技术资料

In calcium imaging studies,Ca 2+ transients are commonly interpreted as neuronal action potentials (APs). However,our findings demonstrate that robust optical Ca 2+ transients primarily stem from complex “AP-Plateaus”,while simple APs lacking underlying depolarization envelopes produce much weaker photonic signatures. Under challenging in vivo conditions,these “AP-Plateaus” are likely to surpass noise levels,thus dominating the Ca 2+ recordings. In spontaneously active neuronal culture,optical Ca 2+ transients (OGB1-AM,GCaMP6f) exhibited approximately tenfold greater amplitude and twofold longer half-width compared to optical voltage transients (ArcLightD). The amplitude of the ArcLightD signal exhibited a strong correlation with the duration of the underlying membrane depolarization,and a weaker correlation with the presence of a fast sodium AP. Specifically,ArcLightD exhibited robust responsiveness to the slow “foot” but not the fast “trunk” of the neuronal AP. Particularly potent stimulators of optical signals in both Ca 2+ and voltage imaging modalities were APs combined with plateau potentials (AP-Plateaus),resembling dendritic Ca 2+ spikes or “UP states” in pyramidal neurons. Interestingly,even the spikeless plateaus (amplitude > 10 mV,duration > 200 ms) could generate conspicuous Ca 2+ optical signals in neurons. Therefore,in certain circumstances,Ca 2+ transients should not be interpreted solely as indicators of neuronal AP firing. Subject terms: Biological techniques,Biophysics,Neuroscience,Physiology View Publication

Poor graft function (PGF),characterized by myelosuppression,represents a significant challenge following allogeneic hematopoietic stem cell transplantation (allo-HSCT) with human cytomegalovirus (HCMV) being established as a risk factor for PGF. However,the underlying mechanism remains unclear. Bone marrow endothelial progenitor cells (BM-EPCs) play an important role in supporting hematopoiesis and their dysfunction contributes to PGF development. We aim to explore the effects of CMV on BM-EPCs and its underlying mechanism. We investigated the compromised functionality of EPCs derived from individuals diagnosed with HCMV viremia accompanied by PGF,as well as after infected by HCMV AD 169 strain in vitro,characterized by decreased cell proliferation,tube formation,migration and hematopoietic support,and increased apoptosis and secretion of TGF-β1. We demonstrated that HCMV-induced TGF-β1 secretion by BM-EPCs played a dominant role in hematopoiesis suppression in vitro experiment. Moreover,HCMV down-regulates Vitamin D receptor (VDR) and subsequently activates p38 MAPK pathway to promote TGF-β1 secretion by BM-EPCs. HCMV could infect BM-EPCs and lead to their dysfunction. The secretion of TGF-β1 by BM-EPCs is enhanced by CMV through the activation of p38 MAPK via a VDR-dependent mechanism,ultimately leading to compromised support for hematopoietic progenitors by BM EPCs,which May significantly contribute to the pathogenesis of PGF following allo-HSCT and provide innovative therapeutic strategies targeting PGF. View Publication

Radiotherapy is widely regarded as the primary therapeutic modality for nasopharyngeal cancer (NPC). Studies have shown that cancer cells with high resistance to radiation,known as radioresistant cancer cells,may cause residual illness,which in turn might contribute to the occurrence of cancer recurrence and metastasis. It has been shown that cancer stem-like cells (CSCs) exhibit resistance to radiation therapy. In the present study,fractionated doses of radiation-induced epithelial-mesenchymal transition (EMT) and ALDH+ CSCs phenotype of NPC tumor spheroids. Furthermore,it has been shown that cells with elevated ALDH activity have increased resistance to the effects of fractionated irradiation. Nuclear factor erythroid-2-related factor 2 (Nrf2) plays a pivotal role in regulating cellular antioxidant systems. A large body of evidence suggests that Nrf2 plays a significant role in the development of radioresistance in cancer. The authors’ research revealed that the application of fractionated irradiation resulted in a decline in Nrf2-dependent reactive oxygen species (ROS) levels,thereby mitigating DNA damage in ALDH+ stem-like NPC cells. In addition,immunofluorescence analysis revealed that subsequent to the process of fractionated irradiation of ALDH+ cells,activated Nrf2 was predominantly localized inside the nucleus. Immunofluorescent analysis also revealed that the presence of the nuclear Nrf2+/NQO1+/ALDH1+ axis might potentially serve as an indicator of poor prognosis and resistance to radiotherapy in patients with NPC. Thus,the authors’ findings strongly suggest that the radioresistance of ALDH-positive NPC CSCs to fractionated irradiation is regulated by nuclear Nrf2 accumulation. Nrf2 exerts its effects through the downstream effector NQO1/ALDH1,which depends on ROS attenuation. View Publication

Natural killer (NK) cells are frontline defenders against cancer and are capable of recognizing and eliminating tumor cells without prior sensitization or antigen presentation. Due to their unique HLA mismatch tolerance,they are ideal for adoptive cell therapy (ACT) because of their ability to minimize graft-versus-host-disease risk. The therapeutic efficacy of NK cells is limited in part by inhibitory immune checkpoint receptors,which are upregulated upon interaction with cancer cells and the tumor microenvironment. Overexpression of inhibitory receptors reduces NK cell-mediated cytotoxicity by impairing the ability of NK cells to secrete effector cytokines and cytotoxic granules. T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT),a well-known checkpoint receptor involved in T-cell exhaustion,has recently been implicated in the exhaustion of NK cells. Overcoming TIGIT-mediated inhibition of NK cells may allow for a more potent antitumor response following ACT. Here,we describe a novel approach to TIGIT inhibition using self-delivering RNAi compounds (INTASYL™) that incorporates the features of RNAi and antisense technology. INTASYL compounds demonstrate potent activity and stability,are rapidly and efficiently taken up by cells,and can be easily incorporated into cell product manufacturing. INTASYL PH-804,which targets TIGIT,suppresses TIGIT mRNA and protein expression in NK cells,resulting in increased cytotoxic capacity and enhanced tumor cell killing in vitro. Delivering PH-804 to NK cells before ACT has emerged as a promising strategy to counter TIGIT inhibition,thereby improving the antitumor response. This approach offers the potential for more potent off-the-shelf products for adoptive cell therapy,particularly for hematological malignancies. The online version contains supplementary material available at 10.1007/s00262-024-03835-x. View Publication

Mutation of tet methylcytosine dioxygenase 2 (encoded by TET2 ) drives myeloid malignancy initiation and progression 1 – 3 . TET2 deficiency is known to cause a globally opened chromatin state and activation of genes contributing to aberrant haematopoietic stem cell self-renewal 4,5 . However,the open chromatin observed in TET2-deficient mouse embryonic stem cells,leukaemic cells and haematopoietic stem and progenitor cells 5 is inconsistent with the designated role of DNA 5-methylcytosine oxidation of TET2. Here we show that chromatin-associated retrotransposon RNA 5-methylcytosine (m 5 C) can be recognized by the methyl-CpG-binding-domain protein MBD6,which guides deubiquitination of nearby monoubiquitinated Lys119 of histone H2A (H2AK119ub) to promote an open chromatin state. TET2 oxidizes m 5 C and antagonizes this MBD6-dependent H2AK119ub deubiquitination. TET2 depletion thereby leads to globally decreased H2AK119ub,more open chromatin and increased transcription in stem cells. TET2- mutant human leukaemia becomes dependent on this gene activation pathway,with MBD6 depletion selectively blocking proliferation of TET2 -mutant leukaemic cells and largely reversing the haematopoiesis defects caused by Tet2 loss in mouse models. Together,our findings reveal a chromatin regulation pathway by TET2 through retrotransposon RNA m 5 C oxidation and identify the downstream MBD6 protein as a feasible target for developing therapies specific against TET2 mutant malignancies. Subject terms: Molecular biology,Chromatin View Publication

Head and neck squamous cell carcinoma (HNSCC) is a highly malignant disease with high death rates that have remained substantially unaltered for decades. Therefore,new treatment approaches are urgently needed. Human papillomavirus-negative tumors harbor areas of terminally differentiated tissue that are characterized by cornification. Dissecting this intrinsic ability of HNSCC cells to irreversibly differentiate into non-malignant cells may have tumor-targeting potential. We modeled the cornification of HNSCC cells in a primary spheroid model and analyzed the mechanisms underlying differentiation by ATAC-seq and RNA-seq. Results were verified by immunofluorescence using human HNSCC tissue of distinct anatomical locations. HNSCC cell differentiation was accompanied by cell adhesion,proliferation stop,diminished tumor-initiating potential in immunodeficient mice,and activation of a wound-healing-associated signaling program. Small promoter accessibility increased despite overall chromatin closure. Differentiating cells upregulated KRT17 and cornification markers. Although KRT17 represents a basal stem cell marker in normal mucosa,we confirm KRT17 to represent an early differentiation marker in HNSCC tissue. Cornification was frequently found surrounding necrotic areas in human tumors,indicating an involvement of pro-inflammatory stimuli. Indeed,inflammatory mediators activated the differentiation program in primary HNSCC cells. In HNSCC tissue,distinct cell differentiation states were found to create a common tissue architecture in normal mucosa and HNSCCs. Our data demonstrate a loss of cell malignancy upon faithful HNSCC cell differentiation,indicating that targeted differentiation approaches may be therapeutically valuable. Moreover,we describe KRT17 to be a candidate biomarker for HNSCC cell differentiation and early tumor detection. Subject terms: Cancer stem cells,Oral cancer View Publication

Immune surveillance by cytotoxic T cells eliminates tumor cells and cells infected by intracellular pathogens. This process relies on the presentation of antigenic peptides by Major Histocompatibility Complex class I (MHC-I) at the cell surface. The loading of these peptides onto MHC-I depends on the peptide loading complex (PLC) at the endoplasmic reticulum (ER). Here,we uncovered that MHC-I antigen presentation is regulated by ER-associated degradation (ERAD),a protein quality control process essential to clear misfolded and unassembled proteins. An unbiased proteomics screen identified the PLC component Tapasin,essential for peptide loading onto MHC-I,as a substrate of the RNF185/Membralin ERAD complex. Loss of RNF185/Membralin resulted in elevated Tapasin steady state levels and increased MHC-I at the surface of professional antigen presenting cells. We further show that RNF185/Membralin ERAD complex recognizes unassembled Tapasin and limits its incorporation into PLC. These findings establish a novel mechanism controlling antigen presentation and suggest RNF185/Membralin as a potential therapeutic target to modulate immune surveillance. Subject terms: Endoplasmic reticulum,ER-associated degradation,MHC class I,Antigen-presenting cells View Publication

The emerging field of synthetic morphogenesis implements synthetic biology tools to investigate the minimal cellular processes sufficient for orchestrating key developmental events. As the field continues to grow,there is a need for new tools that enable scientists to uncover nuances in the molecular mechanisms driving cell fate patterning that emerge during morphogenesis. Here,we present a platform that combines cell engineering with biomaterial design to potentiate artificial signaling in pluripotent stem cells (PSCs). This platform,referred to as PSC-MATRIX,extends the use of programmable biomaterials to PSCs competent to activate morphogen production through orthogonal signaling,giving rise to the opportunity to probe developmental events by initiating morphogenetic programs in a spatially constrained manner through non-native signaling channels. We show that the PSC-MATRIX platform enables temporal and spatial control of transgene expression in response to bulk,soluble inputs in synthetic Notch (synNotch)-engineered human PSCs for an extended culture of up to 11 days. Furthermore,we used PSC-MATRIX to regulate multiple differentiation events via material-mediated artificial signaling in engineered PSCs using the orthogonal ligand green fluorescent protein,highlighting the potential of this platform for probing and guiding fate acquisition. Overall,this platform offers a synthetic approach to interrogate the molecular mechanisms driving PSC differentiation that could be applied to a variety of differentiation protocols. View Publication

Head and neck squamous cell carcinoma (HNSCC) presents a significant challenge worldwide due to its aggressiveness and high recurrence rates post-treatment,often linked to cancer stem cells (CSCs). Melatonin shows promise as a potent tumor suppressor; however,the effects of melatonin on CSCs remain unclear,and the development of models that closely resemble tumor heterogeneity could help to better understand the effects of this molecule. This study developed a tumor scaffold based on patient fibroblast-derived decellularized extracellular matrix that mimics the HNSCC microenvironment. Our study investigates the antitumoral effects of melatonin within this context. We validated its strong antiproliferative effect on HNSCC CSCs and the reduction of tumor invasion and migration markers,even in a strongly chemoprotective environment,as it is required to increase the minimum doses necessary to impact tumor viability compared to the non-scaffolded tumorspheres culture. Moreover,melatonin exhibited no cytotoxic effects on healthy cells co-cultured in the tumor hydrogel. This scaffold-based platform allows an in vitro study closer to HNSCC tumor reality,including CSCs,stromal component,and a biomimetic matrix,providing a new valuable research tool in precision oncology. View Publication

Fatty acid synthase (FASN)-catalyzed endogenous lipogenesis is a hallmark of cancer metabolism. However,whether FASN is an intrinsic mechanism of tumor cell defense against T cell immunity remains unexplored. To test this hypothesis,here we combined bioinformatic analysis of the FASN-related immune cell landscape,real-time assessment of cell-based immunotherapy efficacy in CRISPR/Cas9-based FASN gene knockout ( FASN KO ) cell models,and mathematical and mechanistic evaluation of FASN-driven immunoresistance. FASN expression negatively correlates with infiltrating immune cells associated with cancer suppression,cytolytic activity signatures,and HLA-I expression. Cancer cells engineered to carry a loss-of-function mutation in FASN exhibit an enhanced cytolytic response and an accelerated extinction kinetics upon interaction with cytokine-activated T cells. Depletion of FASN results in reduced carrying capacity,accompanied by the suppression of mitochondrial OXPHOS and strong downregulation of electron transport chain complexes. Targeted FASN depletion primes cancer cells for mitochondrial apoptosis as it synergizes with BCL-2/BCL-X L -targeting BH3 mimetics to render cancer cells more susceptible to T-cell-mediated killing. FASN depletion prevents adaptive induction of PD-L1 in response to interferon-gamma and reduces constitutive overexpression of PD-L1 by abolishing PD-L1 post-translational palmitoylation. FASN is a novel tumor cell-intrinsic metabolic checkpoint that restricts T cell immunity and may be exploited to improve the efficacy of T cell-based immunotherapy. Subject terms: Cancer metabolism,Oncogenesis View Publication

Thyroid hormone receptor alpha (THRα) is a nuclear hormone receptor that binds triiodothyronine (T3) and acts as an important transcription factor in development,metabolism,and reproduction. In mammals,THRα has two major splicing isoforms,THRα1 and THRα2. The better-characterized isoform,THRα1,is a transcriptional stimulator of genes involved in cell metabolism and growth. The less-well-characterized isoform,THRα2,lacks the ligand-binding domain (LBD) and is thought to act as an inhibitor of THRα1 activity. The ratio of THRα1 to THRα2 splicing isoforms is therefore critical for transcriptional regulation in different tissues and during development. However,the expression patterns of both isoforms have not been studied in healthy human tissues or in the developing brain. Given the lack of commercially available isoform-specific antibodies,we addressed this question by analyzing four bulk RNA-sequencing datasets and two scRNA-sequencing datasets to determine the RNA expression levels of human THRA1 and THRA2 transcripts in healthy adult tissues and in the developing brain. We demonstrate how 10X Chromium scRNA-seq datasets can be used to perform splicing-sensitive analyses of isoforms that differ at the 3′-end. In all datasets,we found a strong predominance of THRA2 transcripts at all examined stages of human brain development and in the central nervous system of healthy human adults. View Publication

Drugs work by binding to a specific 3D structure on a protein. Drug discovery has historically been driven by prior knowledge of function,either of a protein or chemical. This knowledge of function then drives investigations to probe chemical/protein interactions. We undertook a different approach. We first identified unique 3D structures,agnostic of function,and investigated whether they could lead us to innovative therapeutics. Using a synchrotron-based X-ray source,we first determined high-resolution structures of hundreds of proteins. With a supercomputer running analytical programs created by us,we identified novel 3D structures and screened for chemicals binding them. We then tested their ability to inhibit cancer growth without damaging normal cells. We identified a potent inhibitor of a deadly cancer,melanoma. It was not toxic to normal cells even at 2100-fold higher doses. It worked by inducing anoikis,a fundamental process of known importance for cancer. Therapeutics that selectively induce anoikis are needed. In summary,we demonstrate the power of using a 3D protein structure as the starting point to discover new biology and drugs. Drug discovery historically starts with an established function,either that of compounds or proteins. This can hamper discovery of novel therapeutics. As structure determines function,we hypothesized that unique 3D protein structures constitute primary data that can inform novel discovery. Using a computationally intensive physics-based analytical platform operating at supercomputing speeds,we probed a high-resolution protein X-ray crystallographic library developed by us. For each of the eight identified novel 3D structures,we analyzed binding of sixty million compounds. Top-ranking compounds were acquired and screened for efficacy against breast,prostate,colon,or lung cancer,and for toxicity on normal human bone marrow stem cells,both using eight-day colony formation assays. Effective and non-toxic compounds segregated to two pockets. One compound,Dxr2-017,exhibited selective anti-melanoma activity in the NCI-60 cell line screen. In eight-day assays,Dxr2-017 had an IC50 of 12 nM against melanoma cells,while concentrations over 2100-fold higher had minimal stem cell toxicity. Dxr2-017 induced anoikis,a unique form of programmed cell death in need of targeted therapeutics. Our findings demonstrate proof-of-concept that protein structures represent high-value primary data to support the discovery of novel acting therapeutics. This approach is widely applicable. View Publication