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Lung adenocarcinoma (LUAD) harboring EGFR mutations prevails in Asian population. However, the inter-patient and intra-tumor heterogeneity has not been addressed at single-cell resolution. Here we performed single-cell RNA sequencing (scRNA-seq) of total 125,674 cells from seven stage-I/II LUAD samples harboring EGFR mutations and five tumor-adjacent lung tissues. We identified diverse cell types within the tumor microenvironment (TME) in which myeloid cells and T cells were the most abundant stromal cell types in tumors and adjacent lung tissues. Within tumors, accompanied by an increase in CD1C + dendritic cells, the tumor-associated macrophages (TAMs) showed pro-tumoral functions without signature gene expression of defined M1 or M2 polarization. Tumor-infiltrating T cells mainly displayed exhausted and regulatory T-cell features. The adenocarcinoma cells can be categorized into different subtypes based on their gene expression signatures in distinct pathways such as hypoxia, glycolysis, cell metabolism, translation initiation, cell cycle, and antigen presentation. By performing pseudotime trajectory, we found that ELF3 was among the most upregulated genes in more advanced tumor cells. In response to secretion of inflammatory cytokines (e.g., IL1B) from immune infiltrates, ELF3 in tumor cells was upregulated to trigger the activation of PI3K/Akt/NF-κB pathway and elevated expression of proliferation and anti-apoptosis genes such as BCL2L1 and CCND1 . Taken together, our study revealed substantial heterogeneity within early-stage LUAD harboring EGFR mutations, implicating complex interactions among tumor cells, stromal cells and immune infiltrates in the TME.

Background Single-cell transcriptome and single-cell methylome technologies have become powerful tools to study RNA and DNA methylation profiles of single cells at a genome-wide scale. A major challenge has been to understand the direct correlation of DNA methylation and gene expression within single-cells. Due to large cell-to-cell variability and the lack of direct measurements of transcriptome and methylome of the same cell, the association is still unclear. Results Here, we describe a novel method (scMT-seq) that simultaneously profiles both DNA methylome and transcriptome from the same cell. In sensory neurons, we consistently identify transcriptome and methylome heterogeneity among single cells but the majority of the expression variance is not explained by proximal promoter methylation, with the exception of genes that do not contain CpG islands. By contrast, gene body methylation is positively associated with gene expression for only those genes that contain a CpG island promoter. Furthermore, using single nucleotide polymorphism patterns from our hybrid mouse model, we also find positive correlation of allelic gene body methylation with allelic expression. Conclusions Our method can be used to detect transcriptome, methylome, and single nucleotide polymorphism information within single cells to dissect the mechanisms of epigenetic gene regulation.

Peripheral nerve injury leads to various injury-induced responses in sensory neurons including physiological pain, neuronal cell death and nerve regeneration. In this study, we performed single-cell RNA-sequencing (scRNA-seq) analysis of mouse nonpeptidergic nociceptors (NP), peptidergic nociceptors (PEP) and large myelinated sensory neurons (LM) under both control and injury conditions at 3 days after sciatic nerve transection (SNT). After performing principle component and weighted gene co-expression network analysis, we categorized dorsal root ganglion (DRG) neurons into different subtypes and discovered co-regulated injury-response genes including novel regeneration associated genes (RAGs) in association with neuronal development, protein translation and cytoplasm transportation. In addition, we found significant up-regulation of the genes associated with cell death such as Pdcd2 in a subset of NP neurons after axotomy, implicating their actions in neuronal cell death upon nerve injury. Our study revealed the distinctive and sustained heterogeneity of transcriptomic responses to injury at single neuron level, implicating the involvement of different gene regulatory networks in nerve regeneration, neuronal cell death and neuropathy in different population of DRG neurons.

Multipotent trophoblasts undergo dynamic morphological movement and cellular differentiation after conceptus implantation to generate placenta. However, the mechanism controlling trophoblast development and differentiation during peri-implantation development in human remains elusive. In this study, we modeled human conceptus peri-implantation development from blastocyst to early postimplantation stages by using an in vitro coculture system and profiled the transcriptome of 476 individual trophoblast cells from these conceptuses. We revealed the genetic networks regulating peri-implantation trophoblast development. While determining when trophoblast differentiation happens, our bioinformatic analysis identified T-box transcription factor 3 (TBX3) as a key regulator for the differentiation of cytotrophoblast (CT) into syncytiotrophoblast (ST). The function of TBX3 in trophoblast differentiation is then validated by a loss-of-function experiment. In conclusion, our results provided a valuable resource to study the regulation of trophoblasts development and differentiation during human peri-implantation development.

BackgroundAlthough the treatment landscape for advanced hepatocellular carcinoma (HCC) has seen significant advancements in the past decade with the introduction of immune checkpoint inhibitors and antiangiogenic drugs, progress has fallen short of expectations. Recently, a novel engineered oncolytic virus (OHSV2) that secretes dual-specific T-cell engagers (DSTEs) targeting the fibroblast activation protein (FAP) was developed and combined with GPC3-targeting CAR-T cells and immunotoxins to exert a synergistic antitumor effect.MethodsOHSV2-DSTEFAP5/CD3 was initially generated by transducing the DSTEs engaging FAP5 on fibroblasts into the backbone of our oncolytic virus OHSV2. An innovative high-order combination was devised in a xenograft mouse model to conceptually explore whether enhanced anti-tumor effects could be achieved. Additionally, the underlying mechanisms of synergistic effects and safety profiles were preliminarily investigated.ResultsOHSV2-DSTEFAP5/CD3 effectively targeted and eliminated fibroblasts in vitro while maintaining cytotoxicity and inducing immune activation compared to parental OHSV2. In vivo , dose-adjusted combination therapy resulted in a remarkable antitumor effect compared to control treatments, leading to tumor regression in 40% of mice without significant toxicity to major organs. Mechanistically, rather than directly depleting fibroblasts, OHSV2-DSTEFAP5/CD3 played an essential role in priming T-cell proliferation, infiltration, and activation, and inhibiting the supportive interaction between cancer cells and fibroblasts.ConclusionsThis high-order combination represents a novel multiple-wave immunotherapeutic approach for HCC. Despite being a conceptual exploration, this strategy has demonstrated promising therapeutic efficacy and acceptable safety profiles.

Tip60 is a key histone acetyltransferase (HAT) enzyme that plays a central role in diverse biological processes critical for general cell function; however, the chromatin-mediated cell-type specific developmental pathways that are dependent exclusively upon the HAT activity of Tip60 remain to be explored. Here, we investigate the role of Tip60 HAT activity in transcriptional control during multicellular development in vivo by examining genome-wide changes in gene expression in a Drosophila model system specifically depleted for endogenous dTip60 HAT function. We show that amino acid residue E431 in the catalytic HAT domain of dTip60 is critical for the acetylation of endogenous histone H4 in our fly model in vivo, and demonstrate that dTip60 HAT activity is essential for multicellular development. Moreover, our results uncover a novel role for Tip60 HAT activity in controlling neuronal specific gene expression profiles essential for nervous system function as well as a central regulatory role for Tip60 HAT function in general metabolism.

Small cell lung cancer (SCLC) originates from pulmonary neuroendocrine cells and accounts for approximately 15% of lung cancer incidents. Patients with SCLC have a very low survival rate due to fast progression and early metastasis. Despite the recent approval of immune checkpoint blockade for treatment of SCLC by US FDA, conventional platinum chemotherapy remains the first‐line treatment which always develops quick drug resistance. To define targets for diagnosis and treatment, SCLC has been categorized by different molecular markers. The most popular markers include genomic mutations in tumour suppressor genes such as TP53 and RB1 and expression of lineage‐specific transcription factors (TFs), that is ASCL1, NEUROD1, POU2F3 and YAP1. As DNA methylation is an important hallmark of cancer, efforts were made to investigate the mechanisms of DNA methylation involved in SCLC progression. Indeed, SCLC has distinct pattern of DNA methylation due to its different features compared to non‐SCLC and other tumours. In this review, we summarized the mechanisms of DNA methylation in SCLC which are associated with lineage‐specific TFs, cell proliferation, anti‐apoptosis, drug resistance, immune evasion and metastasis. We foresaw the challenges of applying DNA methylation in clinical diagnosis and treatment and discussed new approaches and technologies to overcome them. Combined with other gene regulatory information such as transcription and histone modification, DNA methylation/hydroxymethylation in SCLC will be resolved at single‐cell resolution to dissect the tumour microenvironment. The huge multiomics data will be processed and integrated with clinical data such as clinical images and pathological histochemistry by artificial intelligence and cloud computing. Circulating cell‐free DNA methylation will greatly enhance early diagnosis and prognosis prediction. New organoid and organ chip models will break through the obstacles of sampling limitation, faithfully simulate the physiology of human tissues and enable examining spatiotemporal DNA methylation during SCLC progression. Small cell lung cancer (SCLC), a high‐grade neuroendocrine tumor, accounts for approximately 15% of lung cancer incidents. DNA methylation in SCLC involves in disease progression such as lineage‐specific TFs, cell proliferation, anti‐apoptosis, drug resistance, immune evasion, and metastasis. Challenges of applying DNA methylation in clinical diagnosis and treatment can be overcome by new approaches and technologies.

Macrophages play important roles in metabolic dysfunction-associated steatohepatitis (MASH), an advanced and inflammatory stage of metabolic dysfunction-associated steatotic liver disease (MASLD). In humans and mice, the cellular heterogeneity and diverse function of hepatic macrophages in MASH have been investigated by single cell RNA sequencing (scRNA-seq). However, little is known about their roles in rats. Here, we collected liver tissues at the postnatal week 16, when our previously characterized Lep∆I14/∆I14 rats developed MASH phenotypes. By scRNA-seq, we found an increase in the number of macrophages and endothelial cells and a decrease in that of NK and B cells. Hepatic macrophages in rats underwent a unique M1 to M2 transition without expression of the classical markers such as Arg1 and Nos2, except for Cd163. Lipid-associated macrophages (LAMs) were increased, which could be detected by the antibody against Cd63. In the microenvironment, macrophages had an increased number of interactions with hepatocytes, myofibroblasts, T cells, neutrophils, and dendritic cells, while their interaction strengths remained unchanged. Finally, the macrophage migration inhibitory factor (MIF) pathway was identified as the top upregulated cell-communication pathway in MASH. In conclusion, we dissected hepatic macrophage dynamics during MASH at single cell resolution and provided fundamental tools for the investigation of MASH in rat models.

Using the paradigm of in vitro differentiation of hESCs/iPSCs into retinal pigment epithelial (RPE) cells, we have recently profiled mRNA and miRNA transcriptomes to define a set of RPE mRNA and miRNA signature genes implicated in directed RPE differentiation. In this study, in order to understand the role of DNA methylation in RPE differentiation, we profiled genome-scale DNA methylation patterns using the method of reduced representation bisulfite sequencing (RRBS). We found dynamic waves of de novo methylation and demethylation in four stages of RPE differentiation. Integrated analysis of DNA methylation and RPE transcriptomes revealed a reverse-correlation between levels of DNA methylation and expression of a subset of miRNA and mRNA genes that are important for RPE differentiation and function. Gene Ontology (GO) analysis suggested that genes undergoing dynamic methylation changes were related to RPE differentiation and maturation. We further compared methylation patterns among human ESC- and iPSC-derived RPE as well as primary fetal RPE (fRPE) cells, and discovered that specific DNA methylation pattern is useful to classify each of the three types of RPE cells. Our results demonstrate that DNA methylation may serve as biomarkers to characterize the cell differentiation process during the conversion of human pluripotent stem cells into functional RPE cells.

Small cell lung cancer (SCLC) is a deadly neuroendocrine malignancy with high metastasis. However, the heterogeneity of metastatic SCLC at the single‐cell level remains elusive. The single‐cell transcriptome of a total of 24 081 cells in metastatic lymph node samples from seven SCLC patients via endobronchial ultrasound‐guided transbronchial needle aspiration (EBUS‐TBNA) is examined. Genomic alterations are also examined by whole exome sequencing (WES) and the immune infiltration between SCLC and non‐SCLC (NSCLC) is compared using public single‐cell RNA sequencing (scRNA‐seq) data. It is identified that malignant cells in lymph‐node metastatic SCLC have inter‐patient and intra‐tumor heterogeneity characterized by distinct ASCL1 and NEUROD1 expression patterns. High expression of genes such as FZD8 in WNT pathway is associated with drug resistance in malignant cells. Compared to NSCLC, SCLC harbors a unique immunosuppressive tumor microenvironment. Malignant cells exhibit a pattern of attenuated MHC‐I antigen presentation‐related gene expression, which is associated with relatively low proportion of exhausted T cells. Natural killer (NK) cells display impaired antitumor function with high expression of TGFBR2. This work characterizes the inter‐patient and intra‐tumor heterogeneity of metastatic SCLC and uncovers the exhaustion signatures in NK cells, which may pave the way for novel treatments for SCLC including immune checkpoint blockade‐based immunotherapy. Small cell lung cancer (SCLC) is a deadly neuroendocrine malignancy with high metastasis. However, the heterogeneity of metastatic SCLC at the single‐cell level remains elusive. Current study identifies that malignant cells in SCLC have inter‐patient and intra‐tumor heterogeneity, high expression of genes such as FZD8 in WNT pathway is associated with drug resistance, and SCLC harbors a unique immunosuppressive tumor microenvironment.