技术资料

Previous studies have indicated that the presence of cancer stem cells may be a contributing factor to the development of metastasis in colorectal cancer patients. Cancer stem cells represent a small subpopulation within the tumor mass that exhibits heightened resistance to treatment and possesses the capacity for self-replication,epithelial–mesenchymal transition,and the generation of new tumors. The tumor microenvironment secretes and releases several molecules that facilitate the self-renewal of cancer stem cells and provide support for colorectal cancer progression. microRNAs are involved in direct cell-to-cell signaling and paracrine signaling between tumor cells and other tumor microenvironment components. They could act as tumor suppressors or oncomiRs,and their deregulation is involved in colorectal cancer progression and cancer stem cell formation. In our previous studies,we demonstrated the oncosuppressive function of miR-486-5p in colorectal cancer; these findings prompted us to conduct a more detailed investigation into its role in cancer stem cell phenotypes. Background: Colorectal cancer (CRC) is the third diagnosed cancer worldwide. Forty-four percent of metastatic colorectal cancer patients were diagnosed at an early stage. Despite curative resection,approximately 40% of patients will develop metastases within a few years. Previous studies indicate the presence of cancer stem cells (CSCs) and their contribution to CRC progression and metastasis. miRNAs deregulation plays a role in CSCs formation and in tumor development. In light of previous studies,we investigated the role of miR-486-5p to understand its role in CSC better. Methods: The expression of miR-486-5p was assessed in adherent cells and spheres generated from two CRC cell lines to observe the difference in expression in CSC-enriched spheroids. Afterward,we overexpressed and underexpressed this miRNA in adherent and sphere cultures through the transfection of a miR-486-5p mimic and a mimic inhibitor. Results: The results demonstrated that miR-486-5p exhibited a notable downregulation in CSC models,and its overexpression led to a significant decrease in colony size. Conclusions: In this study,we confirmed that miR-486-5p plays an oncosuppressive role in CRC,thereby advancing our understanding of the role of this microRNA in the CSC phenotype. View Publication

Cancer stem cells (CSCs) play a key role in non-small cell lung cancer (NSCLC) chemoresistance and metastasis. In this study,we used two NSCLC cell lines to investigate the regulating effect of hypoxia in the induction and maintenance of CSC traits. Our study demonstrated hypoxia-induced stemness and chemoresistance at levels comparable to those in typical CSC sphere culture. Activation of the NF-κB pathway (by transfection of NF-κB-p65) plays a key role in NSCLC CSCs and chemoresistance. Disulfiram (DS),an anti-alcoholism drug,showed a strong in vitro anti-CSC effect. It blocked cancer cell sphere reformation and clonogenicity,synergistically enhanced the cytotoxicity of four anti-NSCLC drugs (doxorubicin,gemcitabine,oxaliplatin and paclitaxel) and reversed hypoxia-induced resistance. The effect of DS on CSCs is copper-dependent. A very short half-life in the bloodstream is the major limitation for the translation of DS into a cancer treatment. Our team previously developed a poly lactic-co-glycolic acid (PLGA) nanoparticle encapsulated DS (DS-PLGA) with a long half-life in the bloodstream. Intra venous injection of DS-PLGA in combination with the oral application of copper gluconate has strong anticancer efficacy in a metastatic NSCLC mouse model. Further study may be able to translate DS-PLGA into cancer applications. View Publication

Melanoma brain metastasis is linked to dismal prognosis and low overall survival and is detected in up to 80% of patients at autopsy. Circulating tumor cells (CTC) are the smallest functional units of cancer and precursors of fatal metastasis. We previously used an unbiased multilevel approach to discover a unique ribosomal protein large/small subunit (RPL/RPS) CTC gene signature associated with melanoma brain metastasis. In this study,we hypothesized that CTC-driven melanoma brain metastasis secondary metastasis (“metastasis of metastasis” per clinical scenarios) has targeted organ specificity for the liver. We injected parallel cohorts of immunodeficient and newly developed humanized NBSGW (huNBSGW) mice with cells from CTC-derived melanoma brain metastasis to identify secondary metastatic patterns. We found the presence of a melanoma brain–liver metastasis axis in huNBSGW mice. Furthermore,RNA sequencing analysis of tissues showed a significant upregulation of the RPL/RPS CTC gene signature linked to metastatic spread to the liver. Additional RNA sequencing of CTCs from huNBSGW blood revealed extensive CTC clustering with human B cells in these mice. CTC:B-cell clusters were also upregulated in the blood of patients with primary melanoma and maintained either in CTC-driven melanoma brain metastasis or melanoma brain metastasis CTC–derived cells promoting liver metastasis. CTC-generated tumor tissues were interrogated at single-cell gene and protein expression levels (10x Genomics Xenium and HALO spatial biology platforms,respectively). Collectively,our findings suggest that heterotypic CTC:B-cell interactions can be critical at multiple stages of metastasis. This study provides important insights into the relevance of prometastatic CTC:B-cell clusters in melanoma progression,extends the importance of the CTC RPL/RPS gene signature beyond primary metastasis/melanoma brain metastasis driving targeted organ specificity for liver metastasis (“metastasis of metastasis”),and identifies new targets for clinical melanoma metastasis therapies. View Publication

Dishevelled-associated activator of morphogenesis1 (DAAM1) is a member of the evolutionarily conserved Formin family and plays a significant role in the malignant progression of various human cancers. This study aims to explore the clinical and biological significance of DAAM1 in pancreatic cancer. Multiple public datasets and an in-house cohort were utilized to assess the clinical relevance of DAAM1 in pancreatic cancer. The LinkedOmics platform was employed to perform enrichment analysis of DAAM1-associated molecular pathways in pancreatic cancer. Subsequently,a series of in vitro and in vivo experiments were conducted to evaluate the biological roles of DAAM1 in pancreatic cancer cells and its effects on intratumoral T cells. DAAM1 was found to be upregulated in pancreatic cancer tissues,with higher expression levels observed in tumor cells. Additionally,high expression of DAAM1 was associated with poor prognosis. DAAM1 acted as an oncogene in pancreatic cancer,and its inhibition suppressed tumor cell proliferation,migration,and invasion,while promoted apoptosis. Furthermore,DAAM1 was involved in the JAK1/STAT1 signaling pathway and regulated PD-L1 expression in pancreatic cancer cells. The inhibition of DAAM1 also significantly reduced the exhaustion levels of CD8+ T cells. In conclusion,DAAM1 functions as an oncogene and is immunologically implicated in pancreatic cancer,these findings suggest that DAAM1 may serve as a promising therapeutic target for the clinical management of pancreatic cancer. The online version contains supplementary material available at 10.1186/s12935-024-03631-8. View Publication

Chimeric antigen receptor (CAR)-engineered T cell therapy holds promise for treating myeloid malignancies,but challenges remain in bone marrow (BM) infiltration and targeting BM-resident malignant cells. Current autologous CAR-T therapies also face manufacturing and patient selection issues,underscoring the need for off-the-shelf products. In this study,we characterize primary patient samples and identify a unique therapeutic opportunity for CAR-engineered invariant natural killer T (CAR-NKT) cells. Using stem cell gene engineering and a clinically guided culture method,we generate allogeneic CD33-directed CAR-NKT cells with high yield,purity,and robustness. In preclinical mouse models,CAR-NKT cells exhibit strong BM homing and effectively target BM-resident malignant blast cells,including CD33-low/negative leukemia stem and progenitor cells. Furthermore,CAR-NKT cells synergize with hypomethylating agents,enhancing tumor-killing efficacy. These cells also show minimal off-tumor toxicity,reduced graft-versus-host disease and cytokine release syndrome risks,and resistance to allorejection,highlighting their substantial therapeutic potential for treating myeloid malignancies. Subject terms: Cancer therapy,Immunotherapy,Leukaemia View Publication

To fully utilize the potential of human induced pluripotent stem cells (hiPSCs) for allogeneic stem cell–based therapies,efficient and scalable expansion procedures must be developed. For other adherent human cell types,the combination of microcarriers (MCs) and stirred tank bioreactors has been shown to meet these demands. In this study,a hiPSC quasi-perfusion expansion procedure based on MCs was developed at 100-mL scale in spinner flasks. Process development began by assessing various medium exchange strategies and MC coatings,indicating that the hiPSCs tolerated the gradual exchange of medium well when cultivated on Synthemax II–coated MCs. This procedure was therefore scaled-up to the 1.3-L Eppendorf BioBLU 1c stirred tank bioreactor by applying the lower limit of Zwietering’s suspension criterion ( N s 1 u ),thereby demonstrating proof-of-concept when used in combination with hiPSCs for the first time. To better understand the bioreactor and its bioengineering characteristics,computational fluid dynamics and bioengineering investigations were performed prior to hiPSC cultivation. In this manner,improved process understanding allowed an expansion factor of ≈ 26 to be achieved,yielding more than 3 × 10 9 cells within 5 days. Further quality analyses confirmed that the hiPSCs maintained their viability,identity,and differentiation potential throughout cultivation. • N s 1 u can be used as a scale-up criterion for hiPSC cultivations in MC-operated stirred bioreactors • Uniform distribution and attachment of cells to the MCs are crucial for efficient expansion • Perfusion is advantageous and supports the cultivation of hiPSCs The online version contains supplementary material available at 10.1007/s00253-024-13372-3. View Publication

Acute myeloid leukemia (AML) is characterized by the accumulation of immature myeloid cells and a differentiation block,highlighting the urgent need for novel differentiation-inducing therapies. This study evaluated Adina rubella Hance (ARH) stem as a potent differentiation inducer by systematically screening 200 plant extracts. ARH stem promoted phenotypic differentiation in AML cells. In addition to its differentiation-inducing effects,ARH stem exhibited strong antileukemic activities,such as inhibiting cell proliferation,inducing cell death,and enhancing mitochondrial reactive oxygen species (mtROS) levels,the latter of which is critical for its differentiation-promoting activity. Comparative analysis with the extracts from other parts of the plant confirmed the superior efficacy of the stem extract because of its unique chemical composition. Ultra-high-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry analysis identified Picroside III as a major active compound within the stem extract,capable of recapitulating ARH stem-induced differentiation and demonstrating significant antileukemic properties. These findings underscore the therapeutic potential of ARH stem and its active component,Picroside III,as promising agents for differentiation-based treatment strategies in AML. View Publication

Understanding the interaction between genetic and epigenetic variation remains a challenge due to confounding environmental factors. We propose that human induced Pluripotent Stem Cells (iPSCs) are an excellent model to study the relationship between genetic and epigenetic variation while controlling for environmental factors. In this study,we have created a comprehensive resource of high-quality genomic,epigenomic,and transcriptomic data from iPSC lines and three iPSC-derived cell types (neural stem cell (NSC),motor neuron,monocyte) from three healthy donors. We find that epigenetic variation is most strongly associated with genetic variation at the iPSC stage,and that relationship weakens as epigenetic variation increases in differentiated cells. Additionally,cell type is a stronger source of epigenetic variation than genetic variation. Further,we elucidate a utility of studying epigenetic variation in iPSCs and their derivatives for identifying important loci for GWAS studies and the cell types in which they may be acting. Subject terms: Epigenomics,Genomics,Transcriptomics View Publication

Persistence of drug-resistant breast cancer stem cells (brCSCs) after a chemotherapeutic regime correlates with disease recurrence and elevated mortality. Therefore,deciphering mechanisms that dictate their drug-resistant phenotype is imperative for designing targeted and more effective therapeutic strategies. The transcription factor SOX2 has been recognized as a protagonist in brCSC maintenance,and previous studies have confirmed that inhibition of SOX2 purportedly eliminated these brCSCs. However,pharmacological targeting of transcription factors like SOX2 is challenging due to their structural incongruities and intrinsic disorders in their binding interfaces. Therefore,transcriptional co-activators may serve as a feasible alternative for effectively targeting the brCSCs. Incidentally,transcriptional co-activators YAP/TAZ were found to be upregulated in CD44 + /CD24 - /ALDH + cells isolated from patient breast tumors and CSC-enriched mammospheres. Interestingly,it was observed that YAP/TAZ exhibited direct physical interaction with SOX2 and silencing YAP/TAZ attenuated SOX2 expression in mammospheres,leading to significantly reduced sphere forming efficiency and cell viability. YAP/TAZ additionally manipulated redox homeostasis and regulated mitochondrial dynamics by restraining the expression of the mitochondrial fission marker,DRP1. Furthermore,YAP/TAZ inhibition induced DRP1 expression and impaired OXPHOS,consequently inducing apoptosis in mammospheres. In order to enhance clinical relevance of the study,an FDA-approved drug verteporfin (VP),was used for pharmacological inhibition of YAP/TAZ. Surprisingly,VP administration was found to reduce tumor-initiating capacity of the mammospheres,concomitant with disrupted mitochondrial homeostasis and significantly reduced brCSC population. Therefore,VP holds immense potential for repurposing and decisively eliminating the chemoresistant brCSCs,offering a potent strategy for managing tumor recurrence effectively. Subject terms: Cancer stem cells,Cancer stem cells View Publication

Small nucleolar RNAs (snoRNAs) are non-coding RNAs that function in ribosome and spliceosome biogenesis,primarily by guiding modifying enzymes to specific sites on ribosomal RNA (rRNA) and spliceosomal RNA (snRNA). However,many orphan snoRNAs remain uncharacterized,with unidentified or unvalidated targets,and studies on additional snoRNA-associated proteins are limited. We adapted an enhanced chimeric eCLIP approach to comprehensively profile snoRNA-target RNA interactions using both core and accessory snoRNA-binding proteins as baits. Using core snoRNA-binding proteins,we confirmed most annotated snoRNA-rRNA and snoRNA-snRNA interactions in mouse and human cell lines and called novel,high-confidence interactions for orphan snoRNAs. While some of these interactions result in chemical modification,others may have modification-independent functions. We showed that snoRNA ribonucleoprotein complexes containing certain accessory proteins,like WDR43 and NOLC1,enriched for specific subsets of snoRNA-target RNA interactions with distinct roles in ribosome and spliceosome biogenesis. Notably,we discovered that SNORD89 guides 2′-O-methylation at two neighboring sites in U2 snRNA that fine-tune splice site recognition. Chimeric eCLIP of snoRNA-associating proteins enables a comprehensive framework for studying snoRNA-target interactions in an RNA-binding protein-dependent manner,revealing novel interactions and regulatory roles in RNA biogenesis. The online version contains supplementary material available at 10.1186/s13059-025-03508-7. View Publication

The targeting of cancer stem cells (CSCs) has proven to be an effective approach for limiting tumor progression,thus necessitating the identification of new drugs with anti-CSC activity. Through a high-throughput drug repositioning screen,we identify the antibiotic Nifuroxazide (NIF) as a potent anti-CSC compound. Utilizing a click chemistry strategy,we demonstrate that NIF is a prodrug that is specifically bioactivated in breast CSCs. Mechanistically,NIF-induced CSC death is a result of a synergistic action that combines the generation of DNA interstrand crosslinks with the inhibition of the Fanconi anemia (FA) pathway activity. NIF treatment mimics FA-deficiency through the inhibition of STAT3,which we identify as a non-canonical transcription factor of FA-related genes. NIF induces a chemical HRDness (Homologous Recombination Deficiency) in CSCs that (re)sensitizes breast cancers with innate or acquired resistance to PARP inhibitor (PARPi) in patient-derived xenograft models. Our results suggest that NIF may be useful in combination with PARPi for the treatment of breast tumors,regardless of their HRD status. Subject terms: Breast cancer,Mechanisms of disease,Target identification,Cancer stem cells View Publication

The clinical success of the immune checkpoint inhibitor (ICI) targeting programmed cell death protein 1 (PD-1) has revolutionized cancer treatment. However,the full potential of PD-1 blockade therapy remains unrealized,as response rates are still low across many cancer types. Interleukin-2 (IL-2)-based immunotherapies hold promise,as they can stimulate robust T cell expansion and enhance effector function - activities that could synergize potently with PD-1 blockade. Yet,IL-2 therapies also carry a significant drawback: they can trigger severe systemic toxicities and induce immune suppression by expanding regulatory T cells. To overcome the challenges of PD-1 blockade and IL-2 therapies while enhancing safety and efficacy,we have engineered a novel fusion protein,AWT020,combining a humanized anti-PD-1 nanobody and an engineered IL-2 mutein (IL-2c). The IL-2c component of AWT020 has been engineered to exhibit no binding to the IL-2 receptor alpha (IL-2Rα) subunit and attenuated affinity for the IL-2 receptor beta and gamma (IL-2Rβγ) complex,aiming to reduce systemic immune cell activation,thereby mitigating the severe toxicity often associated with IL-2 therapies. The anti-PD-1 antibody portion of AWT020 serves a dual purpose: it precisely delivers the IL-2c payload to tumor-infiltrating T cells while blocking the immune-inhibitory signals mediated by the PD-1 pathway. AWT020 showed significantly enhanced pSTAT5 signaling in PD-1 expressing cells and promoted the proliferation of activated T cells over natural killer (NK) cells. In preclinical studies using both anti-PD-1-sensitive and -resistant mouse tumor models,the mouse surrogate of AWT020 (mAWT020) demonstrated markedly enhanced anti-tumor efficacy compared to an anti-PD-1 antibody,IL-2,or the combination of an anti-PD-1 antibody and IL-2. In addition,the mAWT020 treatment was well-tolerated,with minimal signs of toxicity. Immune profiling revealed that mAWT020 preferentially expands CD8 + T cells within tumors,sparing peripheral T and NK cells. Notably,this selective tumoral T-cell stimulation enables potent tumor-specific T-cell responses,underscoring the molecule’s enhanced efficacy and safety. The AWT020 fusion protein offers a promising novel immunotherapeutic strategy by integrating PD-1 blockade and IL-2 signaling,conferring enhanced anti-tumor activity with reduced toxicity. View Publication