Epigenetic regulators as the foundation for molecular classification of colorectal cancer

Colorectal cancer (CRC) accounts for approximately 10% of newly diagnosed cancer cases and cancer-related deaths worldwide~1. The identification of molecular subtypes of CRCs has significantly advanced treatment strategies,including targeted therapy and immunotherapy.

Single-cell DNA methylome analysis of circulating tumor cells

Objective: Previous investigations of circulating tumor cells(CTCs) have mainly focused on their genomic or transcriptomic features, leaving their epigenetic landscape relatively uncharacterized. Here, we investigated the genome-wide DNA methylome of CTCs with a view to understanding the epigenetic regulatory mechanisms underlying cancer metastasis.Methods: We evaluated single-cell DNA methylome and copy number alteration(CNA) in 196 single cells,including 107 CTCs collected from 17 cancer patients covering six different cancer types. Our single-cell bisulfite sequencing(sc BS-seq) covered on average 11.78% of all Cp G dinucleotides and accurately deduced the CNA patterns at 500 kb resolution.Results: We report distinct subclonal structures and different evolutionary histories of CTCs inferred from CNA and DNA methylation profiles. Furthermore, we demonstrate potential tumor origin classification based on the tissue-specific DNA methylation profiles of CTCs.Conclusions: Our work provides a comprehensive survey of genome-wide DNA methylome in single CTCs and reveals 5-methylcytosine(5-m C) heterogeneity in CTCs, addressing the potential epigenetic regulatory mechanisms underlying cancer metastasis and facilitating the future clinical application of CTCs.

Copy number alteration profiling facilitates differential diagnosis between ossifying fibroma and fibrous dysplasia of the jaws

Ossifying fibroma(OF)and fibrous dysplasia(FD)are two fibro-osseous lesions with overlapping clinicopathological features,making diagnosis challenging.In this study,we applied a whole-genome shallow sequencing approach to facilitate differential diagnosis via precise profiling of copy number alterations(CNAs)using minute amounts of DNA extracted from morphologically correlated microdissected tissue samples.Freshly frozen tissue specimens from OF(n=29)and FD(n=28)patients were obtained for analysis.Lesion fibrous tissues and surrounding normal tissues were obtained by laser capture microdissection(LCM),with~30–50 cells(5000–10000µm2)per sample.We found that the rate of recurrent CNAs in OF cases was much higher(44.8%,13 of 29)than that in FD cases(3.6%,1 of 28).Sixty-nine percent(9 of 13)of the CNA-containing OF cases involved segmental amplifications and deletions on Chrs 7 and 12.We also identified eight CNA-associated genes(HILPDA,CALD1,C1GALT1,MICALL2,PHF14,AIMP2,MDM2,and CDK4)with amplified expression,which was consistent with the copy number changes.We further confirmed a jaw lesion with a previous uncertain diagnosis due to its ambiguous morphological features and the absence of GNAS mutation as OF based on the typical Chr 12 amplification pattern in its CNA profile.Moreover,analysis of a set of longitudinal samples collected from an individual with a cellular lesion in suspicion of OF at the first surgery,recurrence and the latest malignant transformation revealed identical CNA patterns at the three time points,suggesting that copy number profiling can be used as an important tool to identify borderline lesions or lesions with malignant potential.Overall,CNA profiling of fibro-osseous lesions can greatly improve differential diagnosis between OF and FD and help predict disease progression.

Activation-induced pyroptosis contributes to the loss of MAIT cells in chronic HIV-1 infected patients

Background: Mucosal-associated invariant T(MAIT) cells are systemically depleted in human immunodeficiency virus type 1(HIV-1) infected patients and are not replenished even after successful combined antiretroviral therapy(cART).This study aimed to identify the mechanism underlying MAIT cell depletion.Methods: In the present study, we applied flow cytometry, single-cell RNA sequencing and immunohistochemical staining to evaluate the characteristics of pyroptotic MAIT cells in a total of 127 HIV-1 infected individuals, including 69 treatment-naive patients, 28 complete responders, 15 immunological non-responders, and 15 elite controllers, at the Fifth Medical Center of Chinese PLA General Hospital, Beijing, China.Results: Single-cell transcriptomic profiles revealed that circulating MAIT cells from HIV-1 infected subjects were highly activated, with upregulation of pyroptosis-related genes. Further analysis revealed that increased frequencies of pyroptotic MAIT cells correlated with markers of systemic T-cell activation, microbial translocation, and intestinal damage in cART-naive patients and poor CD4+ T-cell recovery in long-term cART patients. Immunohistochemical staining revealed that MAIT cells in the gut mucosa of HIV-1 infected patients exhibited a strong active gasdermin-D(GSDMD, marker of pyroptosis) signal near the cavity side, suggesting that these MAIT cells underwent active pyroptosis in the colorectal mucosa. Increased levels of the proinflammatory cytokines interleukin-12(IL-12) and IL-18 were observed in HIV-1 infected patients. In addition, activated MAIT cells exhibited an increased pyroptotic phenotype after being triggered by HIV-1 virions, T-cell receptor signals, IL-12 plus IL-18, and combinations of these factors, in vitro.Conclusions: Activation-induced MAIT cell pyroptosis contributes to the loss of MAIT cells in HIV-1 infected patients,which could potentiate disease progression and poor immune reconstitution.

Deep learning facilitated whole live cell fast super-resolution imaging

A fully convolutional encoder-decoder network(FCEDN),a deep learning model,was developed and applied to image scanning microscopy(ISM).Super-resolution imaging was achieved with a 78μm×78μm field of view and 12.5 Hz-40 Hz imaging frequency.Mono and dual-color continuous super-resolution images of microtubules and cargo in cells were obtained by ISM.The signal-to-noise ratio of the obtained images was improved from 3.94 to 22.81 and the positioning accuracy of cargoes was enhanced by FCEDN from 15.83±2.79 nm to 2.83±0.83 nm.As a general image enhancement method,FCEDN can be applied to various types of microscopy systems.Application with conventional spinning disk confocal microscopy was demonstrated and significantly improved images were obtained.

Role of ELMOs in cancer cell migration and engulfment

Metastasis is responsible for the majority of deaths related to cancer and results from several interconnected processes including cell proliferation,angiogenesis,chemotaxis,cell adhesion,migration,and invasion into the surrounding tissue.SDF-1αinduced chemotaxis plays an important role in cancer chemotaxis and metastasis.Binding of SDF-1α(CXCL12)to CXCR4 triggers activation of heterotrimeric G proteins that regulate actin polymerization and migration of cancer cells.

Drug repurposing screening and mechanism analysis based on human colorectal cancer organoids

Colorectal cancer(CRC)is a highly heterogeneous cancer and exploring novel therapeutic options is a pressing issue that needs to be addressed.Here,we established human CRC tumor-derived organoids that well represent both morphological and molecular heterogeneities of original tumors.To efficiently identify repurposed drugs for CRC,we developed a robust organoid-based drug screening system.By combining the repurposed drug library and computation-based drug prediction,335 drugs were tested and 34 drugs with anti-CRC effects were identified.More importantly,we conducted a detailed transcriptome analysis of drug responses and divided the drug response signatures into five representative patterns:differentiation induction,growth inhibition,metabolism inhibition,immune response promotion,and cell cycle inhibition.The anticancer activities of drug candidates were further validated in the established patient-derived organoids-based xenograft(PDOX)system in vivo.We found that fedratinib,trametinib,and bortezomib exhibited effective anticancer effects.Furthermore,the concordance and discordance of drug response signatures between organoids in vitro and pairwise PDOX in vivo were evaluated.Our study offers an innovative approach for drug discovery,and the representative transcriptome features of drug responses provide valuable resources for developing novel clinical treatments for CRC.

Blastoids generated purely from embryonic stem cells both in mice and humans

Dear Editor,Embryonic stem cells(ESCs)and trophoblast stem cells(TSCs)are derived from blastocysts(Lu et al.,2001).Blastocyst-like structures(blastoids)are self-assembled structures formed by a combination of ESCs and TSCs(Rivron et al.,2018).To increase the success rate of blastoid formation and its similarity with the features of blastocysts,some reports showed that blastoid could also be derived from a combination of three cell lines,ESCs,TSCs,and extraembryonic endoderm cells(XENs),or novel type of stem cells(Sozen et al.,2018;Weatherbee et al.,2023:Wu et al.,2023;Zhang et al.,2023;Zhang et al.,2019).

Unsynchronized butyrophilin molecules dictate cancer cell evasion of Vγ9Vδ2 T-cell killing

Vγ9Vδ2 T cells are specialized effector cells that have gained prominence as immunotherapy agents due to their ability to target and kill cells with altered pyrophosphate metabolites.In our effort to understand how cancer cells evade the cell-killing activity of Vγ9Vδ2 T cells,we performed a comprehensive genome-scale CRISPR screening of cancer cells.We found that four molecules belonging to the butyrophilin(BTN)family,specifically BTN2A1,BTN3A1,BTN3A2,and BTN3A3,are critically important and play unique,nonoverlapping roles in facilitating the destruction of cancer cells by primary Vγ9Vδ2 T cells.The coordinated function of these BTN molecules was driven by synchronized gene expression,which was regulated by IFN-γsignaling and the RFX complex.Additionally,an enzyme called QPCTL was shown to play a key role in modifying the N-terminal glutamine of these BTN proteins and was found to be a crucial factor in Vγ9Vδ2 T cell killing of cancer cells.Through our research,we offer a detailed overview of the functional genomic mechanisms that underlie how cancer cells escape Vγ9Vδ2 T cells.Moreover,our findings shed light on the importance of the harmonized expression and function of gene family members in modulating T-cell activity.

Rationally designed synthetic peptide as versatile calibrant to improve the accuracy of protein sequence analysis using MALDI mass spectrometry

Matrix-assisted laser desorption/ionization(MALDI)mass spectrometry(MS)plays an indispensable role in analyzing protein covalent structures.The reliable identification of amino acid residues and modifications relies on the mass accuracy,which is highly dependent on calibration.However,the accuracy provided by the currently available calibrants still needs further improvement in terms of compatibility with multiple tandem MS modes or ion polarity modes,calibratable range,and minimizing suppression of and interference with analyte signals.Here aiming at developing a versatile calibrant to solve these problem,we designed a synthetic peptide format of calibrant R_x(GDP_n)_m(referred to as“Gly-Asp-Pro,GDP”)according to the chemical natures of amino acids and polypeptide fragmentation rules in tandem MS.With four types of amino acid residues selected and arranged through rational designs,a GDP peptide produces highly regulated fragments that give rise to evenly spaced signals in each tandem MS mode and is compatible with both positive and negative ion modes.In internal calibration,its regulated fragmentation pattern minimizes interference with analyte signals,and using a single peptide as the input minimizes suppression of the analyte signals.As demonstrated by analyses of proteins including monoclonal antibody and Aβ-42,these features allowed significant increase of the mass accuracy and precision,which improved sequence coverage and sequence resolution in sequence analyses(including de novo sequencing).This rational design strategy may also inspire further development of synthetic calibrants that benefit structural analysis of biomolecules.

Reflective ultrathin light-sheet microscopy with isotropic 3D resolutions

Light-sheet fluorescence microscopy(LSFM)has played an important role in bio-imaging due to its advantages of high photon efficiency,fast speed,and long-term imaging capabilities.The perpendicular layout between LSFM excitation and detection often limits the 3D resolutions as well as their isotropy.Here,we report on a reflective type light-sheet microscope with a mini-prism used as an optical path reflector.The conventional high NA objectives can be used both in excitation and detection with this design.Isotropic resolutions in 3D down to300 nm could be achieved without deconvolution.The proposed method also enables easy transform of a conventional fluorescence microscope to high performance light-sheet microscopy.

DEAD-box helicase 17(DDX17)protects cardiac function by promoting mitochondrial homeostasis in heart failure

DEAD-box helicase 17(DDX17)is a typical member of the DEAD-box family with transcriptional cofactor activity.Although DDX17 is abundantly expressed in the myocardium,its role in heart is not fully understood.We generated cardiomyocyte-specific Ddx17-knockout mice(Ddx17-cKO),cardiomyocyte-specific Ddx17 transgenic mice(Ddx17-Tg),and various models of cardiomyocyte injury and heart failure(HF).DDX17 is downregulated in the myocardium of mouse models of heart failure and cardiomyocyte injury.Cardiomyocyte-specific knockout of Ddx17 promotes autophagic flux blockage and cardiomyocyte apoptosis,leading to progressive cardiac dysfunction,maladaptive remodeling and progression to heart failure.Restoration of DDX17 expression in cardiomyocytes protects cardiac function under pathological conditions.Further studies showed that DDX17 can bind to the transcriptional repressor B-cell lymphoma 6(BCL6)and inhibit the expression of dynamin-related protein 1(DRP1).When DDX17 expression is reduced,transcriptional repression of BCL6 is attenuated,leading to increased DRP1 expression and mitochondrial fission,which in turn leads to impaired mitochondrial homeostasis and heart failure.We also investigated the correlation of DDX17 expression with cardiac function and DRP1 expression in myocardial biopsy samples from patients with heart failure.These findings suggest that DDX17 protects cardiac function by promoting mitochondrial homeostasis through the BCL6-DRP1 pathway in heart failure.

Recent advances in single-cell sequencing technologies

Single-cell omics sequencingwas first achieved for the transcriptome in 2009,whichwas followed by fast development of technologies for profiling the genome,DNA methylome,3D genome architecture,chromatin accessibility,histone modifications,etc.,in an individual cell.In this review we mainly focus on the recent progress in four topics in the single-cell omics field:single-cell epigenome sequencing,single-cell genome sequencing for lineage tracing,spatially resolved single-cell transcriptomics and third-generation sequencing platform-based single-cell omics sequencing.We also discuss the potential applications and future directions of these single-cell omics sequencing technologies for different biomedical systems,especially for the human stem cell field.

Crystal structures of N-terminal WRKY transcription factors and DNA complexes

Dear Editor,Plant-specific WRKY transcription factors(TFs)are among the largest families of TFs in higher plants;they are also found in the unicellular eukaryote Giardia lamblia and the slime mold Dictyostelium discoideum(Ulker and Somssich,2004),but not in animals.WRKY TFs participate in diverse developmental and physiological processes in plants,such as disease resistance,abiotic stress responses,senescence,seed and trichome development,as well as additional developmental and hormone-controlled processes(Agarwal et al.,2011).

Architectural proteins for the formation and maintenance of the 3D genome

Eukaryotic genomes are densely packaged into hierarchical three-dimensional(3D) structures that contain information about gene regulation and many other biological processes. With the development of imaging and sequencing-based technologies, 3D genome studies have revealed that the high-order chromatin structure is composed of hierarchical levels, including chromosome territories, A/B compartments, topologically associated domains, and chromatin loops. However, how this chromatin architecture is formed and maintained is not completely clear. In this review, we introduce experimental methods to investigate the 3D genome, review major architectural proteins that regulate 3D chromatin organization in mammalian cells, such as CTCF(CCCTC-binding factor), cohesin, lamins, and transcription factors, and discuss relevant mechanisms such as phase separation.

Gene editing and its applications in biomedicine

The steady progress in genome editing, especially genome editing based on the use of clustered regularly interspaced short palindromic repeats(CRISPR) and programmable nucleases to make precise modifications to genetic material, has provided enormous opportunities to advance biomedical research and promote human health. The application of these technologies in basic biomedical research has yielded significant advances in identifying and studying key molecular targets relevant to human diseases and their treatment. The clinical translation of genome editing techniques offers unprecedented biomedical engineering capabilities in the diagnosis, prevention, and treatment of disease or disability. Here, we provide a general summary of emerging biomedical applications of genome editing, including open challenges. We also summarize the tools of genome editing and the insights derived from their applications, hoping to accelerate new discoveries and therapies in biomedicine.

In vivo flow cytometry reveals a circadian rhythm of circulating tumor cells

Circulating tumor cells(CTCs)is an established biomarker of cancer metastasis.The circulation dynamics of CTCs are important for understanding the mechanisms underlying tumor cell dissemination.Although studies have revealed that the circadian rhythm may disrupt the growth of tumors,it is generally unclear whether the circadian rhythm controls the release of CTCs.In clinical examinations,the current in vitro methods for detecting CTCs in blood samples are based on a fundamental assumption that CTC counts in the peripheral blood do not change significantly over time,which is being challenged by recent studies.Since it is not practical to draw blood from patients repeatedly,a feasible strategy to investigate the circadian rhythm of CTCs is to monitor them by in vivo detection methods.Fluoresce nee in vivo flow cytometry(IVFC)is a powerful optical technique that is able to detect fluoresce nt circulating cells directly in living animals in a noninvasive manner over a long period of time.In this study,we applied fluorescence IVFC to monitor CTCs noninvasively in an orthotopic mouse model of human prostate cancer.We observed that CTCs exhibited stochastic bursts over cancer progression.The probability of the bursting activity was higher at early stages than at late stages.We longitudinally monitored CTCs over a 24-h period,and our results revealed striking daily oscillations in CTC counts that peaked at the onset of the night(active phase for rodents),suggesting that the release of CTCs might be regulated by the circadian rhythm.

Low-density lipoprotein receptor-related protein 1 is a CROPs-associated receptor for Clostridioides infection toxin B

As the leading cause of worldwide hospital-acquired infection,Clostridioides difficile(C.difficile)infection has caused heavy economic and hospitalized burden,while its pathogenesis is not fully understood.Toxin B(Tcd B)is one of the major virulent factors of C.difficile.Recently,CSPG4 and FZD2 were reported to be the receptors that mediate Tcd B cellular entry.However,genetic ablation of genes encoding these receptors failed to completely block Tcd B entry,implicating the existence of alternative receptor(s)for this toxin.Here,by employing the CRISPR-Cas9 screen in CSPG4-deficient He La cells,we identified LDL receptor-related protein-1(LRP1)as a novel receptor for Tcd B.Knockout of LRP1 in both CSPG4-deficient He La cells and colonic epithelium Caco2 cells conferred cells with increased Tcd B resistance,while LRP1 overexpression sensitized cells to Tcd B at a low concentration.Co-immunoprecipitation assay showed that LRP1 interacts with full-length Tcd B.Moreover,CROPs domain,which is dispensable for Tcd B’s interaction with CSPG4 and FZD2,is sufficient for binding to LRP1.As such,our study provided evidence for a novel mechanism of Tcd B entry and suggested potential therapeutic targets for treating C.difficile infection.

Dissecting Human Gonadal Cell Lineage Specification and Sex Determination Using A Single-cell RNA-seq Approach

Gonadal somatic cells are the main players in gonad development and are important for sex determination and germ cell development.Here,using a time-series single-cell RNA sequencing(scRNA-seq)strategy,we analyzed fetal germ cells(FGCs)and gonadal somatic cells in human embryos and fetuses.Clustering analysis of testes and ovaries revealed several novel cell subsets,including POU5F1^(+)SPARC^(+)FGCs and KRT19^(+) somatic cells.Furthermore,our data indicated that the bone morphogenetic protein(BMP)signaling pathway plays cell type-specific and developmental stage-specific roles in testis development and promotes the gonocyte-to-spermatogonium transition(GST)in late-stage testicular mitotic arrest FGCs.Intriguingly,testosterone synthesis function transitioned from fetal Sertoli cells to adult Leydig cells in a stepwise manner.In our study,potential interactions between gonadal somatic cells were systematically explored and we identified cell type-specific developmental defects in both FGCs and gonadal somatic cells in a Turner syndrome embryo(45,XO).Our work provides a blueprint of the complex yet highly ordered development of and the interactions among human FGCs and gonadal somatic cells.

Gene editing:from technologies to applications in research and beyond

Cutting-edge gene editing technologies enable broadened genomic alternations and accelerate opportunities to use these tools in biomedicine, agriculture, and animal model development. In this issue, Li et al.(2022) reviews the tools of gene editing and highlights key technological developments and its broad applications in biomedicine, hoping to accelerate new discoveries and therapies in biomedicine.