技术资料

Immune checkpoint blockade (ICB) has made remarkable achievements,but newly identified armored and cold tumors cannot respond to ICB therapy. The high prevalence of concomitant medications has huge impact on immunotherapeutic responses,but the clinical effects on the therapeutic outcome of armored and cold tumors are still unclear. In this research,using large-scale transcriptomics datasets,the expression and potential biological functions of angiotensin II receptor 1 (AGTR1),the target of angiotensin receptor blocker (ARB),were investigated. Next,the roles of ARB in tumor cells and tumor microenvironment cells were defined by a series of in vitro and in vivo assays. In addition,the clinical impacts of ARB on ICB therapy were assessed by multicenter cohorts and meta-analysis. AGTR1 was overexpressed in armored and cold tumors and associated with poor response to ICB therapy. ARB,the inhibitor for AGTR1,only suppressed the aggressiveness of tumor cells with high AGTR1 expression,which accounted for a very small proportion. Further analysis revealed that AGTR1 was always highly expressed in cancer-associated fibroblasts (CAFs) and ARB inhibited type I collagen expression in CAFs by suppressing the RhoA-YAP axis. Moreover,ARB could also drastically reverse the phenotype of armored and cold to soft and hot in vivo,leading to a higher response to ICB therapy. In addition,both our in-house cohorts and meta-analysis further supported the idea that ARB can significantly enhance ICB efficacy. Overall,we identify AGTR1 as a novel target in armored and cold tumors and demonstrate the improved therapeutic efficacy of ICB in combination with ARB. These findings could provide novel clinical insight into how to treat patients with refractory armored and cold tumors. View Publication

Reduction of adult hippocampal neurogenesis is an early critical event in Alzheimer’s disease (AD),contributing to progressive memory loss and cognitive decline. Reduced levels of the nucleoporin 153 (Nup153),a key epigenetic regulator of NSC stemness,characterize the neural stem cells isolated from a mouse model of AD (3×Tg) (AD-NSCs) and determine their altered plasticity and gene expression. Nup153-regulated mechanisms contributing to NSC function were investigated: (1) in cultured NSCs isolated from AD and wild type (WT) mice by proteomics; (2) in vivo by lentiviral-mediated delivery of Nup153 or GFP in the hippocampus of AD and control mice analyzing neurogenesis and cognitive function; (3) in human iPSC-derived brain organoids obtained from AD patients and control subjects as a model of neurodevelopment. Proteomic approach identified Nup153 interactors in WT- and AD-NSCs potentially implicated in neurogenesis regulation. Gene ontology (GO) analysis showed that Nup153-bound proteins in WT-NSCs were involved in RNA metabolism,nuclear import and epigenetic mechanisms. Nup153-bound proteins in AD-NSCs were involved in pathways of neurodegeneration,mitochondrial dysfunction,proteasomal processing and RNA degradation. Furthermore,recovery of Nup153 levels in AD-NSCs reduced the levels of oxidative stress markers and recovered proteasomal activity. Lentiviral-mediated delivery of Nup153 in the hippocampal niche of AD mice increased the proliferation of early progenitors,marked by BrdU/DCX and BrdU/PSANCAM positivity and,later,the integration of differentiating neurons in the cell granule layer (BrdU/NeuN + cells) compared with GFP-injected AD mice. Consistently,Nup153-injected AD mice showed an improvement of cognitive performance in comparison to AD-GFP mice at 1 month after virus delivery assessed by Morris Water Maze. To validate the role of Nup153 in neurogenesis we took advantage of brain organoids derived from AD-iPSCs characterized by fewer neuroepithelial progenitor loops and reduced differentiation areas. The upregulation of Nup153 in AD organoids recovered the formation of neural-like tubes and differentiation. Our data suggest that the positive effect of Nup153 on neurogenesis is based on a complex regulatory network orchestrated by Nup153 and that this protein is a valuable disease target. The online version contains supplementary material available at 10.1186/s13287-024-03805-1. View Publication

Recent advances in imaging suggested that spatial organization of hematopoietic cells in their bone marrow microenvironment (niche) regulates cell expansion,governing progression,and leukemic transformation of hematological clonal disorders. However,our ability to interrogate the niche in pre-malignant conditions has been limited,as standard murine models of these diseases rely largely on transplantation of the mutant clones into conditioned mice where the marrow microenvironment is compromised. Here,we leveraged live-animal microscopy and ultralow dose whole body or focal irradiation to capture single cells and early expansion of benign/pre-malignant clones in the functionally preserved microenvironment. 0.5 Gy whole body irradiation (WBI) allowed steady engraftment of cells beyond 30 weeks compared to non-conditioned controls. In-vivo tracking and functional analyses of the microenvironment showed no change in vessel integrity,cell viability,and HSC-supportive functions of the stromal cells,suggesting minimal inflammation after the radiation insult. The approach enabled in vivo imaging of Tet2 + /− and its healthy counterpart,showing preferential localization within a shared microenvironment while forming discrete micro-niches. Notably,stationary association with the niche only occurred in a subset of cells and would not be identified without live imaging. This strategy may be broadly applied to study clonal disorders in a spatial context. View Publication

Astrocytes are essential for the formation and maintenance of neural networks. However,a major technical challenge for investigating astrocyte function and disease-related pathophysiology has been the limited ability to obtain functional human astrocytes. Despite recent advances in human pluripotent stem cell (hPSC) techniques,primary rodent astrocytes remain the gold standard in coculture with human neurons. We demonstrate that a combination of leukemia inhibitory factor (LIF) and bone morphogenetic protein-4 (BMP4) directs hPSC-derived neural precursor cells to a highly pure population of astroglia in 28 d. Using single-cell RNA sequencing,we confirm the astroglial identity of these cells and highlight profound transcriptional adaptations in cocultured hPSC-derived astrocytes and neurons,consistent with their further maturation. In coculture with human neurons,multielectrode array recordings revealed robust network activity of human neurons in a coculture with hPSC-derived or rat astrocytes [3.63 ± 0.44 min −1 (hPSC-derived),2.86 ± 0.64 min −1 (rat); p = 0.19]. In comparison,we found increased spike frequency within network bursts of human neurons cocultured with hPSC-derived astrocytes [56.31 ± 8.56 Hz (hPSC-derived),24.77 ± 4.04 Hz (rat); p < 0.01],and whole-cell patch-clamp recordings revealed an increase of postsynaptic currents [2.76 ± 0.39 Hz (hPSC-derived),1.07 ± 0.14 Hz (rat); p < 0.001],consistent with a corresponding increase in synapse density [14.90 ± 1.27/100 μm 2 (hPSC-derived),8.39 ± 0.63/100 μm 2 (rat); p < 0.001]. Taken together,we show that hPSC-derived astrocytes compare favorably with rat astrocytes in supporting human neural network activity and maturation,providing a fully human platform for investigating astrocyte function and neuronal-glial interactions. View Publication

The inflammatory response to wear particles derived from hip prothesis is considered a hallmark of periprosthetic osteolysis,which can ultimately lead to the need for revision surgery. Exosomes (Exos) have been associated with various bone pathologies,and there is increasing recognition in the literature that they actively transport molecules throughout the body. The role of wear particles in osteoblast-derived Exos is unknown,and the potential contribution of Exos to osteoimmune communication and periprosthetic osteolysis niche is still in its infancy. Given this,we investigate how titanium dioxide nanoparticles (TiO 2 NPs),similar in size and composition to prosthetic wear particles,affect Exos biogenesis. Two osteoblastic cell models commonly used to study the response of osteoblasts to wear particles were selected as a proof of concept. The contribution of Exos to periprosthetic osteolysis was assessed by functional assays in which primary human macrophages were stimulated with bone-derived Exos. We demonstrated that TiO 2 NPs enter multivesicular bodies,the nascent of Exos,altering osteoblast-derived Exos secretion and molecular cargo. No significant differences were observed in Exos morphology and size. However,functional assays reveal that Exos cargo enriched in uPA stimulates macrophages to a mixed M1 and M2 phenotype,inducing the release of pro- and anti-inflammatory signals characteristic of periprosthetic osteolysis. In addition,we demonstrated the expression of uPA in exosomes derived from the urine of patients with osteolysis. These results suggest that uPA can be a potential biomarker of osteolysis. In the future,uPa may serve as a possible non-invasive biomarker to identify patients at risk for peri-implant osteolysis. View Publication

The growth factor Neuregulin-1 (NRG1) has pleiotropic roles in proliferation and differentiation of the stem cell niche in different tissues. It has been implicated in gut,brain and muscle development and repair. Six isoform classes of NRG1 and over 28 protein isoforms have been previously described. Here we report a new class of NRG1,designated NRG1-VII to denote that these NRG1 isoforms arise from a myeloid-specific transcriptional start site (TSS) previously uncharacterized. Long-read sequencing was used to identify eight high-confidence NRG1-VII transcripts. These transcripts presented major structural differences from one another,through the use of cassette exons and alternative stop codons. Expression of NRG1-VII was confirmed in primary human monocytes and tissue resident macrophages and induced pluripotent stem cell-derived macrophages (iPSC-derived macrophages). Isoform switching via cassette exon usage and alternate polyadenylation was apparent during monocyte maturation and macrophage differentiation. NRG1-VII is the major class expressed by the myeloid lineage,including tissue-resident macrophages. Analysis of public gene expression data indicates that monocytes and macrophages are a primary source of NRG1. The size and structure of class VII isoforms suggests that they may be more diffusible through tissues than other NRG1 classes. However,the specific roles of class VII variants in tissue homeostasis and repair have not yet been determined. The online version contains supplementary material available at 10.1186/s12864-024-10723-2. View Publication

Intermediate-length repeat expansions in ATAXIN-2 (ATXN2) are the strongest genetic risk factor for amyotrophic lateral sclerosis (ALS). At the molecular level,ATXN2 intermediate expansions enhance TDP-43 toxicity and pathology. However,whether this triggers ALS pathogenesis at the cellular and functional level remains unknown. Here,we combine patient-derived and mouse models to dissect the effects of ATXN2 intermediate expansions in an ALS background. iPSC-derived motor neurons from ATXN2-ALS patients show altered stress granules,neurite damage and abnormal electrophysiological properties compared to healthy control and other familial ALS mutations. In TDP-43 Tg -ALS mice,ATXN2-Q33 causes reduced motor function,NMJ alterations,neuron degeneration and altered in vitro stress granule dynamics. Furthermore,gene expression changes related to mitochondrial function and inflammatory response are detected and confirmed at the cellular level in mice and human neuron and organoid models. Together,these results define pathogenic defects underlying ATXN2-ALS and provide a framework for future research into ATXN2-dependent pathogenesis and therapy. Subject terms: Amyotrophic lateral sclerosis,Molecular neuroscience,Cellular neuroscience View Publication

Hypomethylating agents (HMAs) are frontline therapies for Myelodysplastic Neoplasms (MDS) and Acute Myeloid Leukemia (AML). However,acquired resistance and treatment failure are commonplace. To address this,we perform a genome-wide CRISPR-Cas9 screen in a human MDS-derived cell line,MDS-L,and identify TOPORS as a loss-of-function target that synergizes with HMAs,reducing leukemic burden and improving survival in xenograft models. We demonstrate that depletion of TOPORS mediates sensitivity to HMAs by predisposing leukemic blasts to an impaired DNA damage response (DDR) accompanied by an accumulation of SUMOylated DNMT1 in HMA-treated TOPORS-depleted cells. The combination of HMAs with targeting of TOPORS does not impair healthy hematopoiesis. While inhibitors of TOPORS are unavailable,we show that inhibition of protein SUMOylation with TAK-981 partially phenocopies HMA-sensitivity and DDR impairment. Overall,our data suggest that the combination of HMAs with inhibition of SUMOylation or TOPORS is a rational treatment option for High-Risk MDS (HR-MDS) or AML. Subject terms: Myelodysplastic syndrome,Acute myeloid leukaemia,Sumoylation View Publication

DNA hypomethylating agents (HMAs) are used for the treatment of myeloid malignancies,although their therapeutic effects have been unsatisfactory. Here we show that CRISPR-Cas9 screening reveals that knockout of topoisomerase 1-binding arginine/serine-rich protein ( TOPORS ),which encodes a ubiquitin/SUMO E3 ligase,augments the efficacy of HMAs on myeloid leukemic cells with little effect on normal hematopoiesis,suggesting that TOPORS is involved in resistance to HMAs. HMAs are incorporated into the DNA and trap DNA methyltransferase-1 (DNMT1) to form DNA-DNMT1 crosslinks,which undergo SUMOylation,followed by proteasomal degradation. Persistent crosslinking is cytotoxic. The TOPORS RING finger domain,which mediates ubiquitination,is responsible for HMA resistance. In TOPORS knockout cells,DNMT1 is stabilized by HMA treatment due to inefficient ubiquitination,resulting in the accumulation of unresolved SUMOylated DNMT1. This indicates that TOPORS ubiquitinates SUMOylated DNMT1,thereby promoting the resolution of DNA-DNMT1 crosslinks. Consistently,the ubiquitination inhibitor,TAK-243,and the SUMOylation inhibitor,TAK-981,show synergistic effects with HMAs through DNMT1 stabilization. Our study provides a novel HMA-based therapeutic strategy that interferes with the resolution of DNA-DNMT1 crosslinks. Subject terms: Myelodysplastic syndrome,Myelodysplastic syndrome View Publication

Three-dimensionality (3D) was proven essential for developing reliable models for different anatomical compartments and many diseases. However,the neuronal compartment still poses a great challenge as we still do not understand precisely how the brain computes information and how the complex chain of neuronal events can generate conscious behavior. Therefore,a comprehensive model of neuronal tissue has not yet been found. The present work was conceived in this framework: we aimed to contribute to what must be a collective effort by filling in some information on possible 3D strategies to pursue. We compared directly different kinds of scaffolds (i.e.,PDMS sponges,thermally crosslinked hydrogels,and glass microbeads) in their effect on neuronal network activity recorded using micro-electrode arrays. While the overall rate of spiking activity remained consistent,the type of scaffold had a notable impact on bursting dynamics. The frequency,density of bursts,and occurrence of random spikes were all affected. The examination of inter-burst intervals revealed distinct burst generation patterns unique to different scaffold types. Network burst propagation unveiled divergent trends among configurations. Notably,it showed the most differences,underlying that functional variations may arise from a different 3D spatial organization. This evidence suggests that not all 3D neuronal constructs can sustain the same level of richness of activity. Furthermore,we commented on the reproducibility,efficacy,and scalability of the methods,where the beads still offer superior performances. By comparing different 3D scaffolds,our results move toward understanding the best strategies to develop functional 3D neuronal units for reliable pre-clinical studies. View Publication

Despite advances in breast cancer screening and treatment,prognosis for metastatic disease remains dismal at 30% five-year survival. This is due,in large,to the failure of current therapeutics to target properties unique to metastatic cells. One of the drivers of metastasis is miR-10b,a small noncoding RNA implicated in cancer cell invasion,migration,viability,and proliferation. We have developed a nanodrug,termed MN-anti-miR10b,that delivers anti-miR-10b antisense oligomers to cancer cells. In mouse models of metastatic triple-negative breast cancer,MN-anti-miR10b has been shown to prevent onset of metastasis and eliminate existing metastases in combination with chemotherapy,even after treatment has been stopped. Recent studies have implicated miR-10b in conferring stem cell-like properties onto cancer cells,such as chemoresistance. In this study,we show transcriptional evidence that inhibition of miR-10b with MN-anti-miR10b activates developmental processes in cancer cells and that stem-like cancer cells have increased miR-10b expression. We then demonstrate that treatment of breast cancer cells with MN-anti-miR10b reduces their stemness,confirming that these properties make metastatic cells susceptible to the nanodrug actions. Collectively,these findings indicate that inhibition of miR-10b functions to impair breast cancer cell stemness,positioning MN-anti-miR10b as an effective treatment option for stem-like breast cancer subtypes. View Publication

Programmed cell death 1 (PD-1) is a premier cancer drug target for immune checkpoint blockade (ICB). Because PD-1 receptor inhibition activates tumor-specific T-cell immunity,research has predominantly focused on T-cell-PD-1 expression and its immunobiology. In contrast,cancer cell-intrinsic PD-1 functional regulation is not well understood. Here,we demonstrate induction of PD-1 in melanoma cells via type I interferon receptor (IFNAR) signaling and reversal of ICB efficacy through IFNAR pathway inhibition. Treatment of melanoma cells with IFN-α or IFN-β triggers IFNAR-mediated Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling,increases chromatin accessibility and resultant STAT1/2 and IFN regulatory factor 9 (IRF9) binding within a PD-1 gene enhancer,and leads to PD-1 induction. IFNAR1 or JAK/STAT inhibition suppresses melanoma-PD-1 expression and disrupts ICB efficacy in preclinical models. Our results uncover type I IFN-dependent regulation of cancer cell-PD-1 and provide mechanistic insight into the potential unintended ICB-neutralizing effects of widely used IFNAR1 and JAK inhibitors. Subject terms: Melanoma,Cancer immunotherapy,Tumour immunology View Publication