Publications

2025

1. Stem Cell Res Ther

   A single-cell transcriptomic atlas of human lens epithelium: identification and functional insights into lens stem/progenitor cells

   Yuzhou Gu, Lu Chen, Shuying Chen, and 14 more authors

   Stem Cell Research & Therapy, Jul 2025

   Abs DOI

   

   Background The existence of stem/progenitor cells in the lens epithelium has been demonstrated, but their identification remains challenging. Accurate identification requires advanced technologies and a comprehensive understanding of lens epithelial cell (LEC) subtypes, presenting a significant challenge in age-related cataract research. Methods Eight pairs of human donor lens epithelium samples were collected for single-cell RNA sequencing (scRNA-seq). This included four non-aged and four aged (65 years) individuals. Subsequent analyses involved cell (sub)type characterization, trajectory inference, and cell-cell communication. Experimental validation was conducted through transcriptome sequencing and immunofluorescence on human lenses, lens organoids, rabbit regenerated lenses, mouse lenses, and cell lines. Results Six groups were identified via UMAP mapping of scRNA-seq data: four LECs, one lens fiber cells (LFCs), and one immune cells. One of the four LEC clusters exhibited a distinct gene expression profile and was identified as transient amplifying cells (TACs). TACs specifically express TOP2A and are localized at the lens equator. CytoTRACE analysis to the LEC and LFC data sets provided a differentiation trajectory. The TAC group was determined as stage 2 in the trajectory and LFCs last. The 3 sub groups were labelled early, mid and late LECs and corresponded to stage 1, 3 and 4 in the path. While cell population demographics remained stable with age, transcriptomic changes in LECs were observed, including weaker intercellular crosstalk and adhesion, and fewer TACs in S phase. Lens progenitor-like cells (LPLCs) were identified as a sub-population in early LECs and express ID1. In addition, pleiotrophin (PTN) signaling was prevalent at all differentiation stages, with a notable weakening of PTN signaling in aged LPLCs. Conclusions This study identified four subclasses of LECs within the human lens epithelium that follow a progressive staged development pathway from progenitor cells to mature LECs. TOP2A can serve as a biomarker for TAC

2. Commun Bio

   

   Reference-informed evaluation of batch correction for single-cell omics data with overcorrection awareness

   Xiaoyue Hu^*, He Li^*, Ming Chen, and 2 more authors

   Communications Biology, Mar 2025

   Abs DOI

   

   Batch effect correction (BEC) is fundamental to integrate multiple single-cell RNA sequencing datasets, and its success is critical to empower in-depth interrogation for biological insights. However, no simple metric is available to evaluate BEC performance with sensitivity to data overcorrection, which erases true biological variations and leads to false biological discoveries. Here, we propose RBET, a reference-informed statistical framework for evaluating the success of BEC. Using extensive simulations and six real data examples including scRNA-seq and scATAC-seq datasets with different numbers of batches, batch effect sizes and numbers of cell types, we demonstrate that RBET evaluates the performance of BEC methods more fairly with biologically meaningful insights from data, while other methods may lead to false results. Moreover, RBET is computationally efficient, sensitive to overcorrection and robust to large batch effect sizes. Thus, RBET provides a robust guideline on selecting case-specific BEC method, and the concept of RBET is extendable to other modalities.

3. JCI insight

   

   Vitamin A treatment restores vision failures arising from Leber’s hereditary optic neuropathy-linked mtDNA mutation

   Cheng Ai^*, Huiying Li^*, Chunyan Wang, and 5 more authors

   JCI Insight, Mar 2025

   Publisher: American Society for Clinical Investigation

   Abs DOI

   

   Leber hereditary optic neuropathy (LHON) is a paradigm for mitochondrial retinopathy due to mitochondrial DNA (mtDNA) mutations. However, the mechanism underlying retinal cell-specific effects of LHON-linked mtDNA mutations remains poorly understood and there has been no effective treatment or cure for this disorder. Using a mice model bearing a LHON-linked ND6P25L mutation, we demonstrated that the mutation caused retinal cell-specific deficiencies, especially in retinal ganglion cells (RGC), rods and Müller cells. Single-cell RNA sequencing revealed cell-specific dysregulation of oxidative phosphorylation and visual signaling pathways in the mutant retina. Strikingly, ND6 mutation-induced dysfunctions caused abnormal vitamin A (VA) metabolism essential for visual function. VA supplementation remarkably alleviated retinal deficiencies, including reduced fundus lesion and retinal thickness, and increasing numbers of RGCs, photoreceptors and Müller cell neurites. The restoration of visual functions with VA treatment were further evidenced by correcting dysregulations of phototransduction cascade and neurotransmitter transmission and restoring electrophysiological properties. Interestingly, VA supplementation markedly rescued the abnormal mitochondrial morphologies and functions in the mutant retina. These findings provide new insight into retina-specific pathophysiology of mitochondrial retinopathy arising from vitamin A deficiency and mitochondrial dysfunction induced by mtDNA mutation and step toward for therapeutic intervention for LHON and other mitochondrial retinopathy.

2024

1. Cancer Res

   

   Cancer-Associated Fibroblasts Expressing Sulfatase 1 Facilitate VEGFA-Dependent Microenvironmental Remodeling to Support Colorectal Cancer

   Huijuan Wang, Jiaxin Chen, Xiaoyu Chen, and 11 more authors

   Cancer Research, Oct 2024

   IF 12.5

   Abs DOI PDF

   

   Abstract Tumor stroma plays a critical role in fostering tumor progression and metastasis. Cancer-associated fibroblasts (CAF) are a major component of the tumor stroma. Identifying the key molecular determinants for the protumor properties of CAFs could enable the development of more effective treatment strategies. In this study, through analyses of single-cell sequencing data, we identified a population of CAFs expressing high levels of sulfatase 1 (SULF1), which was associated with poor prognosis in patients with colorectal cancer. Colorectal cancer models using mice with conditional SULF1 knockout in fibroblasts revealed the tumor-supportive function of SULF1+ CAFs. Mechanistically, SULF1+ CAFs enhanced the release of VEGFA from heparan sulfate proteoglycan. The increased bioavailability of VEGFA initiated the deposition of extracellular matrix and enhanced angiogenesis. In addition, intestinal microbiota–produced butyrate suppressed SULF1 expression in CAFs through its histone deacetylase (HDAC) inhibitory activity. The insufficient butyrate production in patients with colorectal cancer increased the abundance of SULF1+ CAFs, thereby promoting tumor progression. Importantly, tumor growth inhibition by HDAC was dependent on SULF1 expression in CAFs, and patients with colorectal cancer with more SULF1+ CAFs were more responsive to treatment with the HDAC inhibitor chidamide. Collectively, these findings unveil the critical role of SULF1+ CAFs in colorectal cancer and provide a strategy to stratify patients with colorectal cancer for HDAC inhibitor treatment.  Significance: SULF1+ cancer-associated fibroblasts play a tumor-promoting role in colorectal cancer by stimulating extracellular matrix deposition and angiogenesis and can serve as a biomarker for the therapeutic response to HDAC inhibitors in patients.

2. Cancer Disco

   

   Receptor-interacting Protein Kinase 2 Is an Immunotherapy Target in Pancreatic Cancer

   Wenhua Sang, Yiduo Zhou, Haiyan Chen, and 13 more authors

   Cancer Discovery, Feb 2024

   Highlighted by CD, ESI highly cited; IF 28.2

   Abs DOI HTML PDF

   

   Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal malignancy because of its aggressive nature and the paucity of effective treatment options. Almost all registered drugs have proven ineffective in addressing the needs of patients with PDAC. This is the result of a poor understanding of the unique tumor–immune microenvironment (TME) in PDAC. To identify druggable regulators of immunosuppressive TME, we performed a kinome- and membranome-focused CRISPR screening using orthotopic PDAC models. Our data showed that receptor-interacting protein kinase 2 (RIPK2) is a crucial driver of immune evasion of cytotoxic T-cell killing and that genetic or pharmacologic targeting of RIPK2 sensitizes PDAC to anti–programmed cell death protein 1 (anti–PD-1) immunotherapy, leading to prolonged survival or complete regression. Mechanistic studies revealed that tumor-intrinsic RIPK2 ablation disrupts desmoplastic TME and restores MHC class I (MHC-I) surface levels through eliminating NBR1-mediated autophagy-lysosomal degradation. Our results provide a rationale for a novel combination therapy consisting of RIPK2 inhibition and anti–PD-1 immunotherapy for PDAC.

3. Immunity

   

   CD4+ T cell activation distinguishes response to anti-PD-L1+anti-CTLA4 therapy from anti-PD-L1 monotherapy

   Amelie Franken, Michel Bila, Aurelie Mechels, and 23 more authors

   Immunity, Mar 2024

   Abs DOI

   

   Cancer patients often receive a combination of antibodies targeting programmed death-ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen-4 (CTLA4). We conducted a window-of-opportunity study in head and neck squamous cell carcinoma (HNSCC) to examine the contribution of anti-CTLA4 to anti-PD-L1 therapy. Singlecell profiling of on- versus pre-treatment biopsies identified T cell expansion as an early response marker. In tumors, anti-PD-L1 triggered the expansion of mostly CD8+ T cells, whereas combination therapy expanded both CD4+ and CD8+ T cells. Such CD4+ T cells exhibited an activated T helper 1 (Th1) phenotype. CD4+ and CD8+ T cells co-localized with and were surrounded by dendritic cells expressing T cell homing factors or antibody-producing plasma cells. T cell receptor tracing suggests that anti-CTLA4, but not anti-PD-L1, triggers the trafficking of CD4+ naive/central-memory T cells from tumor-draining lymph nodes (tdLNs), via blood, to the tumor wherein T cells acquire a Th1 phenotype. Thus, CD4+ T cell activation and recruitment from tdLNs are hallmarks of early response to anti-PD-L1 plus anti-CTLA4 in HNSCC.

4. Elife

   G protein subunit Gγ13-mediated signaling pathway is critical to the inflammation resolution and functional recovery of severely injured lungs

   Yi-Hong Li, Yi-Sen Yang, Yan-Bo Xue, and 6 more authors

   eLife, Jun 2024

   Abs DOI

   

   Tuft cells are a group of rare epithelial cells that can detect pathogenic microbes and parasites. Many of these cells express signaling proteins initially found in taste buds. It is, however, not well understood how these taste signaling proteins contribute to the response to the invading pathogens or to the recovery of injured tissues. In this study, we conditionally nullified the signaling G protein subunit Gγ13 and found that the number of ectopic tuft cells in the injured lung was reduced following the infection of the influenza virus H1N1. Furthermore, the infected mutant mice exhibited significantly larger areas of lung injury, increased macrophage infiltration, severer pulmonary epithelial leakage, augmented pyroptosis and cell death, greater bodyweight loss, slower recovery, worsened fibrosis and increased fatality. Our data demonstrate that the Gγ13-mediated signal transduction pathway is critical to tuft cells-mediated inflammation resolution and functional repair of the damaged lungs.To our best knowledge, it is the first report indicating subtype-specific contributions of tuft cells to the resolution and recovery.

2023

1. Nat Commun

   PD-1- CD45RA+ effector-memory CD8 T cells and CXCL10+ macrophages are associated with response to atezolizumab plus bevacizumab in advanced hepatocellular carcinoma

   Sarah Cappuyns, Gino Philips, Vincent Vandecaveye, and 16 more authors

   Nature Communications, Nov 2023

   Abs DOI

   

   Abstract The combination of atezolizumab plus bevacizumab (atezo/bev) has dramatically changed the treatment landscape of advanced HCC (aHCC), achieving durable responses in some patients. Using single-cell transcriptomics, we characterize the intra-tumoural and peripheral immune context of patients with aHCC treated with atezo/bev. Tumours from patients with durable responses are enriched for PDL1 CXCL10 + macrophages and, based on cell–cell interaction analysis, express high levels of CXCL9/10/11 and are predicted to attract peripheral CXCR3+ CD8+ effector-memory T cells (CD8 Tem) into the tumour. Based on T cell receptor sharing and pseudotime trajectory analysis, we propose that CD8 T EM preferentially differentiate into clonally-expanded PD1-CD45RA+ effector-memory CD8+ T cells (CD8 Temra) with pronounced cytotoxicity. In contrast, in non-responders, CD8 Tem remain frozen in their effector-memory state. Finally, in responders, CD8 Temra display a high degree of T cell receptor sharing with blood, consistent with their patrolling activity. These findings may help understand the possible mechanisms underlying response to atezo/bev in aHCC.

2022

1. Cell Metab

   

   Tumor-associated macrophages are shaped by intratumoral high potassium via Kir2.1

   Sheng Chen, Wenyu Cui, Zhexu Chi, and 22 more authors

   Cell Metabolism, Nov 2022

   Abs DOI

   

   The tumor microenvironment (TME) is a unique niche governed by constant crosstalk within and across all intratumoral cellular compartments. In particular, intratumoral high potassium (K+) has shown immune-suppressive potency on T cells. However, as a pan-cancer characteristic associated with local necrosis, the impact of this ionic disturbance on innate immunity is unknown. Here, we reveal that intratumoral high K+ suppresses the anti-tumor capacity of tumor-associated macrophages (TAMs). We identify the inwardly rectifying K+ channel Kir2.1 as a central modulator of TAM functional polarization in high K+ TME, and its conditional depletion repolarizes TAMs toward an anti-tumor state, sequentially boosting local anti-tumor immunity. Kir2.1 deficiency disturbs the electrochemically dependent glutamine uptake, engendering TAM metabolic reprogramming from oxidative phosphorylation toward glycolysis. Kir2.1 blockade attenuates both murine tumor- and patient-derived xenograft growth. Collectively, our findings reveal Kir2.1 as a determinant and potential therapeutic target for regaining the anti-tumor capacity of TAMs within ionic-imbalanced TME.

2021

1. Nat Med

   

   A single-cell map of intratumoral changes during anti-PD1 treatment of patients with breast cancer

   Ayse Bassez^*, Hanne Vos^*, Laurien Van Dyck, and 14 more authors

   Nature Medicine, May 2021

   ESI highly cited; IF 87.2

   Abs DOI HTML PDF

   

   Immune-checkpoint blockade (ICB) combined with neoadjuvant chemotherapy improves pathological complete response in breast cancer. To understand why only a subset of tumors respond to ICB, patients with hormone receptor-positive or triple-negative breast cancer were treated with anti-PD1 before surgery. Paired pre- versus on-treatment biopsies from treatment-naive patients receiving anti-PD1 (n = 29) or patients receiving neoadjuvant chemotherapy before anti-PD1 (n = 11) were subjected to single-cell transcriptome, T cell receptor and proteome profiling. One-third of tumors contained PD1-expressing T cells, which clonally expanded upon anti-PD1 treatment, irrespective of tumor subtype. Expansion mainly involved CD8+ T cells with pronounced expression of cytotoxic-activity (PRF1, GZMB), immune-cell homing (CXCL13) and exhaustion markers (HAVCR2, LAG3), and CD4+ T cells characterized by expression of T-helper-1 (IFNG) and follicular-helper (BCL6, CXCR5) markers. In pre-treatment biopsies, the relative frequency of immunoregulatory dendritic cells (PD-L1+), specific macrophage phenotypes (CCR2+ or MMP9+) and cancer cells exhibiting major histocompatibility complex class I/II expression correlated positively with T cell expansion. Conversely, undifferentiated pre-effector/memory T cells (TCF7+, GZMK+) or inhibitory macrophages (CX3CR1+, C3+) were inversely correlated with T cell expansion. Collectively, our data identify various immunophenotypes and associated gene sets that are positively or negatively correlated with T cell expansion following anti-PD1 treatment. We shed light on the heterogeneity in treatment response to anti-PD1 in breast cancer.

2. Cell Res

   

   Discriminating mild from critical COVID-19 by innate and adaptive immune single-cell profiling of bronchoalveolar lavages.

   Els Wauters, Pierre Van Mol, Abhishek Dinkarnath Garg, and 26 more authors

   Cell research, Mar 2021

   Highlighted by Nat Rev Immunol; Top Article Cell Res; ESI highly cited; IF 46.4

   Abs DOI HTML PDF

   

   How the innate and adaptive host immune system miscommunicate to worsen COVID-19 immunopathology has not been fully elucidated. Here, we perform single-cell deep-immune profiling of bronchoalveolar lavage (BAL) samples from 5 patients with mild and 26 with critical COVID-19 in comparison to BALs from non-COVID-19 pneumonia and normal lung. We use pseudotime inference to build T-cell and monocyte-to-macrophage trajectories and model gene expression changes along them. In mild COVID-19, CD8+ resident-memory (TRM) and CD4+ T-helper-17 (TH17) cells undergo active (presumably antigen-driven) expansion towards the end of the trajectory, and are characterized by good effector functions, while in critical COVID-19 they remain more naïve. Vice versa, CD4+ T-cells with T-helper-1 characteristics (TH1-like) and CD8+ T-cells expressing exhaustion markers (TEX-like) are enriched halfway their trajectories in mild COVID-19, where they also exhibit good effector functions, while in critical COVID-19 they show evidence of inflammation-associated stress at the end of their trajectories. Monocyte-to-macrophage trajectories show that chronic hyperinflammatory monocytes are enriched in critical COVID-19, while alveolar macrophages, otherwise characterized by anti-inflammatory and antigen-presenting characteristics, are depleted. In critical COVID-19, monocytes contribute to an ATP-purinergic signaling-inflammasome footprint that could enable COVID-19 associated fibrosis and worsen disease-severity. Finally, viral RNA-tracking reveals infected lung epithelial cells, and a significant proportion of neutrophils and macrophages that are involved in viral clearance.

3. Genom Med

   High-grade serous tubo-ovarian cancer refined with single-cell RNA sequencing: specific cell subtypes influence survival and determine molecular subtype classification

   Siel Olbrecht, Pieter Busschaert, Junbin Qian, and 10 more authors

   Genome Medicine, Dec 2021

   Publisher: Genome Medicine ISBN: 0000100498

   DOI

   
4. STTT

   BCL9 regulates CD226 and CD96 checkpoints in CD8+ T cells to improve PD-1 response in cancer

   Mei Feng, Zhongen Wu, Yan Zhou, and 16 more authors

   Signal Transduction and Targeted Therapy, Aug 2021

   Abs DOI

   

   Abstract To date, the overall response rate of PD-1 blockade remains unsatisfactory, partially due to limited understanding of tumor immune microenvironment (TIME). B-cell lymphoma 9 (BCL9), a key transcription co-activator of the Wnt pathway, is highly expressed in cancers. By genetic depletion and pharmacological inhibition of BCL9 in tumors, we found that BCL9 suppression reduced tumor growth, promoted CD8 + T cell tumor infiltration, and enhanced response to anti-PD-1 treatment in mouse colon cancer models. To determine the underlying mechanism of BCL9’s role in TIME regulation, single-cell RNA-seq was applied to reveal cellular landscape and transcription differences in the tumor immune microenvironment upon BCL9 inhibition. CD155-CD226 and CD155-CD96 checkpoints play key roles in cancer cell/CD8 + T cell interaction. BCL9 suppression induces phosphorylation of VAV1 in CD8 + T cells and increases GLI1 and PATCH expression to promote CD155 expression in cancer cells. In The Cancer Genome Atlas database analysis, we found that BCL9 expression is positively associated with CD155 and negatively associated with CD226 expression. BCL9 is also linked to adenomatous polyposis coli (APC) mutation involved in patient survival following anti-PD-1 treatment. This study points to cellular diversity within the tumor immune microenvironment affected by BCL9 inhibition and provides new insights into the role of BCL9 in regulating CD226 and CD96 checkpoints

5. Cancer Immunol Res

   IL1β Promotes Immune Suppression in the Tumor Microenvironment Independent of the Inflammasome and Gasdermin D

   Máté Kiss, Lieselotte Vande Walle, Pedro H.V. Saavedra, and 25 more authors

   Cancer Immunology Research, Mar 2021

   Abs DOI

   

   Abstract IL1β is a central mediator of inflammation. Secretion of IL1β typically requires proteolytic maturation by the inflammasome and formation of membrane pores by gasdermin D (GSDMD). Emerging evidence suggests an important role for IL1β in promoting cancer progression in patients, but the underlying mechanisms are ill-defined. Here, we have shown a key role for IL1β in driving tumor progression in two distinct mouse tumor models. Notably, activation of the inflammasome, caspase-8, as well as the pore-forming proteins GSDMD and mixed lineage kinase domain–like protein in the host were dispensable for the release of intratumoral bioactive IL1β. Inflammasome-independent IL1β release promoted systemic neutrophil expansion and fostered accumulation of T-cell–suppressive neutrophils in the tumor. Moreover, IL1β was essential for neutrophil infiltration triggered by antiangiogenic therapy, thereby contributing to treatment-induced immunosuppression. Deletion of IL1β allowed intratumoral accumulation of CD8+ effector T cells that subsequently activated tumor-associated macrophages. Depletion of either CD8+ T cells or macrophages abolished tumor growth inhibition in IL1β-deficient mice, demonstrating a crucial role for CD8+ T-cell–macrophage cross-talk in the antitumor immune response. Overall, these results support a tumor-promoting role for IL1β through establishing an immunosuppressive microenvironment and show that inflammasome activation is not essential for release of this cytokine in tumors.

6. Front Immunol

   Pro-Inflammatory Signature in Decidua of Recurrent Pregnancy Loss Regardless of Embryonic Chromosomal Abnormalities

   Zaigui Wu, Miaomiao Wang, Guanmian Liang, and 7 more authors

   Frontiers in Immunology, Dec 2021

   Abs DOI

   

   Recurrent pregnancy loss (RPL), especially the unexplained RPL, is associated with the disruption of maternal immune tolerance. However, little is known about the immune status at the decidua of RPL with embryonic chromosomal aberrations. Herein, mass cytometry (CyTOF) was used to interrogate the immune atlas at the decidua which was obtained from 15 RPL women—six with normal chromosome and nine with chromosomal aberrations—and five controls. The total frequency of CCR2−CD11chigh macrophages increased, while CD39high NK cells and CCR2−CD11clow macrophages decrease significantly in RPL when RPLs were stratified, compared with controls. Proinflammatory subsets of CD11chigh macrophages increased, while less proinflammatory or suppressive subsets decreased statistically in RPL decidua whenever RPLs were stratified or not. However, CD11chigh NK and CD161highCD8+ T cells increased only in RPL with normal chromosome, while the inactivated and naive CD8+/ CD4+ T cells were enriched only in RPL with chromosomal aberrations. A proinflammatory signature is observed in RPL decidua; however, differences exist between RPL with and without chromosomal abnormalities.

7. Nat Commun

   Monocyte-driven atypical cytokine storm and aberrant neutrophil activation as key mediators of COVID-19 disease severity

   L. Vanderbeke, P. Van Mol, Y. Van Herck, and 52 more authors

   Nature Communications, Dec 2021

   Publisher: Springer US

   Abs DOI

   

   Epidemiological and clinical reports indicate that SARS-CoV-2 virulence hinges upon the triggering of an aberrant host immune response, more so than on direct virus-induced cellular damage. To elucidate the immunopathology underlying COVID-19 severity, we perform cytokine and multiplex immune profiling in COVID-19 patients. We show that hypercytokinemia in COVID-19 differs from the interferon-gamma-driven cytokine storm in macrophage activation syndrome, and is more pronounced in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels paired with deep-immune profiling shows that classical monocytes drive this hyper-inflammatory phenotype and that a reduction in T-lymphocytes correlates with disease severity, with CD8+ cells being disproportionately affected. Antigen presenting machinery expression is also reduced in critical disease. Furthermore, we report that neutrophils contribute to disease severity and local tissue damage by amplification of hypercytokinemia and the formation of neutrophil extracellular traps. Together our findings suggest a myeloid-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity.

8. Nat Commun

   Clinical practices underlie COVID-19 patient respiratory microbiome composition and its interactions with the host

   Verónica Lloréns-Rico, Ann C Gregory, Johan Van Weyenbergh, and 24 more authors

   Nature Communications, Dec 2021

   Abs DOI

   

   Understanding the pathology of COVID-19 is a global research priority. Early evidence suggests that the respiratory microbiome may be playing a role in disease progression, yet current studies report contradictory results. Here, we examine potential confounders in COVID-19 respiratory microbiome studies by analyzing the upper ( n = 58) and lower ( n = 35) respiratory tract microbiome in well-phenotyped COVID-19 patients and controls combining microbiome sequencing, viral load determination, and immunoprofiling. We find that time in the intensive care unit and type of oxygen support, as well as associated treatments such as antibiotic usage, explain the most variation within the upper respiratory tract microbiome, while SARS-CoV-2 viral load has a reduced impact. Specifically, mechanical ventilation is linked to altered community structure and significant shifts in oral taxa previously associated with COVID-19. Single-cell transcriptomics of the lower respiratory tract of COVID-19 patients identifies specific oral bacteria in physical association with proinflammatory immune cells, which show higher levels of inflammatory markers. Overall, our findings suggest confounders are driving contradictory results in current COVID-19 microbiome studies and careful attention needs to be paid to ICU stay and type of oxygen support, as bacteria favored in these conditions may contribute to the inflammatory phenotypes observed in severe COVID-19 patients.

2020

1. Cell Res

   

   A pan-cancer blueprint of the heterogeneous tumor microenvironment revealed by single-cell profiling

   Junbin Qian, Siel Olbrecht, Bram Boeckx, and 21 more authors

   Cell Research, Sep 2020

   Cover, Recommended by F1000, ESI highly cited; IF 46.4

   Abs DOI HTML PDF

   

   The stromal compartment of the tumor microenvironment consists of a heterogeneous set of tissue-resident and tumor-infiltrating cells, which are profoundly moulded by cancer cells. An outstanding question is to what extent this heterogeneity is similar between cancers affecting different organs. Here, we profile 233,591 single cells from patients with lung, colorectal, ovary and breast cancer (n = 36) and construct a pan-cancer blueprint of stromal cell heterogeneity using different single-cell RNA and protein-based technologies. We identify 68 stromal cell populations, of which 46 are shared between cancer types and 22 are unique. We also characterise each population phenotypically by highlighting its marker genes, transcription factors, metabolic activities and tissue-specific expression differences. Resident cell types are characterised by substantial tissue specificity, while tumor-infiltrating cell types are largely shared across cancer types. Finally, by applying the blueprint to melanoma tumors treated with checkpoint immunotherapy and identifying a naïve CD4+ T-cell phenotype predictive of response to checkpoint immunotherapy, we illustrate how it can serve as a guide to interpret scRNA-seq data. In conclusion, by providing a comprehensive blueprint through an interactive web server, we generate the first panoramic view on the shared complexity of stromal cells in different cancers.

2. Cancer Cell

   An Integrated Gene Expression Landscape Profiling Approach to Identify Lung Tumor Endothelial Cell Heterogeneity and Angiogenic Candidates

   Jermaine Goveia, Katerina Rohlenova, Federico Taverna, and 46 more authors

   Cancer Cell, Jan 2020

   Abs DOI

   

   Heterogeneity of lung tumor endothelial cell (TEC) phenotypes across patients, species (human/mouse), and models (in vivo/in vitro) remains poorly inventoried at the single-cell level. We single-cell RNA (scRNA)-sequenced 56,771 endothelial cells from human/mouse (peri)-tumoral lung and cultured human lung TECs, and detected 17 known and 16 previously unrecognized phenotypes, including TECs putatively regulating immune surveillance. We resolved the canonical tip TECs into a known migratory tip and a putative basement-membrane remodeling breach phenotype. Tip TEC signatures correlated with patient survival, and tip/breach TECs were most sensitive to vascular endothelial growth factor blockade. Only tip TECs were congruent across species/models and shared conserved markers. Integrated analysis of the scRNA-sequenced data with orthogonal multi-omics and meta-analysis data across different human tumors, validated by functional analysis, identified collagen modification as a candidate angiogenic pathway.

3. Cancer Cell

   Lineage-dependent gene expression programs influence the immune landscape of colorectal cancer

   Hae-Ock Lee, Yourae Hong, Hakki Emre Etlioglu, and 34 more authors

   Nature Genetics, Jun 2020

   DOI

   
4. Mol Biol Cell

   Co-regulation of the antagonistic RepoMan:Aurora-B pair in proliferating cells

   Maria Giulia Manzione, Jan Rombouts, Mikhail Steklov, and 5 more authors

   Molecular Biology of the Cell, Mar 2020

   Abs DOI PDF

   

   We found that the counteracting enzymes RepoMan and Aurora B co-oscillate during the cell cycle. The co–up-regulation of RepoMan and Aurora B in human tumors correlates with poor prognosis. Moreover, cancer cells with high RepoMan concentration are more sensitive to treatment with Aurora-B inhibitors.

5. Genom Biol

   DNA methylation repels binding of hypoxia-inducible transcription factors to maintain tumor immunotolerance

   Flora D’Anna, Laurien Van Dyck, Jieyi Xiong, and 22 more authors

   Genome Biology, Dec 2020

   Abs DOI

   

   Background: Hypoxia is pervasive in cancer and other diseases. Cells sense and adapt to hypoxia by activating hypoxia-inducible transcription factors (HIFs), but it is still an outstanding question why cell types differ in their transcriptional response to hypoxia. Results: We report that HIFs fail to bind CpG dinucleotides that are methylated in their consensus binding sequence, both in in vitro biochemical binding assays and in vivo studies of differentially methylated isogenic cell lines. Based on in silico structural modeling, we show that 5-methylcytosine indeed causes steric hindrance in the HIF binding pocket. A model wherein cell-type-specific methylation landscapes, as laid down by the differential expression and binding of other transcription factors under normoxia, control cell-type-specific hypoxia responses is observed. We also discover ectopic HIF binding sites in repeat regions which are normally methylated. Genetic and pharmacological DNA demethylation, but also cancer-associated DNA hypomethylation, expose these binding sites, inducing HIF-dependent expression of cryptic transcripts. In line with such cryptic transcripts being more prone to cause double-stranded RNA and viral mimicry, we observe low DNA methylation and high cryptic transcript expression in tumors with high immune checkpoint expression, but not in tumors with low immune checkpoint expression, where they would compromise tumor immunotolerance. In a low-immunogenic tumor model, DNA demethylation upregulates cryptic transcript expression in a HIF-dependent manner, causing immune activation and reducing tumor growth. Conclusions: Our data elucidate the mechanism underlying cell-type-specific responses to hypoxia and suggest DNA methylation and hypoxia to underlie tumor immunotolerance.

2019

1. BioEssays

   

   Coordination of Timers and Sensors in Cell Signaling

   Junbin Qian^†, Lendert Gelens^†, and Mathieu Bollen^†

   BioEssays, Mar 2019

   Abs DOI PDF

   

   Timers and sensors are common devices that make our daily life safer, more convenient, and more efficient. In a cellular context, they arguably play an even more crucial role as they ensure the survival of cells in the presence of various extrinsic and intrinsic stresses. Biological timers and sensors generate distinct signaling profiles, enabling them to produce different types of cellular responses. Recent data suggest that they can work together to guarantee correct timing and responsiveness. By exploring examples of cellular stress signaling from mitosis, DNA damage, and hypoxia, the authors discuss the common architecture of timer-sensor integration, and how its added features contribute to the generation of desired signaling profiles when dealing with stresses of variable duration and strength. The authors propose timer-sensor integration as a widespread mechanism with profound biological implications and therapeutic potential.

2. J Cell Biochem

   LINC00461 promotes cell migration and invasion in breast cancer through miR-30a-5p/integrin β3 axis

   Lifeng Dong, Junbin Qian, Fangfang Chen, and 2 more authors

   Journal of Cellular Biochemistry, Mar 2019

   Abs DOI

   

   Mounting evidence has demonstrated that long noncoding RNAs (lncRNAs) are dysregulated and implicated in the occurrence and development of a wide range of human malignancies. LINC00461, a novel cancer-related lncRNA, has been reported to be highly expressed and serve as oncogene in glioma; however, its biological role in breast cancer (BC) remains obscure. This study aimed to explore the role of LINC00461 in BC and elucidate the potential molecular mechanisms involved. In the current study, LINC00461 was found to be significantly upregulated in both BC tissues and cell lines. Besides, we found that high LINC00461 expression was associated with TNM stage and differentiation. Furthermore, functional studies demonstrated that LINC00461 expedited BC cell migration and invasion. Notably, LINC00461 was observed to enhance the expression of vimentin and zinc-finger E-box binding homeobox factor 1, suppress the expression of E-cadherin, and promote the activation of extracellular signal-regulated kinase and AKT signaling pathways. Mechanical investigations revealed that LINC00461 positively modulated integrin β3 (ITGB3) expression as miR-30a-5p sponge in BC cells. Taken together, LINC00461 exerts an oncogenic role in BC through miR-30a-5p/ITGB3 axis. Our data indicate that LINC00461 may be used to be a novel candidate therapeutic target and a valuable diagnostic biomarker for BC. © 2019 Wiley Periodicals, Inc.

2018

1. Trends Cell Biol

   The Importance of Kinase–Phosphatase Integration: Lessons from Mitosis

   Lendert Gelens, Junbin Qian, Mathieu Bollen, and 1 more author

   Trends in Cell Biology, Jan 2018

   Publisher: Elsevier Ltd

   Abs DOI

   

   Kinases and phosphatases work antagonistically to control the behaviour of individual substrate molecules. This can be incorrectly extrapolated to imply that they also work antagonistically on the signals or processes that these molecules control. In fact, in many situations kinases and phosphatases work together to positively drive signal responses. We explain how this ‘cooperativity’ is critical for setting the amplitude, localisation, timing, and shape of phosphorylation signals. We use mitosis to illustrate why these properties are important for controlling mitotic entry, sister chromatid cohesion, kinetochore–microtubule attachments, the spindle assembly checkpoint, mitotic spindle elongation, and mitotic exit. These examples provide a rationale to explain how complex signalling behaviour could rely on similar types of integration within many other biological processes.

2. Nat Methods

   

   A proximity-tagging system to identify membrane protein–protein interactions

   Qiang Liu, Jun Zheng, Weiping Sun, and 5 more authors

   Nature Methods, Jan 2018

   Publisher: Springer US

   Abs DOI

   The communication between cells and between cellular organelles is often controlled by the interaction of membrane proteins. Although many methods for the detection of protein–protein interactions (PPIs) exist, membrane PPIs remain difficult to detect. Here we developed a proximity-based tagging system, PUP-IT (pupylation-based interaction tagging), to identify membrane protein interactions. In this approach, a small protein tag, Pup, is applied to proteins that interact with a PafA-fused bait, enabling transient and weak interactions to be enriched and detected by mass spectrometry. Pup does not diffuse from the enzyme, which allows high-specificity labeling. We applied this approach to CD28, a critical costimulatory receptor for T lymphocyte activation, and identified known CD28 binding partners and multiple potential interacting proteins. In addition, we demonstrated that this method can identify the interaction between a cell surface receptor and its ligand.

2017

1. Mol Cell

   

   An Attachment-Independent Biochemical Timer of the Spindle Assembly Checkpoint.

   Junbin Qian^†, Maria Adelaida García-Gimeno, Monique Beullens, and 7 more authors

   Molecular cell, Nov 2017

   Abs DOI PDF

   

   The spindle assembly checkpoint (SAC) generates a diffusible protein complex that prevents anaphase until all chromosomes are properly attached to spindle microtubules. A key step in SAC initiation is the recruitment of MAD1 to kinetochores, which is generally thought to be governed by the microtubule-kinetochore (MT-KT) attachment status. However, we demonstrate that the recruitment of MAD1 via BUB1, a conserved kinetochore receptor, is not affected by MT-KT interactions in human cells. Instead, BUB1:MAD1 interaction depends on BUB1 phosphorylation, which is controlled by a biochemical timer that integrates counteracting kinase and phosphatase effects on BUB1 into a pulse-generating incoherent feedforward loop. We propose that this attachment-independent timer serves to rapidly activate the SAC at mitotic entry, before the attachment-sensing MAD1 receptors have become fully operational. The BUB1-centered timer is largely impervious to conventional anti-mitotic drugs, and it is, therefore, a promising therapeutic target to induce cell death through permanent SAC activation.

2. FEBS Lett

   

   Split-BioID: a proximity biotinylation assay for dimerization-dependent protein interactions

   Sofie De Munter, Janina Görnemann, Rita Derua, and 7 more authors

   FEBS Letters, Jan 2017

   ISBN: 4955139574

   DOI PDF

   

2015

1. Nat Commun

   

   Cdk1 orders mitotic events through coordination of a chromosome-associated phosphatase switch.

   Junbin Qian, Monique Beullens, Jin Huang, and 3 more authors

   Nature Communications, Dec 2015

   Publisher: Nature Publishing Group

   Abs DOI PDF

   

   RepoMan is a scaffold for signalling by mitotic phosphatases at the chromosomes. During (pro)metaphase, RepoMan-associated protein phosphatases PP1 and PP2A-B56 regulate the chromosome targeting of Aurora-B kinase and RepoMan, respectively. Here we show that this task division is critically dependent on the phosphorylation of RepoMan by protein kinase Cyclin-dependent kinase 1 (Cdk1), which reduces the binding of PP1 but facilitates the recruitment of PP2A-B56. The inactivation of Cdk1 in early anaphase reverses this phosphatase switch, resulting in the accumulation of PP1-RepoMan to a level that is sufficient to catalyse its own chromosome targeting in a PP2A-independent and irreversible manner. Bulk-targeted PP1-RepoMan also inactivates Aurora B and initiates nuclear-envelope reassembly through dephosphorylation-mediated recruitment of Importin β. Bypassing the Cdk1 regulation of PP1-RepoMan causes the premature dephosphorylation of its mitotic-exit substrates in prometaphase. Hence, the regulation of RepoMan-associated phosphatases by Cdk1 is essential for the timely dephosphorylation of their mitotic substrates.

2013

1. Curr Biol

   

   Aurora B Defines Its Own Chromosomal Targeting by Opposing the Recruitment of the Phosphatase Scaffold Repo-Man

   Junbin Qian, Monique Beullens, Bart Lesage, and 1 more author

   Current Biology, Jun 2013

   Abs DOI PDF

   

   Aurora B is the catalytic subunit of the chromosomal passenger complex (CPC), which coordinates mitotic processes through phosphorylation of key regulatory proteins [1]. In prometaphase, the CPC is enriched at the centromeres to regulate the spindle checkpoint and kinetochore-microtubule interactions. Centromeric CPC binds to histone H3 that is phosphorylated at T3 (H3T3ph) by Aurora B-stimulated Haspin [2–5]. PP1/Repo-Man acts antagonistically to Haspin and dephosphorylates H3T3ph at the chromosome arms but is somehow prevented from causing a net dephosphorylation of centromeric H3T3ph during prometaphase [6, 7]. Here, we show that Aurora B phosphorylates Repo-Man at S893, preventing its recruitment by histones. We also identify PP2A as a mitotic interactor of Repo-Man that dephosphorylates S893 and thereby promotes the targeting of Repo-Man to chromosomes and the dephosphorylation of H3T3ph by PP1. Thus, Repo-Man-associated PP1 and PP2A collaborate to oppose the chromosomal targeting of Aurora B. We propose that the reciprocal feedback regulation of Haspin and Repo-Man by Aurora B generates a robust bistable response that culminates in the centromeric targeting of the CPC during prometaphase.

2. Curr Opin Cell Biol

   

   4D-networking by mitotic phosphatases.

   Junbin Qian^*, Claudia Winkler^*, and Mathieu Bollen

   Current opinion in cell biology, Dec 2013

   Publisher: Elsevier Ltd

   Abs DOI PDF

   

   Faithful progression through mitosis is critically dependent on the timely phosphorylation and dephosphorylation of a host of proteins. The involved protein kinases and phosphatases are embedded in interconnected feedback and feedforward circuits that ensure swift and robust phase transitions. Here we review recent evidence showing that protein phosphatases are modulators of the mitotic entry but also organize the mitotic exit through an orderly dephosphorylation of their substrates. In addition, phosphatases spatiotemporally restrict the phosphorylation of key regulatory proteins and oppose kinases to control highly dynamic mitotic processes, including chromosome congression and checkpoint signaling. In accordance with their important role as nodes in phosphorylation networks, mitotic protein phosphatases are tightly regulated in four dimensions.

2011

1. Curr Biol

   

   PP1/Repo-Man Dephosphorylates Mitotic Histone H3 at T3 and Regulates Chromosomal Aurora B Targeting

   Junbin Qian^*, Bart Lesage^*, Monique Beullens, and 2 more authors

   Current Biology, May 2011

   Abs DOI PDF

   

   The transient mitotic histone H3 phosphorylation by various protein kinases regulates chromosome condensation and segregation, but the counteracting phosphatases have been poorly characterized [1-8]. We show here that PP1γ is the major histone H3 phosphatase acting on the mitotically phosphorylated (ph) residues H3T3ph, H3S10ph, H3T11ph, and H3S28ph. In addition, we identify Repo-Man, a chromosome-bound interactor of PP1γ [9], as a selective regulator of H3T3ph and H3T11ph dephosphorylation. Repo-Man promotes H3T11ph dephosphorylation by an indirect mechanism but directly and specifically targets H3T3ph for dephosphorylation by associated PP1γ. The PP1γ/Repo-Man complex opposes the protein kinase Haspin-mediated spreading of H3T3ph to the chromosome arms until metaphase and catalyzes the net dephosphorylation of H3T3ph at the end of mitosis. Consistent with these findings, Repo-Man modulates in a PP1-dependent manner the H3T3ph-regulated chromosomal targeting of Aurora kinase B and its substrate MCAK. Our study defines a novel mechanism by which PP1 counteracts Aurora B.

2. Curr Biol

   

   Spindle Checkpoint Silencing: PP1 Tips the Balance

   Bart Lesage^*, Junbin Qian^*, and Mathieu Bollen

   Current Biology, Nov 2011

   Abs DOI PDF

   

   The spindle checkpoint is a mitotic surveillance mechanism that delays anaphase until all sister chromatids are correctly attached to microtubules from opposite poles. Recent studies reveal that protein kinase Aurora B is a key regulator of spindle checkpoint activation whereas protein phosphatase PP1 antagonizes Aurora B and induces checkpoint silencing. Chromosome biorientation stretches the kinetochores and spatially separates centromeric Aurora B from its kinetochore substrates, comprising several PP1-interacting proteins (PIPs). The ensuing dephosphorylation of these PIPs creates docking sites for the bulk recruitment of PP1 to the kinetochores. We propose that this tension-induced targeting of PP1 triggers checkpoint silencing by the dephosphorylation of kinetochore and checkpoint components, including Aurora B substrates. In addition, PP1 also directly inactivates a kinetochore-associated pool of Aurora B and silences checkpoint signaling by opposing the centromeric targeting of Aurora B.