Tissues were obtained from normal lung, lung tumor, and multiple mediastinal LNs, and processed with scRNA-seq along with the Total-Seq C panel to quantify the levels of 130 cell surface proteins. In total, 98,337 cells (4,661 normal lung; 52,119 tumor; 42,117 LN) were identified with a median of 1,401 genes and 23 protein features detected per cell. Weighted-Nearest Neighbor analysis from the Seurat R package was applied to integrate the CITE-seq and RNA-seq level data for clustering cells into subpopulations. Results: Six broad cell populations were identified including T/NK, myeloid (CD14+), B (CD19+), mast (TPSAB1+), pDC (IRF8+), and epithelial (EPCAM+) cells. Protein expression measured with CITE-seq revealed additional T cell populations not captured by scRNA-seq. Preliminary results have identified cell populations enriched in LNs, which include naive CD4+ (CD4, FHIT, LEF1, CCR7) and CD8+ T cells (CD8A, TCF7, LEF1, CD27, CCR7). Additionally, we have identified numerous other CD8+ T-cell subpopulations enriched in tumors, including resident memory CD8+ T cells (KLRC1, ITGAE, ITGA1, VIM, JUN). Furthermore, immune populations enriched in LNs were similarly shared across different patients, while those enriched in tumors displayed patient-level specificity. Conclusion: Single-cell profiling reveals diversity in immune cell populations between LNs, tumor, and adjacent normal tissue in early-stage LUAD. The results suggest the composition of immune cell type is consistent across LNs but more heterogeneous in the tumor and adjacent normal tissue. In the future, we aim to determine if these markers for various immune subpopulations are associated with survival, recurrence, aggressiveness, and predict responses for neoadjuvant treatments, which could improve prognosis and patient quality of life. Citation Format: Zhan Xi, Yusuke Koga, Shannon McDermott, Jennifer E. Beane, Sarah A. Mazzilli, Kei Suzuki, Joshua D. Campbell. Comparison of the tumor and lymph node immune microenvironment in early non-small cell lung cancer through multimodal single cell sequencing. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P075.