技术资料

microRNAs (miRNAs) are small,non-coding RNAs that regulate expression of multiple genes. MiR-193a-3p functions as a tumor suppressor in many cancer types,but its effect on inducing specific anti-tumor immune responses is unclear. Therefore,we examined the effect of our lipid nanoparticle (LNP) formulated,chemically modified,synthetic miR-193a-3p mimic (INT-1B3) on anti-tumor immunity. INT-1B3 inhibited distant tumor metastasis and significantly prolonged survival. INT-1B3-treated animals were fully protected against challenge with autologous tumor cells even in absence of treatment indicating long-term immunization. Protection against autologous tumor cell challenge was hampered upon T cell depletion and adoptive T cell transfer abrogated tumor growth. Transfection of tumor cells with our miR-193a-3p mimic (1B3) resulted in tumor cell death and apoptosis accompanied by increased expression of DAMPs. Co-culture of 1B3-transfected tumor cells and immature DC led to DC maturation and these mature DC were able to stimulate production of type 1 cytokines by CD4+ and CD8+ T cells. CD4-CD8- T cells also produced type 1 cytokines,even in response to 1B3-transfected tumor cells directly. Live cell imaging demonstrated PBMC-mediated cytotoxicity against 1B3-transfected tumor cells. These data demonstrate for the first time that miR-193a-3p induces long-term immunity against tumor development via modulation of the tumor microenvironment and induction of immunogenic cell death. View Publication

Chimeric antigen receptor (CAR) T cells show suboptimal efficacy in acute myeloid leukemia (AML). We find that CAR T cells exposed to myeloid leukemia show impaired activation and cytolytic function,accompanied by impaired antigen receptor downstream calcium,ZAP70,ERK,and C-JUN signaling,compared to those exposed to B-cell leukemia. These defects are caused in part by the high expression of CD155 by AML. Overexpressing C-JUN,but not other antigen receptor downstream components,maximally restores anti-tumor function. C-JUN overexpression increases costimulatory molecules and cytokines through reinvigoration of ERK or transcriptional activation,independent of anti-exhaustion. We conduct an open-label,non-randomized,single-arm,phase I trial of C-JUN-overexpressing CAR-T in AML ( NCT04835519 ) with safety and efficacy as primary and secondary endpoints,respectively. Of the four patients treated,one has grade 4 (dose-limiting toxicity) and three have grade 1–2 cytokine release syndrome. Two patients have no detectable bone marrow blasts and one patient has blast reduction after treatment. Thus,overexpressing C-JUN endows CAR-T efficacy in AML. Subject terms: Translational research,Leukaemia View Publication

Immunodeficiency,Centromeric instability and Facial anomalies (ICF) syndrome is a rare genetic disorder characterized by variable immunodeficiency. More than half of the affected individuals show mild to severe intellectual disability at early onset. This disorder is genetically heterogeneous and ZBTB24 is the causative gene of the subtype 2,accounting for about 30% of the ICF cases. ZBTB24 is a multifaceted transcription factor belonging to the Zinc-finger and BTB domain-containing protein family,which are key regulators of developmental processes. Aberrant DNA methylation is the main molecular hallmark of ICF syndrome. The functional link between ZBTB24 deficiency and DNA methylation errors is still elusive. Here,we generated a novel ICF2 disease model by deriving induced pluripotent stem cells (iPSCs) from peripheral CD34 + -blood cells of a patient homozygous for the p.Cys408Gly mutation,the most frequent missense mutation in ICF2 patients and which is associated with a broad clinical spectrum. The mutation affects a conserved cysteine of the ZBTB24 zinc-finger domain,perturbing its function as transcriptional activator. ICF2-iPSCs recapitulate the methylation defects associated with ZBTB24 deficiency,including centromeric hypomethylation. We validated that the mutated ZBTB24 protein loses its ability to directly activate expression of CDCA7 and other target genes in the patient-derived iPSCs. Upon hematopoietic differentiation,ICF2-iPSCs showed decreased vitality and a lower percentage of CD34 + /CD43 + /CD45 + progenitors. Overall,the ICF2-iPSC model is highly relevant to explore the role of ZBTB24 in DNA methylation homeostasis and provides a tool to investigate the early molecular events linking ZBTB24 deficiency to the ICF2 clinical phenotype. View Publication

Olanzapine (OLZ) reverses chronic stress-induced anxiety. Chronic stress promotes cancer development via abnormal neuro-endocrine activation. However,how intervention of brain-body interaction reverses chronic stress-induced tumorigenesis remains elusive. Kras LSL−G12D/WT lung cancer model and LLC1 syngeneic tumor model were used to study the effect of OLZ on cancer stemness and anxiety-like behaviors. Cancer stemness was evaluated by qPCR,western-blotting,immunohistology staining and flow-cytometry analysis of stemness markers,and cancer stem-like function was assessed by serial dilution tumorigenesis in mice and extreme limiting dilution analysis in primary tumor cells. Anxiety-like behaviors in mice were detected by elevated plus maze and open field test. Depression-like behaviors in mice were detected by tail suspension test. Anxiety and depression states in human were assessed by Hospital Anxiety and Depression Scale (HADS). Chemo-sensitivity of lung cancer was assessed by in vivo syngeneic tumor model and in vitro CCK-8 assay in lung cancer cell lines. In this study,we found that OLZ reversed chronic stress-enhanced lung tumorigenesis in both Kras LSL−G12D/WT lung cancer model and LLC1 syngeneic tumor model. OLZ relieved anxiety and depression-like behaviors by suppressing neuro-activity in the mPFC and reducing norepinephrine (NE) releasing under chronic stress. NE activated ADRB2-cAMP-PKA-CREB pathway to promote CLOCK transcription,leading to cancer stem-like traits. As such,CLOCK-deficiency or OLZ reverses NE/chronic stress-induced gemcitabine (GEM) resistance in lung cancer. Of note,tumoral CLOCK expression is positively associated with stress status,serum NE level and poor prognosis in lung cancer patients. We identify a new mechanism by which OLZ ameliorates chronic stress-enhanced tumorigenesis and chemoresistance. OLZ suppresses mPFC-NE-CLOCK axis to reverse chronic stress-induced anxiety-like behaviors and lung cancer stemness. Decreased NE-releasing prevents activation of ADRB2-cAMP-PKA-CREB pathway to inhibit CLOCK transcription,thus reversing lung cancer stem-like traits and chemoresistance under chronic stress. The online version contains supplementary material available at 10.1186/s12964-024-01747-y. View Publication

Riboflavin transporter deficiency type 2 (RTD2) is a rare neurodegenerative autosomal recessive disease caused by mutations in the SLC52A2 gene encoding the riboflavin transporters,RFVT2. Riboflavin (Rf) is the precursor of FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide),which are involved in different redox reactions,including the energetic metabolism processes occurring in mitochondria. To date,human induced pluripotent stem cells (iPSCs) have given the opportunity to characterize RTD2 motoneurons,which reflect the most affected cell type. Previous works have demonstrated mitochondrial and peroxisomal altered energy metabolism as well as cytoskeletal derangement in RTD2 iPSCs and iPSC-derived motoneurons. So far,no attention has been dedicated to astrocytes. Here,we demonstrate that in vitro differentiation of astrocytes,which guarantee trophic and metabolic support to neurons,from RTD2 iPSCs is not compromised. These cells do not exhibit evident morphological differences nor significant changes in the survival rate when compared to astrocytes derived from iPSCs of healthy individuals. These findings indicate that differently from what had previously been documented for neurons,RTD2 does not compromise the morpho-functional features of astrocytes. 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

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

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

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

In recent years,Raman spectroscopy has garnered growing interest in the field of biomedical research. It offers a non-invasive and label-free approach to defining the molecular fingerprint of immune cells. We utilized Raman spectroscopy on optically trapped immune cells to investigate their molecular compositions. While numerous immune cell types have been studied in the past,the characterization of living human CD3/CD28-stimulated T cell subsets remains incomplete. In this study,we demonstrate the capability of Raman spectroscopy to readily distinguish between naïve and stimulated CD4 and CD8 cells. Additionally,we compared these cells with monocytes and discovered remarkable similarities between stimulated T cells and monocytes. This paper contributes to expanding our knowledge of Raman spectroscopy of immune cells and serves as a launching point for future clinical applications. View Publication

Paulownin,a natural compound derived from Paulownia tomentosa wood,exhibits various physiological functions,including anti-bacterial and anti-fungal effects. However,the impact of paulownin on natural killer (NK) cell immune activity remains largely unknown. In this study,we investigated the effect of paulownin on NK cell activity both in vitro and in vivo,and explored its potential mechanisms. NK-92 cells were used for in vitro experiments and a BALB/c mouse model with B16F10 cells injected subcutaneously were used for in vivo anti-tumor analysis. We found that paulownin enhanced the cytolytic activity of NK-92 cells against leukemia,human colon,and human lung cancer cell lines. Paulownin treatment increased the expression of the degranulation marker protein CD107a and cytolytic granules,including granzyme B and perforin in NK-92 cells. Moreover,these enhancements of cytotoxicity and the expression of cytolytic granules induced by paulownin were also observed in human primary NK cells. Signaling studies showed that paulownin promoted the phosphorylation of JNK. The increased perforin expression and elevated cytotoxic activity induced by paulownin were effectively inhibited by pre-treatment with a JNK inhibitor. In vivo studies demonstrated that the administration of paulownin suppressed the growth of B16F10 melanoma cells allografted into mice. Paulownin administration promoted the activation of NK cells in the spleen of mice,resulting in enhanced cytotoxicity against YAC-1 cells. Moreover,the anti-tumor effects of paulownin were reduced upon the depletion of NK cells. Therefore,these results suggest that paulownin enhances NK cell cytotoxicity by activating the JNK signaling pathway and provide significant implications for developing new strategies for cancer immunotherapy. View Publication

Automation and quality control (QC) are critical in manufacturing safe and effective cell and gene therapy products. However,current QC methods,reliant on molecular staining,pose difficulty in in-line testing and can increase manufacturing costs. Here we demonstrate the potential of using label-free ghost cytometry (LF-GC),a machine learning-driven,multidimensional,high-content,and high-throughput flow cytometry approach,in various stages of the cell therapy manufacturing processes. LF-GC accurately quantified cell count and viability of human peripheral blood mononuclear cells (PBMCs) and identified non-apoptotic live cells and early apoptotic/dead cells in PBMCs (ROC-AUC: area under receiver operating characteristic curve = 0.975),T cells and non-T cells in white blood cells (ROC-AUC = 0.969),activated T cells and quiescent T cells in PBMCs (ROC-AUC = 0.990),and particulate impurities in PBMCs (ROC-AUC ≧ 0.998). The results support that LF-GC is a non-destructive label-free cell analytical method that can be used to monitor cell numbers,assess viability,identify specific cell subsets or phenotypic states,and remove impurities during cell therapy manufacturing. Thus,LF-GC holds the potential to enable full automation in the manufacturing of cell therapy products with reduced cost and increased efficiency. Subject terms: Biotechnology,Cell biology,Immunology,Biomedical engineering View Publication