ABSTRACT
Hepatocellular carcinoma (HCC) has high relapse and low 5-year survival rates. Single-cell profiling in relapsed HCC may aid in the design of effective anticancer therapies, including immunotherapies. We profiled the transcriptomes of â¼17,000 cells from 18 primary or early-relapse HCC cases. Early-relapse tumors have reduced levels of regulatory T cells, increased dendritic cells (DCs), and increased infiltrated CD8+ T cells, compared with primary tumors, in two independent cohorts. Remarkably, CD8+ T cells in recurrent tumors overexpressed KLRB1 (CD161) and displayed an innate-like low cytotoxic state, with low clonal expansion, unlike the classical exhausted state observed in primary HCC. The enrichment of these cells was associated with a worse prognosis. Differential gene expression and interaction analyses revealed potential immune evasion mechanisms in recurrent tumor cells that dampen DC antigen presentation and recruit innate-like CD8+ T cells. Our comprehensive picture of the HCC ecosystem provides deeper insights into immune evasion mechanisms associated with tumor relapse.
Subject(s)
Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology , Neoplasm Recurrence, Local/pathology , Single-Cell Analysis , CD8-Positive T-Lymphocytes/immunology , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/immunology , Gene Expression Regulation, Neoplastic , Humans , Killer Cells, Natural/immunology , Liver Neoplasms/genetics , Liver Neoplasms/immunology , Myeloid Cells/metabolism , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/immunology , Phenotype , RNA-Seq , Tumor MicroenvironmentABSTRACT
Peripheral blood mononuclear cells (PBMCs) reflect systemic immune response during cancer progression. However, a comprehensive understanding of the composition and function of PBMCs in cancer patients is lacking, and the potential of these features to assist cancer diagnosis is also unclear. Here, the compositional and status differences between cancer patients and healthy donors in PBMCs were investigated by single-cell RNA sequencing (scRNA-seq), involving 262,025 PBMCs from 68 cancer samples and 14 healthy samples. We observed an enhanced activation and differentiation of most immune subsets in cancer patients, along with reduction of naïve T cells, expansion of macrophages, impairment of NK cells and myeloid cells, as well as tumor promotion and immunosuppression. Based on characteristics including differential cell type abundances and/or hub genes identified from weight gene co-expression network analysis (WGCNA) modules of each major cell type, we applied logistic regression to construct cancer diagnosis models. Furthermore, we found that the above models can distinguish cancer patients and healthy donors with high sensitivity. Our study provided new insights into using the features of PBMCs in non-invasive cancer diagnosis.
Subject(s)
Leukocytes, Mononuclear , Neoplasms , Humans , Single-Cell Gene Expression Analysis , Neoplasms/diagnosis , Neoplasms/genetics , Cell Differentiation , Cell Transformation, NeoplasticABSTRACT
Endocrine-resistant ER+HER2- breast cancer (BC) is particularly aggressive and leads to poor clinical outcomes. Effective therapeutic strategies against endocrine-resistant BC remain elusive. Here, analysis of the RNA-sequencing data from ER+HER2- BC patients receiving neoadjuvant endocrine therapy and spatial transcriptomics analysis both show the downregulation of innate immune signaling sensing cytosolic DNA, which primarily occurs in endocrine-resistant BC cells, not immune cells. Indeed, compared with endocrine-sensitive BC cells, the activity of sensing cytosolic DNA through the cGAS-STING pathway is attenuated in endocrine-resistant BC cells. Screening of kinase inhibitor library show that this effect is mainly mediated by hyperactivation of AKT1 kinase, which binds to kinase domain of TBK1, preventing the formation of a trimeric complex TBK1/STING/IRF3. Notably, inactivation of cGAS-STING signaling forms a positive feedback loop with hyperactivated AKT1 to promote endocrine resistance, which is physiologically important and clinically relevant in patients with ER+HER2- BC. Blocking the positive feedback loop using the combination of an AKT1 inhibitor with a STING agonist results in the engagement of innate and adaptive immune signaling and impairs the growth of endocrine-resistant tumors in humanized mice models, providing a potential strategy for treating patients with endocrine-resistant BC.
ABSTRACT
Introduction: Helicobacter pylori (H.pylori, Hp) affects billions of people worldwide. However, the emerging resistance of Hp to antibiotics challenges the effectiveness of current treatments. Investigating the genotype-phenotype connection for Hp using next-generation sequencing could enhance our understanding of this resistance. Methods: In this study, we analyzed 52 Hp strains collected from various hospitals. The susceptibility of these strains to five antibiotics was assessed using the agar dilution assay. Whole-genome sequencing was then performed to screen the antimicrobial resistance (AMR) genotypes of these Hp strains. To model the relationship between drug resistance and genotype, we employed univariate statistical tests, unsupervised machine learning, and supervised machine learning techniques, including the development of support vector machine models. Results: Our models for predicting Amoxicillin resistance demonstrated 66% sensitivity and 100% specificity, while those for Clarithromycin resistance showed 100% sensitivity and 100% specificity. These results outperformed the known resistance sites for Amoxicillin (A1834G) and Clarithromycin (A2147), which had sensitivities of 22.2% and 87%, and specificities of 100% and 96%, respectively. Discussion: Our study demonstrates that predictive modeling using supervised learning algorithms with feature selection can yield diagnostic models with higher predictive power compared to models relying on single single-nucleotide polymorphism (SNP) sites. This approach significantly contributes to enhancing the precision and effectiveness of antibiotic treatment strategies for Hp infections. The application of whole-genome sequencing for Hp presents a promising pathway for advancing personalized medicine in this context.
Subject(s)
Helicobacter Infections , Helicobacter pylori , Humans , Clarithromycin/pharmacology , Clarithromycin/therapeutic use , Helicobacter Infections/drug therapy , Helicobacter Infections/genetics , Helicobacter pylori/genetics , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Amoxicillin/pharmacology , Amoxicillin/therapeutic use , Drug Resistance, Microbial , Machine Learning , Whole Genome Sequencing , Drug Resistance, Bacterial/genetics , Microbial Sensitivity TestsABSTRACT
Dissecting and understanding the cancer ecosystem, especially that around the tumor margins, which have strong implications for tumor cell infiltration and invasion, are essential for exploring the mechanisms of tumor metastasis and developing effective new treatments. Using a novel tumor border scanning and digitization model enabled by nanoscale resolution-SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq), we identified a 500 µm-wide zone centered around the tumor border in patients with liver cancer, referred to as "the invasive zone". We detected strong immunosuppression, metabolic reprogramming, and severely damaged hepatocytes in this zone. We also identified a subpopulation of damaged hepatocytes with increased expression of serum amyloid A1 and A2 (referred to collectively as SAAs) located close to the border on the paratumor side. Overexpression of CXCL6 in adjacent malignant cells could induce activation of the JAK-STAT3 pathway in nearby hepatocytes, which subsequently caused SAAs' overexpression in these hepatocytes. Furthermore, overexpression and secretion of SAAs by hepatocytes in the invasive zone could lead to the recruitment of macrophages and M2 polarization, further promoting local immunosuppression, potentially resulting in tumor progression. Clinical association analysis in additional five independent cohorts of patients with primary and secondary liver cancer (n = 423) showed that patients with overexpression of SAAs in the invasive zone had a worse prognosis. Further in vivo experiments using mouse liver tumor models in situ confirmed that the knockdown of genes encoding SAAs in hepatocytes decreased macrophage accumulation around the tumor border and delayed tumor growth. The identification and characterization of a novel invasive zone in human cancer patients not only add an important layer of understanding regarding the mechanisms of tumor invasion and metastasis, but may also pave the way for developing novel therapeutic strategies for advanced liver cancer and other solid tumors.
Subject(s)
Ecosystem , Liver Neoplasms , Mice , Animals , Humans , Liver Neoplasms/pathology , Hepatocytes/metabolism , Immunosuppression Therapy , Cell Line, TumorABSTRACT
In recent years, spatial transcriptomics (ST) technologies have developed rapidly and have been widely used in constructing spatial tissue atlases and characterizing spatiotemporal heterogeneity of cancers. Currently, ST has been used to profile spatial heterogeneity in multiple cancer types. Besides, ST is a benefit for identifying and comprehensively understanding special spatial areas such as tumor interface and tertiary lymphoid structures (TLSs), which exhibit unique tumor microenvironments (TMEs). Therefore, ST has also shown great potential to improve pathological diagnosis and identify novel prognostic factors in cancer. This review presents recent advances and prospects of applications on cancer research based on ST technologies as well as the challenges.
ABSTRACT
Antibodies targeting the programmed cell death protein-1 (PD-1) molecule have been reported to hold promising antitumor activities in patients with nasopharyngeal carcinoma (NPC). However, only a small subset of NPC patients benefits from the anti-PD-1 monotherapy and factors that affect the treatment response need further investigation. This study aimed to examine the impact of multiple genetic and environmental factors on outcome of anti-PD-1 immunotherapy by identifying tumor size, tumor mutation burden (TMB) based on whole exon sequencing, human leukocyte antigen class I (HLA-I) homo-/heterozygosity and supertypes, blood Epstein-Barr virus (EBV) DNA load, T cell proportions, and interferon-γ(IFN-γ) levels in a cohort of 57 NPC patients that received Nivolumab or Camrelizumab treatment. Moreover, we profiled the longitudinal changes in gut microbiota composition using shotgun metagenomics sequencing. We observed that high TMB combined with HLA-I heterozygosity was associated with improved clinical outcomes. In agreement with previous studies, we found that patients with higher plasma EBV DNA load showed worse progression-free survival. We found no evidence for an effect of gut bacterial diversity on the treatment response, but identified a higher abundance of seven specific gut bacteria at baseline of non-responders, including Blautia wexlera and Blautia obeum, as well as four other bacteria belonging to the Clostridiales order, and one Erysipelatoclostridium. Combined, this study provides insight into the influence of several genetic and environmental factors on anti-PD-1 immunotherapy responses in NPC patients.
ABSTRACT
Hepatocellular carcinoma (HCC) is the most common liver cancer with a high rate of metastasis. However, the molecular mechanisms that drive metastasis remain unclear. We combined single-cell transcriptomic, proteomic, and chromatin accessibility data to investigate how heterogeneous phenotypes contribute to metastatic potential in five HCC cell lines. We confirmed that the prevalence of a mesenchymal state and levels of cell proliferation are linked to the metastatic potential. We also identified a rare hypoxic subtype that has a higher capacity for glycolysis and exhibits dormant, invasive, and malignant characteristics. This subtype has increased metastatic potential. We further identified a robust 14-gene panel representing this hypoxia signature and this hypoxia signature could serve as a prognostic index. Our data provide a valuable data resource, facilitate a deeper understanding of metastatic mechanisms, and may help diagnosis of metastatic potential in individual patients, thus supporting personalized medicine.
ABSTRACT
Single cell approaches have increased our knowledge about the cell type composition of the non-human primate (NHP), but a detailed characterization of area-specific regulatory features remains outstanding. We generated single-cell transcriptomic and chromatin accessibility (single-cell ATAC) data of 358,237 cells from prefrontal cortex (PFC), primary motor cortex (M1) and primary visual cortex (V1) of adult female cynomolgus monkey brain, and integrated this dataset with Stereo-seq (spatial enhanced resolution omics-sequencing) of the corresponding cortical areas to assign topographic information to molecular states. We identified area-specific chromatin accessible sites and their targeted genes, including the cell type-specific transcriptional regulatory network associated with excitatory neurons heterogeneity. We reveal calcium ion transport and axon guidance genes related to specialized functions of PFC and M1, identified the similarities and differences between adult macaque and human oligodendrocyte trajectories, and mapped the genetic variants and gene perturbations of human diseases to NHP cortical cells. This resource establishes a transcriptomic and chromatin accessibility combinatory regulatory landscape at a single-cell and spatially resolved resolution in NHP cortex.
Subject(s)
Neurons , Prefrontal Cortex , Animals , Female , Macaca fascicularis/genetics , Neurons/metabolism , Prefrontal Cortex/metabolism , Gene Regulatory Networks , Chromatin/genetics , Chromatin/metabolismABSTRACT
Little is known about the transcriptomic plasticity and adaptive mechanisms of circulating tumor cells (CTCs) during hematogeneous dissemination. Here we interrogate the transcriptome of 113 single CTCs from 4 different vascular sites, including hepatic vein (HV), peripheral artery (PA), peripheral vein (PV) and portal vein (PoV) using single-cell full-length RNA sequencing in hepatocellular carcinoma (HCC) patients. We reveal that the transcriptional dynamics of CTCs were associated with stress response, cell cycle and immune-evasion signaling during hematogeneous transportation. Besides, we identify chemokine CCL5 as an important mediator for CTC immune evasion. Mechanistically, overexpression of CCL5 in CTCs is transcriptionally regulated by p38-MAX signaling, which recruites regulatory T cells (Tregs) to facilitate immune escape and metastatic seeding of CTCs. Collectively, our results reveal a previously unappreciated spatial heterogeneity and an immune-escape mechanism of CTC, which may aid in designing new anti-metastasis therapeutic strategies in HCC.
Subject(s)
Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/immunology , Genetic Heterogeneity , Immune Evasion , Liver Neoplasms/genetics , Liver Neoplasms/immunology , Neoplastic Cells, Circulating/immunology , Aged , Animals , Biomarkers, Tumor/metabolism , Cell Cycle , Cell Line, Tumor , Chemokine CCL5/metabolism , Gene Expression Regulation, Neoplastic , Humans , Male , Mice , Mice, Inbred C57BL , Middle Aged , Neoplasm Metastasis , Neoplastic Cells, Circulating/metabolism , Prognosis , RNA-Seq , Transcriptome , Tumor MicroenvironmentABSTRACT
Recent studies show that non-coding RNAs (ncRNAs) can regulate the expression of protein-coding genes and play important roles in mammalian development. Previous studies have revealed that during C. elegans (Caenorhabditis elegans) embryo development, numerous genes in each cell are spatiotemporally regulated, causing the cell to differentiate into distinct cell types and tissues. We ask whether ncRNAs participate in the spatiotemporal regulation of genes in different types of cells and tissues during the embryogenesis of C. elegans. Here, by using marker-free full-length high-depth single-cell RNA sequencing (scRNA-seq) technique, we sequence the whole transcriptomes from 1031 embryonic cells of C. elegans and detect 20,431 protein-coding genes, including 22 cell-type-specific protein-coding markers, and 9843 ncRNAs including 11 cell-type-specific ncRNA markers. We induce a ncRNAs-based clustering strategy as a complementary strategy to the protein-coding gene-based clustering strategy for single-cell classification. We identify 94 ncRNAs that have never been reported to regulate gene expressions, are co-expressed with 1208 protein-coding genes in cell type specific and/or embryo time specific manners. Our findings suggest that these ncRNAs could potentially influence the spatiotemporal expression of the corresponding genes during the embryogenesis of C. elegans.
Subject(s)
Caenorhabditis elegans/genetics , Embryonic Development/genetics , RNA, Untranslated/genetics , Transcriptome/genetics , Animals , Caenorhabditis elegans/growth & development , Caenorhabditis elegans Proteins/genetics , Gene Expression Regulation, Developmental/genetics , RNA, Untranslated/classification , Single-Cell AnalysisSubject(s)
Genomics , Cluster Analysis , Genomics/methods , Humans , Algorithms , Computational Biology/methodsABSTRACT
Active retrotransposons play important roles during evolution and continue to shape our genomes today, especially in genetic polymorphisms underlying a diverse set of diseases. However, studies of human retrotransposon insertion polymorphisms (RIPs) based on whole-genome deep sequencing at the population level have not been sufficiently undertaken, despite the obvious need for a thorough characterization of RIPs in the general population. Herein, we present a novel and efficient computational tool called Specific Insertions Detector (SID) for the detection of non-reference RIPs. We demonstrate that SID is suitable for high-depth whole-genome sequencing data using paired-end reads obtained from simulated and real datasets. We construct a comprehensive RIP database using a large population of 90 Han Chinese individuals with a mean ×68 depth per individual. In total, we identify 9342 recent RIPs, and 8433 of these RIPs are novel compared with dbRIP, including 5826 Alu, 2169 long interspersed nuclear element 1 (L1), 383 SVA, and 55 long terminal repeats. Among the 9342 RIPs, 4828 were located in gene regions and 5 were located in protein-coding regions. We demonstrate that RIPs can, in principle, be an informative resource to perform population evolution and phylogenetic analyses. Taking the demographic effects into account, we identify a weak negative selection on SVA and L1 but an approximately neutral selection for Alu elements based on the frequency spectrum of RIPs. SID is a powerful open-source program for the detection of non-reference RIPs. We built a non-reference RIP dataset that greatly enhanced the diversity of RIPs detected in the general population, and it should be invaluable to researchers interested in many aspects of human evolution, genetics, and disease. As a proof of concept, we demonstrate that the RIPs can be used as biomarkers in a similar way as single nucleotide polymorphisms.
Subject(s)
Genome, Human , High-Throughput Nucleotide Sequencing/methods , Polymorphism, Genetic , Retroelements , Whole Genome Sequencing/methods , Asian People/genetics , HumansABSTRACT
Chronic lymphocytic leukaemia (CLL) is a frequent B-cell malignancy, characterized by recurrent somatic chromosome alterations and a low level of point mutations. Here we present single-nucleotide polymorphism microarray analyses of a single CLL patient over 29 years of observation and treatment, and transcriptome and whole-genome sequencing at selected time points. We identify chromosome alterations 13q14-, 6q- and 12q+ in early cell clones, elimination of clonal populations following therapy, and subsequent appearance of a clone containing trisomy 12 and chromosome 10 copy-neutral loss of heterogeneity that marks a major population dominant at death. Serial single-cell RNA sequencing reveals an expression pattern with high FOS, JUN and KLF4 at disease acceleration, which resolves following therapy, but reoccurs following relapse and death. Transcriptome evolution indicates complex changes in expression occur over time. In conclusion, CLL can evolve gradually during indolent phases, and undergo rapid changes following therapy.
Subject(s)
Clonal Evolution/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Polymorphism, Single Nucleotide , Aged , Chromosome Aberrations , Chromosome Disorders , Chromosomes, Human, Pair 12/genetics , Chromosomes, Human, Pair 13/genetics , Chromosomes, Human, Pair 6/genetics , DNA/genetics , Female , Humans , Immune System/metabolism , Kruppel-Like Factor 4 , Oligonucleotide Array Sequence Analysis , RNA/geneticsABSTRACT
BACKGROUND: Viral infection causes multiple forms of human cancer, and HPV infection is the primary factor in cervical carcinomas. Recent single-cell RNA-seq studies highlight the tumor heterogeneity present in most cancers, but virally induced tumors have not been studied. HeLa is a well characterized HPV+ cervical cancer cell line. RESULT: We developed a new high throughput platform to prepare single-cell RNA on a nanoliter scale based on a customized microwell chip. Using this method, we successfully amplified full-length transcripts of 669 single HeLa S3 cells and 40 of them were randomly selected to perform single-cell RNA sequencing. Based on these data, we obtained a comprehensive understanding of the heterogeneity of HeLa S3 cells in gene expression, alternative splicing and fusions. Furthermore, we identified a high diversity of HPV-18 expression and splicing at the single-cell level. By co-expression analysis we identified 283 E6, E7 co-regulated genes, including CDC25, PCNA, PLK4, BUB1B and IRF1 known to interact with HPV viral proteins. CONCLUSION: Our results reveal the heterogeneity of a virus-infected cell line. It not only provides a transcriptome characterization of HeLa S3 cells at the single cell level, but is a demonstration of the power of single cell RNA-seq analysis of virally infected cells and cancers.