Single-cell Transcriptional Profiling Research Articles

Blockade of IL-1β and PD-1 with combination chemotherapy reduces systemic myeloid suppression in metastatic pancreatic cancer with heterogeneous effects in the tumor.

Innate inflammation promotes tumor development, although the role of innate inflammatory cytokines in established human tumors is unclear. Here we report clinical and translational results from a phase Ib trial testing whether IL-1β blockade in human pancreatic cancer would alleviate myeloid immunosuppression and reveal antitumor T-cell responses to PD-1 blockade. Patients with treatment-naïve advanced pancreatic ductal adenocarcinoma (n=10) were treated with canakinumab, a high-affinity monoclonal human anti-interleukin-1β (IL-1β), the PD-1 blocking antibody spartalizumab, and gemcitabine/n(ab)paclitaxel. Analysis of paired peripheral blood from patients in the trial versus patients receiving multiagent chemotherapy showed a modest increase in HLA-DR+CD38+ activated CD8+ T cells and a decrease in circulating monocytic myeloid-derived suppressor cells (MDSCs) by flow cytometry for patients in the trial, but not in controls. Similarly, we used patient serum to differentiate monocytic MDSCs in vitro and showed that functional inhibition of T-cell proliferation was reduced when using on-treatment serum samples from patients in the trial but not when using serum from patients treated with chemotherapy alone. Within the tumor we observed few changes in suppressive myeloid-cell populations or activated T cells as assessed by single-cell transcriptional profiling or multiplex immunofluorescence, although increases in CD8+ T cells suggest that improvements in the tumor immune microenvironment might be revealed by a larger study. Overall, the data indicate that exposure to PD-1 and IL-1β blockade induced a modest reactivation of peripheral CD8+ T cells and decreased circulating monocytic MDSCs; however, these changes did not lead to similarly uniform alterations in the tumor microenvironment.

Using Human Intestinal Organoids to Understand the Small Intestine Epithelium at the Single Cell Transcriptional Level.

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid cultures provide ex vivo model systems that bridge the gap between animal models and clinical studies. The application of single cell transcriptomics to human intestinal organoid (HIO) models is revealing previously unrecognized cell biology, biochemistry, and physiology of the GI tract. The advanced single cell transcriptomics platforms use microfluidic partitioning and barcoding to generate cDNA libraries. These barcoded cDNAs can be easily sequenced by next generation sequencing platforms and used by various visualization tools to generate maps. Here, we describe methods to culture and differentiate human small intestinal HIOs in different formats and procedures for isolating viable cells from these formats that are suitable for use in single-cell transcriptional profiling platforms. These protocols and procedures facilitate the use of small intestinal HIOs to obtain an increased understanding of the cellular response of human intestinal epithelium at the transcriptional level in the context of a variety of different environments.

Transient stabilization of human cardiovascular progenitor cells from hPSCs in vitro reflects stage-specific heart development in vivo.

Understanding the molecular identity of human pluripotent stem cell (hPSC)-derived cardiac progenitors and mechanisms controlling their proliferation and differentiation, is valuable for developmental biology and regenerative medicine. Here we show that chemical modulation of Histone Acetyl Transferases (HATs; by IQ-1) and WNT (by CHIR99021), synergistically enable the transient and reversible block of directed cardiac differentiation progression on hPSCs. The resulting stabilized cardiovascular progenitors (SCPs) are characterized by ISL1pos/KI-67pos/NKX2-5neg expression. In the presence of the chemical inhibitors, SCPs maintain a proliferation quiescent state. Upon small molecules removal SCPs resume proliferation and concomitant NKX2-5 upregulation triggers cell-autonomous differentiation into cardiomyocytes. Directed differentiation of SCPs into the endothelial and smooth muscle lineages confirms their full developmental potential typical of bona fide cardiovascular progenitors. Single-cell RNAseq-based transcriptional profiling of our in vitro generated human SCPs notably reflects the dynamic cellular composition of E8.25-E9.25 posterior second heart field (pSHF) of mouse hearts, hallmarked by NR2F2 expression. Investigating molecular mechanisms of SCP stabilization, we found that the cell-autonomously regulated Retinoic Acid (RA) and BMP signaling is governing SCPs transition from quiescence towards proliferation and cell-autonomous differentiation, reminiscent of a niche-like behavior. The chemically defined and reversible nature our stabilization approach provides an unprecedented opportunity to dissect mechanisms of cardiovascular progenitors' specification and reveal their cellular and molecular properties.

Unveiling gene regulatory networks during cellular state transitions without linkage across time points

Time-stamped cross-sectional data, which lack linkage across time points, are commonly generated in single-cell transcriptional profiling. Many previous methods for inferring gene regulatory networks (GRNs) driving cell-state transitions relied on constructing single-cell temporal ordering. Introducing COSLIR (COvariance restricted Sparse LInear Regression), we presented a direct approach to reconstructing GRNs that govern cell-state transitions, utilizing only the first and second moments of samples between two consecutive time points. Simulations validated COSLIR’s perfect accuracy in the oracle case and demonstrated its robust performance in real-world scenarios. When applied to single-cell RT-PCR and RNAseq datasets in developmental biology, COSLIR competed favorably with existing methods. Notably, its running time remained nearly independent of the number of cells. Therefore, COSLIR emerges as a promising addition to GRN reconstruction methods under cell-state transitions, bypassing the single-cell temporal ordering to enhance accuracy and efficiency in single-cell transcriptional profiling.

Single-cell transcriptional profiling in osteosarcoma and the effect of neoadjuvant chemotherapy on the tumor microenvironment

Osteosarcoma (OS), a malignant tumor, originates from the bone marrow. Currently, treatment for OS remains limited, making it urgent to understand the immune response in the tumor microenvironment of patients with OS. A comprehensive bioinformatics analysis was performed, including cell clustering subgroups, differential expression genes screening, proposed temporal order, and genomic variant analysis on single-cell RNA-sequencing data, from ten pre-chemotherapy patients and eleven post-chemotherapy patients. Subsequently, we analyzed the differentiation trajectories of osteoblasts, osteoclasts, fibroblasts, myeloid cells, and tumor-infiltrating lymphocytes (TILs) in detail to compare the changes in cell proportions and differential genes pre- and post-chemotherapy. The nine cell types were identified, including fibroblasts, myeloid cells, osteoblasts, TILs, osteoclasts, proliferative osteoblasts, pericytes, endothelial cells, and B cells. Post-chemotherapy treatment, the proportions of myeloid cells and TILs in OS were declined, while the number of osteoblasts was elevated. Besides, a decrease was observed in CD74, FTL, FTH1, MT1X and MT2A, and an increase in PTN, COL3A1, COL1A1, IGFBP7 and FN1. Meanwhile, EMT, DNA repair, G2M checkpoint, and E2F targets were highly enriched post-chemotherapy. Furthermore, there was a down-regulation in the proportions of CD14 monocytes, Tregs, NK cells and CD1C-/CD141-DCs, while an up-regulation was observed in the proportions of SELENOP macrophages, IL7R macrophages, COL1A1 macrophages, CD1C DCs, CD4+ T cells and CD8+ T cells. Overall, these findings revealed changes in the tumor microenvironment of OS post-chemotherapy treatment, providing a new direction for investigating OS treatment.

Abstract PO4-01-02: High throughput analysis in HER2 positive locally advanced breast cancer: pCR and mutational status

Abstract Background Pathologic complete response (pCR) after neoadjuvant treatment (NAD) is a strong prognostic biomarker associated with improved survival in breast cancer patients, especially with HER2-positive and Triple negative subtypes. PIK3CA is mutated in up to 20% of HER2 positive breast cancers, contributes to anti-HER-2 resistance and may be predictive of the lack of response to anti HER2 NAD. PIK3CA mutations in breast cancer occur primarily at hotspots E545K at exon 9 and H1047R at exon 20. Next generation sequencing has improved our knowledge regarding the biology of the mechanisms behind treatment resistance. The aim of this study is to evaluate the genomic landscape of tissue samples obtained from locally advanced breast cancer patients treated in an neoadjuvant setting, in order to correlate the genomic analysis to response and clinical outcome. Methods DNA extracted from the archival 84 formalin-fixed paraffin-embedded (FFPE) samples from core needle biopsies was subjected to deep sequencing using the TruSight Oncology (TSO) 500 panel (Illumina, San Diego, USA; 523 genes, size: 1.94 Mb), following the manufacturer’s protocol, which assesses microsatellite instability (MSI) status, tumor mutation burden (TMB), recurrent somatic copy number variations (sCNV), somatic single nucleotide variants (SNVs). Libraries were sequenced on a NovaSeq 6000 instrument (Illumina) to reach a minimum of 500 × read depth. All samples were evaluated for RNA single cell transcriptional profile. 73 of 84 patients were treated with traditional neoadjuvant chemotherapy plus trastuzumab meanwhile 13 of 84 received neoadjuvant chemotherapy plus trastuzumab and pertuzumab. Statistical analysis was performed to detect the genes whose mutation involved in the progression and/or non-response of the disease using the Statistical Package for Social Science (SPSS), release 23.0. Continuous variables were expressed as mean ± SD or median [range], categorical variables were displayed as frequencies and the appropriate parametric (Student‘s t-test) non-parametric test (X2-test or Mann-Whitney test) was used to assess significance of the differences between subgroups (patients with or without pCR). Results In total we analyzed samples of 84 patients. The median age was 48 years. 64 of 84 (76.2%) were triple positive, 66 (78.6%) with G3 and 29 (34.5%) with stage III. The most common frequently mutated genes were PIK3CA (16/84, 19%) and BRCA1 (7/84, 8.3%). The pathogenic variant of BRCA1 (sBRCA1) had a significant association with resistance to treatment: we found a pCR in only 14.3% of sBRCA1 versus 62.3% in sBRCA1 wild type (wt) patients (p=0.014). We also identified 11 somatic mutations in hereditary genes (ATM, BRCA1, BRCA2, PALB2, CHEK2, RAD51C, RAD51D). From a germline standpoint we detected 7 pathogenic variants (two affecting BRCA1, two affecting CHEK2, one affecting PALB2, 7/84, 8.3%), which will be clinically confirmed. Among patients with a mutated PI3KCA, 43.8% obtained a pCR versus 61.8% of patients with PI3KCA wt (p=0.19). This data probably presents an imbalance due to the sample size A PTEN mutation was identified in 2% of pts (2/84) which obtained no benefit in terms of pCR after NAD versus 59.8% in wt patients (p=0.09). Conclusions In this preliminary report, genetic drivers such as BRCA1 status may have a clinical implication for prognosis and treatment response after NAD in locally advanced HER2 positive breast cancer. More samples are needed to evaluate the significance of PI3K and PTEN mutations in this patient setting. We are waiting for the results of the RNA single cell transcriptional profile of these samples. Event free survival data is not yet mature at this moment. We would like to acknowledge the contribution of Multi-specialistic Biobank Research Core Facility G-STeP, Fondazione Policlinico Universitario “A. Gemelli” IRCCS (Biobank-FPG) who provided the bio-resources. Citation Format: Ida Paris, Angelo Minucci, Daniele Generali, simona Nanni, Tatiana D'angelo, Francesco Pavese, Alessandra Fabi, Maria De Bonis, Katia Cannita, Alessia Piermattei, Giordana Tiberi, Paola Fuso, Luisa Carbognin, Silvia Rotondaro, Camilla Nero, Flavia Giacomini, Tina Pasciuto, Maria Marrazzo, Luciano Giaco', Antonino Mulè, Riccardo Masetti, Gianluca Franceschini, Giorgia Garganese, Diana Giannarelli, Giovanni Scambia. High throughput analysis in HER2 positive locally advanced breast cancer: pCR and mutational status [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-01-02.

Transcription factor Tox2 is required for metabolic adaptation and tissue residency of ILC3 in the gut

Group 3 innate lymphoid cells (ILC3) are the major subset of gut-resident ILC with essential roles in infections and tissue repair, but how they adapt to the gut environment to maintain tissue residency is unclear. We report that Tox2 is critical for gut ILC3 maintenance and function. Gut ILC3 highly expressed Tox2, and depletion of Tox2 markedly decreased ILC3 in gut but not at central sites, resulting in defective control of Citrobacter rodentium infection. Single-cell transcriptional profiling revealed decreased expression of Hexokinase-2 in Tox2-deficient gut ILC3. Consistent with the requirement for hexokinases in glycolysis, Tox2-/- ILC3 displayed decreased ability to utilize glycolysis for protein translation. Ectopic expression of Hexokinase-2 rescued Tox2-/- gut ILC3 defects. Hypoxia and interleukin (IL)-17A each induced Tox2 expression in ILC3, suggesting a mechanism by which ILC3 adjusts to fluctuating environments by programming glycolytic metabolism. Our results reveal the requirement for Tox2 to support the metabolic adaptation of ILC3 within the gastrointestinal tract.

Single-Cell Transcriptional Profile Construction of Rat Pituitary Glands before and after Sexual Maturation and Identification of Novel Marker Spp1 in Gonadotropes.

The mammalian pituitary gland drives highly conserved physiological processes such as somatic cell growth, pubertal transformation, fertility, and metabolism by secreting a variety of hormones. Recently, single-cell transcriptomics techniques have been used in pituitary gland research. However, more studies have focused on adult pituitary gland tissues from different species or different sexes, and no research has yet resolved cellular differences in pituitary gland tissue before and after sexual maturation. Here, we identified a total of 15 cell clusters and constructed single-cell transcriptional profiles of rats before and after sexual maturation. Furthermore, focusing on the gonadotrope cluster, 106 genes were found to be differentially expressed before and after sexual maturation. It was verified that Spp1, which is specifically expressed in gonadotrope cells, could serve as a novel marker for this cell cluster and has a promotional effect on the synthesis and secretion of follicle-stimulating hormone. The results provide a new resource for further resolving the regulatory mechanism of pituitary gland development and pituitary hormone synthesis and secretion.

Distinct features of the regenerating heart uncovered through comparative single-cell profiling.

Adult humans respond to heart injury by forming a permanent scar, yet other vertebrates are capable of robust and complete cardiac regeneration. Despite progress towards characterizing the mechanisms of cardiac regeneration in fish and amphibians, the large evolutionary gulf between mammals and regenerating vertebrates complicates deciphering which cellular and molecular features truly enable regeneration. To better define these features, we compared cardiac injury responses in zebrafish and medaka, two fish species that share similar heart anatomy and common teleost ancestry but differ in regenerative capability. We used single-cell transcriptional profiling to create a time-resolved comparative cell atlas of injury responses in all major cardiac cell types across both species. With this approach, we identified several key features that distinguish cardiac injury response in the non-regenerating medaka heart. By comparing immune responses to injury, we found altered cell recruitment and a distinct pro-inflammatory gene program in medaka leukocytes, and an absence of the injury-induced interferon response seen in zebrafish. In addition, we found a lack of pro-regenerative signals, including nrg1 and retinoic acid, from medaka endothelial and epicardial cells. Finally, we identified alterations in the myocardial structure in medaka, where they lack primordial layer cardiomyocytes and fail to employ a cardioprotective gene program shared by regenerating vertebrates. Our findings reveal notable variation in injury response across nearly all major cardiac cell types in zebrafish and medaka, demonstrating how evolutionary divergence influences the hidden cellular features underpinning regenerative potential in these seemingly similar vertebrates.

GPX8+ cancer-associated fibroblast, as a cancer-promoting factor in lung adenocarcinoma, is related to the immunosuppressive microenvironment

BackgroundCancer-associated fibroblasts (CAFs) play a crucial role in the tumor microenvironment of lung adenocarcinoma (LUAD) and are often associated with poorer clinical outcomes. This study aimed to screen for CAF-specific genes that could serve as promising therapeutic targets for LUAD.MethodsWe established a single-cell transcriptional profile of LUAD, focusing on genetic changes in fibroblasts. Next, we identified key genes associated with fibroblasts through weighted gene co-expression network analysis (WGCNA) and univariate Cox analysis. Then, we evaluated the relationship between glutathione peroxidase 8 (GPX8) and clinical features in multiple independent LUAD cohorts. Furthermore, we analyzed immune infiltration to shed light on the relationship between GPX8 immune microenvironment remodeling. For clinical treatment, we used the tumor immune dysfunction and exclusion (TIDE) algorithm to assess the immunotherapy prediction efficiency of GPX8. After that, we screened potential therapeutic drugs for LUAD by the connectivity map (cMAP). Finally, we conducted a cell trajectory analysis of GPX8+ CAFs to show their unique function.ResultsFibroblasts were found to be enriched in tumor tissues. Then we identified GPX8 as a key gene associated with CAFs through comprehensive bioinformatics analysis. Further analysis across multiple LUAD cohorts demonstrated the relationship between GPX8 and poor prognosis. Additionally, we found that GPX8 played a role in inducing the formation of an immunosuppressive microenvironment. The TIDE method indicated that patients with low GPX8 expression were more likely to be responsive to immunotherapy. Using the cMAP, we identified beta-CCP as a potential drug-related to GPX8. Finally, cell trajectory analysis provided insights into the dynamic process of GPX8+ CAFs formation.ConclusionsThis study elucidates the association between GPX8+ CAFs and poor prognosis, as well as the induction of immunosuppressive formation in LUAD. These findings suggest that targeting GPX8+ CAFs could potentially serve as a therapeutic strategy for the treatment of LUAD.

Circulating hematopoietic stem/progenitor cell subsets contribute to human hematopoietic homeostasis

AbstractIn physiological conditions, few circulating hematopoietic stem/progenitor cells (cHSPCs) are present in the peripheral blood, but their contribution to human hematopoiesis remain unsolved. By integrating advanced immunophenotyping, single-cell transcriptional and functional profiling, and integration site (IS) clonal tracking, we unveiled the biological properties and the transcriptional features of human cHSPC subpopulations in relationship to their bone marrow (BM) counterpart. We found that cHSPCs reduced in cell count over aging and are enriched for primitive, lymphoid, and erythroid subpopulations, showing preactivated transcriptional and functional state. Moreover, cHSPCs have low expression of multiple BM-retention molecules but maintain their homing potential after xenotransplantation. By generating a comprehensive human organ-resident HSPC data set based on single-cell RNA sequencing data, we detected organ-specific seeding properties of the distinct trafficking HSPC subpopulations. Notably, circulating multi-lymphoid progenitors are primed for seeding the thymus and actively contribute to T-cell production. Human clonal tracking data from patients receiving gene therapy (GT) also showed that cHSPCs connect distant BM niches and participate in steady-state hematopoietic production, with primitive cHSPCs having the highest recirculation capability to travel in and out of the BM. Finally, in case of hematopoietic impairment, cHSPCs composition reflects the BM-HSPC content and might represent a biomarker of the BM state for clinical and research purposes. Overall, our comprehensive work unveiled fundamental insights into the in vivo dynamics of human HSPC trafficking and its role in sustaining hematopoietic homeostasis. GT patients’ clinical trials were registered at ClinicalTrials.gov (NCT01515462 and NCT03837483) and EudraCT (2009-017346-32 and 2018-003842-18).

ASCL1 Drives Tolerance to Osimertinib in EGFR Mutant Lung Cancer in Permissive Cellular Contexts.

Analysis of residual disease following tyrosine kinase inhibitor treatment identified heterogeneous and context-specific mechanisms of drug tolerance in lung cancer that could lead to the development of strategies to forestall drug resistance. See related commentary by Rumde and Burns, p. 1188.

Abstract A079: Loss of BPTF restores estrogen-regulated programs and suppress metastatic development of mammary tumors

Abstract Chromatin remodeling controls transcriptional programs that drive mammary epithelial lineage commitment and cellular differentiation. This process is also important for the regulation of highly dynamic cellular states during mammary tumorigenesis, and perturbing the chromatin landscape of cancer cells can halt cellular and molecular mechanisms of mammary cancer development and progression. However, this paradigm assumes that all cancer cells that develop in the mammary tissue bear the same chromatin dependencies, and rely on similar programs throughout cancer progression. Here, we show that molecular dependencies regulated by the epigenetic factor BPTF control tumor development and progression across distinct mammary cancer cell states. With the utilization of classical models of mammary tumorigenesis, single cell transcriptional profiling, and organoid cultures, we demonstrate that BPTF loss results in the development of mammary tumors with a longer latency of ER•+ expression and tamoxifen sensitivity and the inhibition of lung metastasis development. While gain of ER• expression was associated with gain of luminal markers in Krt5+ BPTF KO mammary epithelial cells (MECs), metastasis-like signatures were significantly perturbed in cells with defined luminal fate. In addition, we detected a partial rescue of metastatic features via the activation of transcriptional programs controlled by TGF-•, thus indicating that BPTF sits atop a canonical metastatic pathway. Collectively, our studies illustrate how BPTF-specific chromatin remodeling inhibition induces phenotypes that differentially affect tumor development and progression, while allowing for the development of a murine model of ER•+ mammary tumorigenesis. Citation Format: Chen Chen, Michael F. Ciccone, Marygrace c. Trousdell, Deeptiman Chatterjee, Yixin Zhao, Steven M. Lewis, Dhivya Anandan, John E. Wilkinson, William C. K. Pomerantz, Adam Siepel, Camila O. dos Santos. Loss of BPTF restores estrogen-regulated programs and suppress metastatic development of mammary tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A079.

Robust detection of clinically relevant features in single-cell RNA profiles of patient-matched fresh and formalin-fixed paraffin-embedded (FFPE) lung cancer tissue.

Single-cell transcriptional profiling reveals cell heterogeneity and clinically relevant traits in intra-operatively collected patient-derived tissue. So far, single-cell studies have been constrained by the requirement for prospectively collected fresh or cryopreserved tissue. This limitation might be overcome by recent technical developments enabling single-cell analysis of FFPE tissue. We benchmark single-cell profiles from patient-matched fresh, cryopreserved and archival FFPE cancer tissue. We find that fresh tissue and FFPE routine blocks can be employed for the robust detection of clinically relevant traits on the single-cell level. Specifically, single-cell maps of fresh patient tissues and corresponding FFPE tissue blocks could be integrated into common low-dimensional representations, and cell subtype clusters showed highly correlated transcriptional strengths of signaling pathway, hallmark, and clinically useful signatures, although expression of single genes varied due to technological differences. FFPE tissue blocks revealed higher cell diversity compared to fresh tissue. In contrast, single-cell profiling of cryopreserved tissue was prone to artifacts in the clinical setting. Our analysis highlights the potential of single-cell profiling in the analysis of retrospectively and prospectively collected archival pathology cohorts and increases the applicability in translational research.

Single-Cell Transcription Mapping of Murine and Human Mammary Organoids Responses to Female Hormones

During female adolescence and pregnancy, rising levels of hormones result in a cyclic source of signals that control the development of mammary tissue. While such alterations are well understood from a whole-gland perspective, the alterations that such hormones bring to organoid cultures derived from mammary glands have yet to be fully mapped. This is of special importance given that organoids are considered suitable systems to understand cross species breast development. Here we utilized single-cell transcriptional profiling to delineate responses of murine and human normal breast organoid systems to female hormones across evolutionary distinct species. Collectively, our study represents a molecular atlas of epithelial dynamics in response to estrogen and pregnancy hormones.

Single-cell transcriptional profiling of porcine muscle satellite cells and myoblasts during myogenesis

Here, we unravel the transcriptional mechanisms that govern porcine muscle myogenesis, a crucial aspect of pork production. Through single cell RNA-sequencing of longissimus dorsi (LD) muscle from 3-day-old piglets, we delineated 5 major cell types from 14,002 cells—muscle cells, fibro-adipogenic progenitors, mural cells, endothelial cells, and immune cells. Within the muscle cell clusters, we specifically identified muscle satellite cells and myoblasts, with 5,388 differentially expressed genes distinguishing the cell populations. Furthermore, our investigation led to the identification of novel cell surface markers for porcine muscle satellite cells, namely ITGA7, SDC2, and SDC4. In addition, we found 55 transcription factors, including AHCTF1, CEBPD, and MAX, which contribute to the regulation of muscle development. Through predictive receptor–ligand analysis, we uncovered 16 secretory factors with associated cellular communication channels influencing porcine skeletal muscle development. Notably, we identified the secretory factor ANGPTL4 as an inhibitor of porcine muscle satellite cell proliferation and differentiation, and confirmed its function through the in vitro treatment of cell lines. We propose that ANGTPL4 is a potential target for the treatment of myopathy. Taken together, our findings contribute to the identification and characterization of myogenic genes and regulatory networks, which enhances our understanding of porcine muscle satellite cell myogenesis and offers potential avenues for optimizing pork production.

Integrating bulk-RNA sequencing and single-cell sequencing analyses to characterize adenosine-enriched tumor microenvironment landscape and develop an adenosine-related prognostic signature predicting immunotherapy in lung adenocarcinoma.

The adenosine-signaling axis has been recognized as an important immunomodulatory pathway in tumor immunity. However, the biological role of the adenosine-signaling axis in the remodeling of the tumor microenvironment (TME) in lung adenocarcinoma (LUAD) remains unclear. Here, we quantified adenosine signaling (ado_sig) in LUAD samples using the GSVA method and assessed the prognostic value of adenosine in LUAD. Afterward, we explored the heterogeneity of the tumor-immune microenvironment at different adenosine levels. In addition, we analyzed the potential biological pathways engaged by adenosine. Next, we established single-cell transcriptional profiles of LUAD and analyzed cellular composition and cell-cell communication analysis under different adenosine microenvironments. Moreover, we established adenosine-related prognostic signatures (ARS) based on comprehensive bioinformatics analysis and evaluated the efficacy of ARS in predicting immunotherapy. The results demonstrated that adenosine signaling adversely impacted the survival of immune-enriched LUAD. The high-adenosine microenvironment exhibited elevated pro-tumor-immune infiltration, including M2 macrophages and displayed notably increased epithelial-mesenchymal transition (EMT) transformation. Furthermore, adenosine signaling displayed significant associations with the expression patterns and prognostic value of immunomodulators within theTME. Single-cell sequencing data revealed increased fibroblast occupancy and a prominent activation of the SPP1 signaling pathway in the high adenosine-signaling microenvironment. The ARS exhibited promising effectiveness in prognostication and predicting immunotherapy response in LUAD. In summary,overexpression of adenosine can cause a worsened prognosis in the LUAD with abundant immune infiltration. Moreover, increased adenosine levels are associated with pro-tumor-immune infiltration, active EMT transformation, pro-tumor angiogenesis, and other factors promoting cancer progression, which collectively contribute to the formation of an immunosuppressive microenvironment. Importantly, the ARS developed in this study demonstrate high efficacy in evaluating the response to immunotherapy.

Chromatin accessibility and cell cycle progression are controlled by the HDAC-associated Sin3B protein in murine hematopoietic stem cells

BackgroundBlood homeostasis requires the daily production of millions of terminally differentiated effector cells that all originate from hematopoietic stem cells (HSCs). HSCs are rare and exhibit unique self-renewal and multipotent properties, which depend on their ability to maintain quiescence through ill-defined processes. Defective control of cell cycle progression can eventually lead to bone marrow failure or malignancy. In particular, the molecular mechanisms tying cell cycle re-entry to cell fate commitment in HSCs remain elusive. Previous studies have identified chromatin coordination as a key regulator of differentiation in embryonic stem cells.ResultsHere, we utilized genetic inactivation of the chromatin-associated Sin3B protein to manipulate cell cycle control and found dysregulated chromatin accessibility and cell cycle progression in HSCs. Single cell transcriptional profiling of hematopoietic stem and progenitor cells (HSPCs) inactivated for Sin3B reveals aberrant progression through the G1 phase of the cell cycle, which correlates with the engagement of specific signaling pathways, including aberrant expression of cell adhesion molecules and the interferon signaling program in LT-HSCs. In addition, we uncover the Sin3B-dependent accessibility of genomic elements controlling HSC differentiation, which points to cell cycle progression possibly dictating the priming of HSCs for differentiation.ConclusionsOur findings provide new insights into controlled cell cycle progression as a potential regulator of HSC lineage commitment through the modulation of chromatin features.

Macro CD5L+ deteriorates CD8+T cells exhaustion and impairs combination of Gemcitabine-Oxaliplatin-Lenvatinib-anti-PD1 therapy in intrahepatic cholangiocarcinoma

Intratumoral immune status influences tumor therapeutic response, but it remains largely unclear how the status determines therapies for patients with intrahepatic cholangiocarcinoma. Here, we examine the single-cell transcriptional and TCR profiles of 18 tumor tissues pre- and post- therapy of gemcitabine plus oxaliplatin, in combination with lenvatinib and anti-PD1 antibody for intrahepatic cholangiocarcinoma. We find that high CD8 GZMB+ and CD8 proliferating proportions and a low Macro CD5L+ proportion predict good response to the therapy. In patients with a poor response, the CD8 GZMB+ and CD8 proliferating proportions are increased, but the CD8 GZMK+ proportion is decreased after the therapy. Transition of CD8 proliferating and CD8 GZMB+ to CD8 GZMK+ facilitates good response to the therapy, while Macro CD5L+–CD8 GZMB+ crosstalk impairs the response by increasing CTLA4 in CD8 GZMB+. Anti-CTLA4 antibody reverses resistance of the therapy in intrahepatic cholangiocarcinoma. Our data provide a resource for predicting response of the combination therapy and highlight the importance of CD8+T-cell status conversion and exhaustion induced by Macro CD5L+ in influencing the response, suggesting future avenues for cancer treatment optimization.

Single-cell transcriptional profiling reveals aberrant gene expression patterns and cell states in autoimmune diseases

Multiple sclerosis(MS), primary Sjögren syndrome (pSS), and systemic lupus erythematosus (SLE) share numerous clinical symptoms and serological characteristics. We analyzed 153550 cells of scRNA-seq data of 17 treatment-naive patients (5 MS, 5 pSS, and 7 SLE) and 10 healthy controls, and we examined the enrichment of biological processes, differentially expressed genes (DEGs), immune cell types, and their subpopulations, and cell-cell communication in peripheral blood mononuclear cells (PBMCs). The percentage of B cells, megakaryocytes, monocytes, and proliferating T cells presented significant changes in autoimmune diseases. The enrichment of cell types based on gene expression revealed an elevated monocyte. MIF, MK, and GALECTIN signaling networks were obvious differences in autoimmune diseases. Taken together, our analysis provides a comprehensive map of the cell types and states of ADs patients at the single-cell level to understand better the pathogenesis and treatment of these ADs.