技术资料

The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic,but pericellular oxygen levels,which are affected by oxygen diffusivity and consumption,are rarely reported. Here,we provide evidence that several cell types in culture actually experience local hypoxia,with important implications for cell metabolism and function. We focused initially on adipocytes,as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions,cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria,lowered HIF1α activity,and resulted in widespread transcriptional rewiring. Functionally,adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli,recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages,hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types,and that considering pericellular oxygen improves the quality,reproducibility and translatability of culture models. View Publication

Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitro generation of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using leukemia inhibitory factor (LIF),which is not expressed in guided cortical organoids,we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP,LIFR,and HOPX,closely matching human fetal oRG. Finally,incorporating neural crest-derived LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely. View Publication

Hematopoietic stem and progenitor cells (HSPCs) that have impaired differentiation can transform into leukemic blasts. However,the mechanism that controls differentiation remains elusive. Here,we show that the genetic elimination of Proteinase 3 (PRTN3) in mice led to spontaneous myeloid differentiation. Mechanistically,our findings indicate that PRTN3 interacts with the N-terminal of STAT3,serving as a negative regulator of STAT3-dependent myeloid differentiation. Specifically,PRTN3 promotes STAT3 ubiquitination and degradation,while simultaneously reducing STAT3 phosphorylation and nuclear translocation during G-CSF-stimulated myeloid differentiation. Strikingly,pharmacological inhibition of STAT3 (Stattic) partially counteracted the effects of PRTN3 deficiency on myeloid differentiation. Moreover,the deficiency of PRTN3 in primary AML blasts promotes the differentiation of those cells into functional neutrophils capable of chemotaxis and phagocytosis,ultimately resulting in improved overall survival rates for recipients. These findings indicate PRTN3 exerts an inhibitory effect on STAT3-dependent myeloid differentiation and could be a promising therapeutic target for the treatment of acute myeloid leukemia. Subject terms: Signal transduction,Haematological diseases View Publication

Alcohol consumption increases the risk of breast cancer and promotes cancer progression. Alcohol exposure could affect both processes of the mammary carcinogenesis,namely,the cell transformation and onset of tumorigenesis as well as cancer aggressiveness including metastasis and drug resistance/recurrence. However,the cellular and molecular mechanisms underlying alcohol tumor promotion remain unclear. There are four members of the mammalian p38 mitogen-activated protein kinase (MAPK) family,namely,p38α,p38β,p38γ and p38δ. We have previously demonstrated alcohol exposure selectively activated p38γ MAPK in breast cancer cells in vitro and in vivo . Pirfenidone (PFD),an antifibrotic compound approved for the treatment of idiopathic pulmonary fibrosis,is also a pharmacological inhibitor of p38γ MAPK. This study aimed to determine whether PFD is useful to inhibit alcohol-induced promotion of breast cancer. Female adolescent (5 weeks) MMTV-Wnt1 mice were exposed to alcohol with a liquid diet containing 6.7% ethanol. Some mice received intraperitoneal (IP) injection of PFD (100 mg/kg) every other day. After that,the effects of alcohol and PFD on mammary tumorigenesis and metastasis were examined. Alcohol promoted the progression of mammary tumors in adolescent MMTV-Wnt1 mice. Treatment of PFD blocked tumor growth and alcohol-promoted metastasis. It also significantly inhibited alcohol-induced tumorsphere formation and cancer stem cell (CSC) population. PFD inhibited mammary tumor growth and alcohol-promoted metastasis. Since PFD is an FDA-approved drug,the current findings may be helpful to re-purpose its application in treating aggressive breast cancer and alcohol-promoted mammary tumor progression. View Publication

Tumor cells have the ability to invade and form small clusters that protrude into adjacent tissues,a phenomenon that is frequently observed at the periphery of a tumor as it expands into healthy tissues. The presence of these clusters is linked to poor prognosis and has proven challenging to treat using conventional therapies. We previously reported that p60AmotL2 expression is localized to invasive colon and breast cancer cells. In vitro,p60AmotL2 promotes epithelial cell invasion by negatively impacting E-cadherin/AmotL2-related mechanotransduction. Using epithelial cells transfected with inducible p60AmotL2,we employed a phenotypic drug screening approach to find compounds that specifically target invasive cells. The phenotypic screen was performed by treating cells for 72 h with a library of compounds with known antitumor activities in a dose-dependent manner. After assessing cell viability using CellTiter-Glo,drug sensitivity scores for each compound were calculated. Candidate hit compounds with a higher drug sensitivity score for p60AmotL2-expressing cells were then validated on lung and colon cell models,both in 2D and in 3D,and on colon cancer patient-derived organoids. Nascent RNA sequencing was performed after BET inhibition to analyse BET-dependent pathways in p60AmotL2-expressing cells. We identified 60 compounds that selectively targeted p60AmotL2-expressing cells. Intriguingly,these compounds were classified into two major categories: Epidermal Growth Factor Receptor (EGFR) inhibitors and Bromodomain and Extra-Terminal motif (BET) inhibitors. The latter consistently demonstrated antitumor activity in human cancer cell models,as well as in organoids derived from colon cancer patients. BET inhibition led to a shift towards the upregulation of pro-apoptotic pathways specifically in p60AmotL2-expressing cells. BET inhibitors specifically target p60AmotL2-expressing invasive cancer cells,likely by exploiting differences in chromatin accessibility,leading to cell death. Additionally,our findings support the use of this phenotypic strategy to discover novel compounds that can exploit vulnerabilities and specifically target invasive cancer cells. The online version contains supplementary material available at 10.1186/s13046-024-03031-w. View Publication

Chimeric antigen receptor (CAR) T cell therapies targeting B cell-restricted antigens CD19,CD20,or CD22 can produce potent clinical responses for some B cell malignancies,but relapse remains common. Camelid single-domain antibodies (sdAbs or nanobodies) are smaller,simpler,and easier to recombine than single-chain variable fragments (scFvs) used in most CARs,but fewer sdAb-CARs have been reported. Thus,we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein,sdAb-cDNA library construction,and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. Changing CD28- to CD8-transmembrane design increased CAR persistence and expression in vitro . CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro,although only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies,a lead CD22sdCAR-T was selected,achieving comparable complete responses to a benchmark short linker m971-scFv CAR-T in high-dose experiments. Finally,immunohistochemistry and flow cytometry confirm tissue and cellular-level specificity of the lead CD22-sdAb. This presents a complete report on preclinical development of a novel CD22sdCAR therapeutic. View Publication

Pulmonary macrophage transplantation (PMT) is a gene and cell transplantation approach in development as therapy for hereditary pulmonary alveolar proteinosis (hPAP),a surfactant accumulation disorder caused by mutations in CSF2RA/B (and murine homologs). We conducted a toxicology study of PMT of Csf2ra gene-corrected macrophages (mGM-Rα + Mϕs) or saline-control intervention in Csf2ra KO or wild-type (WT) mice including single ascending dose and repeat ascending dose studies evaluating safety,tolerability,pharmacokinetics,and pharmacodynamics. Lentiviral-mediated Csf2ra cDNA transfer restored GM-CSF signaling in mGM-Rα + Mϕs. Following PMT,mGM-Rα + Mϕs engrafted,remained within the lungs,and did not undergo uncontrolled proliferation or result in bronchospasm,pulmonary function abnormalities,pulmonary or systemic inflammation,anti-transgene product antibodies,or pulmonary fibrosis. Aggressive male fighting caused a similarly low rate of serious adverse events in saline- and PMT-treated mice. Transient,minor pulmonary neutrophilia and exacerbation of pre-existing hPAP-related lymphocytosis were observed 14 days after PMT of the safety margin dose but not the target dose (5,000,000 or 500,000 mGM-Rα + Mϕs,respectively) and only in Csf2ra KO mice but not in WT mice. PMT reduced lung disease severity in Csf2ra KO mice. Results indicate PMT of mGM-Rα + Mϕs was safe,well tolerated,and therapeutically efficacious in Csf2ra KO mice,and established a no adverse effect level and 10-fold safety margin. View Publication

Microglia,the brain’s resident immune cells,play vital roles in brain development,and disorders like Alzheimer’s disease (AD). Human iPSC-derived microglia (iMG) provide a promising model to study these processes. However,existing iMG generation protocols face challenges,such as prolonged differentiation time,lack of detailed characterization,and limited gene function investigation via CRISPR-Cas9. Our integrated toolkit for in-vitro microglia functional genomics optimizes iPSC differentiation into iMG through a streamlined two-step,20-day process,producing iMG with a normal karyotype. We confirmed the iMG’s authenticity and quality through single-cell RNA sequencing,chromatin accessibility profiles (ATAC-Seq),proteomics and functional tests. The toolkit also incorporates a drug-dependent CRISPR-ON/OFF system for temporally controlled gene expression. Further,we facilitate the use of multi-omic data by providing online searchable platform that compares new iMG profiles to human primary microglia: https://sherlab.shinyapps.io/IPSC-derived-Microglia/ . Our method generates iMG that closely align with human primary microglia in terms of transcriptomic,proteomic,and chromatin accessibility profiles. Functionally,these iMG exhibit Ca2 + transients,cytokine driven migration,immune responses to inflammatory signals,and active phagocytosis of CNS related substrates including synaptosomes,amyloid beta and myelin. Significantly,the toolkit facilitates repeated iMG harvesting,essential for large-scale experiments like CRISPR-Cas9 screens. The standalone ATAC-Seq profiles of our iMG closely resemble primary microglia,positioning them as ideal tools to study AD-associated single nucleotide variants (SNV) especially in the genome regulatory regions. Our advanced two-step protocol rapidly and efficiently produces authentic iMG. With features like the CRISPR-ON/OFF system and a comprehensive multi-omic data platform,our toolkit equips researchers for robust microglial functional genomic studies. By facilitating detailed SNV investigation and offering a sustainable cell harvest mechanism,the toolkit heralds significant progress in neurodegenerative disease drug research and therapeutic advancement. The online version contains supplementary material available at 10.1186/s13287-024-03700-9. View Publication

Chemotherapy remains the mainstay treatment for triple-negative breast cancer (TNBC) due to the lack of specific targets. Given a modest response of immune checkpoint inhibitors in TNBC patients,improving immunotherapy is an urgent and crucial task in this field. CD73 has emerged as a novel immunotherapeutic target,given its elevated expression on tumor,stromal,and specific immune cells,and its established role in inhibiting anti-cancer immunity. CD73-generated adenosine suppresses immunity by attenuating tumor-infiltrating T- and NK-cell activation,while amplifying regulatory T cell activation. Chemotherapy often leads to increased CD73 expression and activity,further suppressing anti-tumor immunity. While debulking the tumor mass,chemotherapy also enriches heterogenous cancer stem cells (CSC),potentially leading to tumor relapse. Therefore,drugs targeting both CD73,and CSCs hold promise for enhancing chemotherapy efficacy,overcoming treatment resistance,and improving clinical outcomes. However,safe and effective inhibitors of CD73 have not been developed as of now. We used in silico docking to screen compounds that may be repurposed for inhibiting CD73. The efficacy of these compounds was investigated through flow cytometry,RT-qPCR,CD73 activity,cell viability,tumorsphere formation,and other in vitro functional assays. For assessment of clinical translatability,TNBC patient-derived xenograft organotypic cultures were utilized. We also employed the ovalbumin-expressing AT3 TNBC mouse model to evaluate tumor-specific lymphocyte responses. We identified quercetin and luteolin,currently used as over-the-counter supplements,to have high in silico complementarity with CD73. When quercetin and luteolin were combined with the chemotherapeutic paclitaxel in a triple-drug regimen,we found an effective downregulation in paclitaxel-enhanced CD73 and CSC-promoting pathways YAP and Wnt. We found that CD73 expression was required for the maintenance of CD44 high CD24 low CSCs,and co-targeting CD73,YAP,and Wnt effectively suppressed the growth of human TNBC cell lines and patient-derived xenograft organotypic cultures. Furthermore,triple-drug combination inhibited paclitaxel-enriched CSCs and simultaneously improved lymphocyte infiltration in syngeneic TNBC mouse tumors. Conclusively,our findings elucidate the significance of CSCs in impairing anti-tumor immunity. The high efficacy of our triple-drug regimen in clinically relevant platforms not only underscores the importance for further mechanistic investigations but also paves the way for potential development of new,safe,and cost-effective therapeutic strategies for TNBC. View Publication

Prenatal lethality associated with mouse knockout of Mettl16,a recently identified RNA N6-methyladenosine (m 6 A) methyltransferase,has hampered characterization of the essential role of METTL16-mediated RNA m 6 A modification in early embryonic development. Here,using cross-species single-cell RNA sequencing analysis,we found that during early embryonic development,METTL16 is more highly expressed in vertebrate hematopoietic stem and progenitor cells (HSPCs) than other methyltransferases. In Mettl16-deficient zebrafish,proliferation capacity of embryonic HSPCs is compromised due to G1/S cell cycle arrest,an effect whose rescue requires Mettl16 with intact methyltransferase activity. We further identify the cell-cycle transcription factor mybl2b as a directly regulated by Mettl16-mediated m 6 A modification. Mettl16 deficiency resulted in the destabilization of mybl2b mRNA,likely due to lost binding by the m 6 A reader Igf2bp1 in vivo. Moreover,we found that the METTL16-m 6 A- MYBL2 -IGF2BP1 axis controlling G1/S progression is conserved in humans. Collectively,our findings elucidate the critical function of METTL16-mediated m 6 A modification in HSPC cell cycle progression during early embryonic development. View Publication

Non-small cell lung cancer (NSCLC) is known for high relapse rates despite resection in early stages. Here,we present the results of a phase I clinical trial in which a dendritic cell (DC) vaccine targeting patient-individual neoantigens is evaluated in patients with resected NSCLC. Vaccine manufacturing is feasible in six of 10 enrolled patients. Toxicity is limited to grade 1–2 adverse events. Systemic T cell responses are observed in five out of six vaccinated patients,with T cell responses remaining detectable up to 19 months post vaccination. Single-cell analysis indicates that the responsive T cell population is polyclonal and exhibits the near-entire spectrum of T cell differentiation states,including a naive-like state,but excluding exhausted cell states. Three of six vaccinated patients experience disease recurrence during the follow-up period of 2 years. Collectively,these data support the feasibility,safety,and immunogenicity of this treatment in resected NSCLC. View Publication

MicroRNAs (miRNAs) are short non-coding RNAs that play crucial roles in gene regulation,exerting post-transcriptional silencing,thereby influencing cellular function,development,and disease. Traditional loss-of-function methods for studying miRNA functions,such as miRNA inhibitors and sponges,present limitations in terms of specificity,transient effects,and off-target effects. Similarly,CRISPR/Cas9-based editing of miRNAs using single guide RNAs (sgRNAs) also has limitations in terms of design space for generating effective gRNAs. In this study,we introduce a novel approach that utilizes CRISPR/Cas9 with dual guide RNAs (dgRNAs) for the rapid and efficient generation of short deletions within miRNA genomic regions. Through the expression of dgRNAs through single-copy lentiviral integration,this approach achieves over a 90% downregulation of targeted miRNAs within a week. We conducted a comprehensive analysis of various parameters influencing efficient deletion formation. In addition,we employed doxycycline (Dox)-inducible expression of Cas9 from the AAVS1 locus,enabling homogeneous,temporal,and stage-specific editing during cellular differentiation. Compared to miRNA inhibitory methods,the dgRNA-based approach offers higher specificity,allowing for the deletion of individual miRNAs with similar seed sequences,without affecting other miRNAs. Due to the increased design space,the dgRNA-based approach provides greater flexibility in gRNA design compared to the sgRNA-based approach. We successfully applied this approach in two human cell lines,demonstrating its applicability for studying the mechanisms of human erythropoiesis and pluripotent stem cell (iPSC) biology and differentiation. Efficient deletion of miR-451 and miR-144 resulted in blockage of erythroid differentiation,and the deletion of miR-23a and miR-27a significantly affected iPSC survival. We have validated the highly efficient deletion of genomic regions by editing protein-coding genes,resulting in a significant impact on protein expression. This protocol has the potential to be extended to delete multiple miRNAs within miRNA clusters,allowing for future investigations into the cooperative effects of the cluster members on cellular functions. The protocol utilizing dgRNAs for miRNA deletion can be employed to generate efficient pooled libraries for high-throughput comprehensive analysis of miRNAs involved in different biological processes. View Publication