Biology subjects

Wu, S. Z.

Publications and source records attributed to Wu, S. Z..

15 recordsLinked to original sources

bioRxiv2026

Fibroblast TGF-β3 promotes tissue-residency and survival of CD8 T cells in barrier tissues and tumors

Fibroblasts are key organizers of tissue architecture and immune cell homeostasis, yet how they shape adaptive immune function within non-lymphoid tissues remains incompletely understood. CD8+ tissue-resident memory T cells (TRM) provide localized protection against pathogens and contribute to tumor control, but the microenvironmental signals that maintain their persistence and survival are poorly defined. Here, we identify fibroblast-derived TGF-{beta}3 as a conserved stromal niche factor that specifically sustains CD8+ TRM in both steady-state and disease settings. Across human single-cell cross-tissue atlases, CD8+ TRM preferentially correlated with fibroblast abundance in healthy barrier tissues and multiple tumor types, and TGFB3 emerged as a key fibroblast-enriched candidate mediator. In human and murine co-culture systems, fibroblast-derived TGF-{beta}3 promoted CD8 TRM-like differentiation in vitro. Using a novel genetic in vivo model, inducible fibroblast-specific deletion of Tgfb3 reduced CD8 TRM across barrier tissues at steady state and impaired antigen-specific CD8 TRM formation following viral infection. In tumor models, genetic loss or antibody mediated neutralization of TGF-{beta}3 impaired CD8 T cell residency and cytotoxicity, induced dysfunction via proteotoxic stress and apoptotic programs, and accelerated tumor growth. These findings provide mechanistic insight into the limited efficacy of pan-TGF-{beta} blockade in cancer therapy. Collectively, we describe a novel fibroblast-CD8 T cell axis mediated by TGF-{beta}3 that sustains residency and restrains proteotoxic stress in barrier tissues and tumors.

immunology↗

bioRxiv2025

Inferring cellular communication through mapping cells in space using Tangram 2

Cell-to-cell communication (CCC) shapes development, immunity, and disease, yet current spatial transcriptomics (SRT) platforms rarely achieve both single-cell resolution and high-quality transcriptome-wide coverage to accurately characterize CCC in the tissue microenvironment. Tangram2 bridges this gap by integrating single-cell RNA sequencing (scRNA-seq) with SRT to identify genes whose expression changes as a function of neighboring cell types. By accurately mapping cells in tissue space, Tangram2 disentangles interaction-driven transcriptional shifts from intrinsic identity markers, yielding mechanistic CCC maps. Validation across diverse settings--including Slide-tags, co-cultured experiments, human lymph nodes, and simulations--demonstrates high accuracy. Applied to triple-negative breast cancer (TNBC) and cutaneous squamous cell carcinoma (cSCC), Tangram2 recapitulates known biology and uncovers new hypotheses, including various immunosuppressive mechanisms in TNBC and a macrophage-regulatory T-cell circuit associated with survival in cSCC.

genomics↗

bioRxiv2025

ROCK2 inhibition has a dual role in reducing ECM remodelling and cell growth, while impairing migration and invasion

The Rho-associated coiled-coil containing protein kinases 1/2 (ROCK1/2) are key signalling proteins involved in the regulation of the actin cytoskeleton and control a variety of cellular processes. This includes cell proliferation, stemness, cell migration and invasion as well as actomyosin contraction and extracellular matrix (ECM) remodelling. Due to a lack of ROCK2 specific inhibitors, previous pharmacological studies of ROCK function have relied on pan-ROCK1/2 inhibition. Here, we use the ROCK2 specific inhibitor, GV101, in the context of breast cancer (BC) models to uncouple the effects of ROCK2 pharmacological inhibition on both epithelial and stromal fibroblast cell populations. We demonstrate that ROCK2 inhibition with GV101 reduces fibroblast-mediated remodelling of pre-existing and de novo synthesised ECM resulting in a reduction in biomechanical stiffness, while also limiting epithelial cell growth in 2D and 3D settings. We also demonstrate that ROCK2 inhibition exposes epithelial cell vulnerability to fluid flow-induced shear stress. Furthermore, using 3D co-cultures we reveal that GV101 reduces single-cell migratory capacity, while the disruption of stromal ECM architecture is sufficient to impede collective cell migration and invasion. Finally, we assessed ROCK2 expression in human BC patient tumours revealing that ROCK2 expression is up-regulated during BC progression and that high ROCK2 expression correlates with poor patient outcome in the triple-negative subtype of BC. Together, these results demonstrate that ROCK2 specific inhibition disrupts key functions in both epithelial cell and stromal fibroblast compartments warranting further assessment of ROCK2 in highly proliferative, fibrotic or metastatic diseases, such as BC. HighlightsO_LIFibroblast-mediated remodelling of pre-existing and de novo synthesised extracellular matrix is reduced upon ROCK2 specific targeting. C_LIO_LIROCK2 inhibition impairs epithelial cancer cell growth and colony formation in 2D and 3D contexts, while also limiting single-cell migration and cell survival upon fluid flow-induced shear stress. C_LIO_LIROCK2 targeting during fibroblast-mediated ECM remodelling is sufficient to disrupt collective cell migration as well as 3D invasion into organotypic fibroblast-contracted collagen matrices. C_LIO_LIROCK2 expression is up-regulated during breast cancer progression and high expression correlates with poor patient outcome in triple-negative breast cancer. C_LI

cell biology↗

bioRxiv2024

Dermatopontin-expressing fibroblasts mediate an essential skin macrophage niche

Fibroblasts are present in all tissues and are crucial for maintaining tissue homeostasis. We previously identified fibroblasts marked by Dermatopontin (Dpt) but their role in supporting macrophage homeostasis remains unclear. Here, we generated novel mesenchymal lineage-restricted genetic tools to target Dpt expressing fibroblasts and elucidate their role in supporting skin macrophages. Transcriptional profiling, flow cytometry, and in situ hybridization uncovered two broad populations of F4/80-expressing skin macrophages, denoted by high expression of CD206 and CD64 (CD206hiCD64+), or CD11c. Targeted depletion of Dpt+ fibroblasts resulted in a profound loss of both macrophage populations. Conditional deletion of colony-stimulating factor-1 (Csf1) in Dpt+ fibroblasts revealed that CD206hiCD64+, and not CD11c+, macrophages are acutely dependent on fibroblast-derived Csf1, consistent with their higher expression of the Csf1 receptor. Following Csf1 deletion in Dpt+ fibroblasts, loss of CD206hiCD64+ macrophages were observed across the dermis, dermal white adipose tissue (dWAT), and adventitia, accompanied by a modest upregulation of fibroblast-related and extracellular matrix (ECM) genes and structural changes to the skin. Alterations to the skin network upon loss of fibroblast-derived Csf1 and CD206hiCD64+ macrophages led to a significant delay in wound healing. We also demonstrate the CSF1-CSF1R signaling pathway is functionally relevant in human systemic sclerosis, or scleroderma, as elevated levels of CSF1 produced by fibroblasts and an increased abundance of macrophages both correlate with disease severity. Our findings demonstrate the role of Dpt+ fibroblasts in regulating a Csf1-dependent macrophage niche in skin and orchestrating responses in injury and disease.

immunology↗

bioRxiv2024

Profiling of epithelial functional states and fibroblast phenotypes in hormone therapy-naive localised prostate cancer

Localised prostate cancers (PCa) are heterogeneous and multifocal, with diverse outcomes. Current prognostic methods are epithelium-centric, overlooking the complex cellular landscape within the tumour microenvironment (TME), which remains incompletely characterised. We performed a comprehensive analysis of cancerous and adjacent-benign cores from 24 patients with hormone therapy-naive localised PCa using single-cell RNA-sequencing. By integrating copy number variation and transcriptional signatures, we classified epithelial cells across a malignant spectrum, revealing widespread molecular perturbation. We found an expansion of Club cell phenotypes, suggestive of Luminal dedifferentiation. We also performed a detailed annotation of stromal phenotypes, focusing on fibroblasts, and identified a novel peri-neural fibroblast population. Spatial transcriptomics elucidated the precise anatomical distribution of CAFs within the PCa TME. This study provides a valuable foundation for advancing our understanding of PCa pathobiology and developing a comprehensive cellular model of the disease. Statement of SignificanceOur study leverages single-cell RNA-sequencing and spatial transcriptomics to provide a comprehensive cellular annotation of hormone therapy-naive localised PCa. We reveal widespread molecular perturbations in epithelial cells and map distinct fibroblast populations to specific anatomical niches. Notably, we identify a novel peri-neural phenotype associated with nerves, which merits further functional characterisation and exploration as a potential therapeutic target.

cancer biology↗

bioRxiv2024

Fibroblastic FLT3L supports lymph node dendritic cells in the interfollicular niche

Dendritic cell (DC) homeostasis is maintained in secondary lymphoid organs (SLOs) by Fms-like tyrosine kinase 3 ligand (FLT3L). The specific niche providing this DC growth factor within human and mouse SLOs is unclear. Here, we show that Gremlin1 (Grem1)-expressing lymph node fibroblastic reticular cells (FRCs) support DC homeostasis via provision of FLT3L. Grem1 FRCs co-localize with DCs and express FLT3L in human and mouse lymph nodes. Using a new genetic model, we provide evidence that FLT3L produced by GREM1 FRCs maintains lymph node preDCs, cDCs, and plasmacytoid DCs (pDCs). Spatial transcriptomics and cytofluorometry reveal that Grem1 FRC-derived FLT3L supports not only proliferation, but also survival of lymph node cDCs within the interfollicular zone (IFZ). Functionally, loss of Grem1 FRC-derived FLT3L impairs cDC priming of antigen-specific T cell responses. These findings provide key mechanistic insights underlying stromal cell support of DC homeostasis and function.

immunology↗

bioRxiv2024

Systematic perturbation screens decode regulators of inflammatory macrophage states and identify a role for TNF mRNA m6A modification

Macrophages adopt dynamic cell states with distinct effector functions to maintain tissue homeostasis and respond to environmental challenges. During chronic inflammation, macrophage polarization is subverted towards sustained inflammatory states which contribute to disease, but there is limited understanding of the regulatory mechanisms underlying these disease-associated states. Here, we describe a systematic functional genomics approach that combines genome-wide phenotypic screening in primary murine macrophages with transcriptional and cytokine profiling of genetic perturbations in primary human monocyte-derived macrophages (hMDMs) to uncover regulatory circuits of inflammatory macrophage states. This process identifies regulators of five distinct inflammatory states associated with key features of macrophage function. Among these, the mRNA m6A writer components emerge as novel inhibitors of a TNF-driven cell state associated with multiple inflammatory pathologies. Loss of m6A writer components in hMDMs enhances TNF transcript stability, thereby elevating macrophage TNF production. A PheWAS on SNPs predicted to impact m6A installation on TNF revealed an association with cystic kidney disease, implicating an m6A-mediated regulatory mechanism in human disease. Thus, systematic phenotypic characterization of primary human macrophages describes the regulatory circuits underlying distinct inflammatory states, revealing post-transcriptional control of TNF mRNA stability as an immunosuppressive mechanism in innate immunity.

immunology↗

bioRxiv2022

Integration of single-cell RNA and protein data identifies novel clinically-relevant lymphocyte phenotypes in breast cancers

Immune cells are critical determinants of solid tumour aetiology, but the diverse phenotypes of intra-tumoural immune cells remain incompletely characterised. We applied integrated single cell RNA sequencing (scRNA-Seq) and highly multiplexed protein epitope analysis to a cohort of breast cancer samples to resolve cell states within the tumour microenvironment. We reveal novel protein markers for resting and activated tumour infiltrating lymphocytes, and show that high expression of CD103 primarily marks exhausted CD8 rather than tissue resident CD8 T-cells in human breast cancers. We identify two distinct states of activated CD4+ T follicular helper (Tfh) cells. A population resembling conventional Tfh (cTfh) cells were localised primarily to lymphoid aggregates by spatial transcriptomics. In contrast, cancer associated Tfh (caTfh) cells expressing markers of tissue residency and exhaustion co-localized with cancer foci and signalled to macrophages. Importantly, increased caTfh : cTfh ratio associated with improved disease outcome and response to checkpoint immunotherapy.

immunology↗

bioRxiv2021

DNA barcoding reveals ongoing immunoediting of clonal cancer populations during metastatic progression and in response to immunotherapy

Cancers evade the immune system in order to grow or metastasise through the process of cancer immunoediting. While immune checkpoint inhibitors have been effective for reactivating tumour immunity in some types of cancer, many other solid cancers, including breast cancer, remain largely non-responsive. Understanding the way non-responsive cancers evolve to evade immunity, what resistance pathways are activated and whether this occurs at the clonal level will improve immunotherapeutic design. We tracked cancer cell clones during the immunoediting process and determined clonal transcriptional profiles that allow immune evasion in murine mammary tumour growth in response to immunotherapy with anti-PD1 and anti-CTLA4. Clonal diversity was significantly restricted by immunotherapy treatment in both primary tumours and metastases. These findings demonstrate that immunoediting selects for pre-existing breast cancer cell populations and that immunoediting is not static, it is ongoing during metastasis and immunotherapy treatment. Isolation of immunotherapy resistant clones revealed unique and overlapping transcriptional signatures. The overlapping gene signature was associated with poor survival of basal-like breast cancer patients in two cohorts. At least one of these overlapping genes has an existing small molecule that can potentially be used to improve immunotherapy response.

cancer biology↗

bioRxiv2020

Spatial Deconvolution of HER2-positive Breast Tumors Reveals Novel Intercellular Relationships

In the past decades, transcriptomic studies have revolutionized cancer treatment and diagnosis. However, tumor sequencing strategies typically result in loss of spatial information, critical to understand cell interactions and their functional relevance. To address this, we investigate spatial gene expression in HER2-positive breast tumors using Spatial Transcriptomics technology. We show that expression-based clustering enables data-driven tumor annotation and assessment of intra-and interpatient heterogeneity; from which we discover shared gene signatures for immune and tumor processes. We integrate and spatially map tumor-associated types from single cell data to find: segregated epithelial cells, interactions between B and T-cells and myeloid cells, co-localization of macrophage and T-cell subsets. A model is constructed to infer presence of tertiary lymphoid structures, applicable across tissue types and technical platforms. Taken together, we combine different data modalities to define novel interactions between tumor-infiltrating cells in breast cancer and provide tools generalizing across tissues and diseases.

cancer biology↗

bioRxiv2020

Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis

BackgroundHigh throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity amongst complex human cancers. scRNA-Seq studies using fresh human surgical tissue is logistically difficult, precludes histopathological triage of samples and limits the ability to perform batch processing. This hinderance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers, and on high throughput scRNA-Seq platforms. ResultsWe show that the viable cryopreservation of human cancers provides high quality single-cell transcriptomes using the Chromium 10X platform. We sequenced a total of [~]120,000 cells from fresh and cryopreserved replicates across three breast cancers, two prostate cancers and a cutaneous melanoma. Importantly, tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We then show that cryopreservation had minimal impacts on results of downstream analyses such as biological pathway enrichment. Further, we demonstrate the advantage of cryopreserving whole-cells for immunophenotyping methods such as CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. ConclusionsOur study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.

cancer biology↗

bioRxiv2020

Single-cell analysis reveals diverse stromal subsets associated with immune evasion in triple-negative breast cancer

The tumour stroma regulates nearly all stages of carcinogenesis. Stromal heterogeneity in human triple-negative breast cancers (TNBCs) remains poorly understood, limiting the development of stromal-targeted therapies. Single cell RNA-sequencing of five TNBCs revealed two cancer-associated fibroblast (CAF) and two perivascular-like (PVL) subpopulations. CAFs clustered into two states, the first with features of myofibroblasts and the second characterised by high expression of growth factors and immunomodulatory molecules. PVL cells clustered into two states consistent with a differentiated and immature phenotype. We showed that these stromal states have distinct morphologies, spatial relationships and functional properties in regulating the extracellular matrix. Using cell-signalling predictions, we provide evidence that stromal-immune crosstalk acts via a diverse array of immunoregulatory molecules. Importantly, the investigation of gene signatures from inflammatory-CAFs and differentiated-PVL cells in independent TNBC patient cohorts revealed strong associations with cytotoxic T-cell dysfunction and exclusion, respectively. Such insights present promising candidates to further investigate for new therapeutic strategies in the treatment of TNBCs.

cancer biology↗

bioRxiv2020

ID4 represses myoepithelial differentiation of mammary stem cells through its interaction with HEB

Differentiation of stem cells embedded within the mammary epithelium is orchestrated by lineage-specifying transcription factors. Unlike the well-defined luminal hierarchy, dissection of the basal lineage has been hindered by a lack of specific markers. Inhibitor of Differentiation 4 (ID4) is a basally-restricted helix-loop-helix (HLH) transcription factor essential for mammary development. Here we show that ID4 is highly expressed in basal stem cells and decreases during myoepithelial differentiation. By integrating transcriptomic, proteomic, and ChIP-sequencing data, we reveal that ID4 is required to suppress myoepithelial gene expression and cell fate. We identify the bHLH protein HEB as a direct binding partner of ID4, and describe a previously-unknown role for this regulator in mammary development. HEB binds to E-boxes in regulatory elements of developmental genes, negatively regulated by ID4, involved in extracellular matrix synthesis and cytoskeletal contraction. Together our findings support a model whereby ID4 binds to HEB and blocks it from promoting myoepithelial specialisation. These new insights expand our current understanding into control of myoepithelial differentiation and mammary gland morphogenesis.

developmental biology↗

bioRxiv2018

A novel role for the HLH protein Inhibitor of Differentiation 4 (ID4) in the DNA damage response in basal-like breast cancer.

Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies. Inhibitor of Differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator required for mammary gland development. ID4 is overexpressed in a subset of BLBC patients, associating with a stem-like poor prognosis phenotype, and is necessary for the growth of cell line models of BLBC, through unknown mechanisms. Here, we have defined a molecular mechanism of action for ID4 in BLBC and the related disease highgrade serous ovarian cancer (HGSOV), by combining RIME proteomic analysis and ChIP-Seq mapping of genomic binding sites. Remarkably, these studies have revealed novel interactions with DNA damage response proteins, in particular, mediator of DNA damage checkpoint protein 1 (MDC1). Through MDC1, ID4 interacts with other DNA repair proteins ({gamma}H2AX and BRCA1) at fragile chromatin sites. ID4 does not affect transcription at these sites, instead binding to chromatin following DNA damage and regulating DNA damage signalling. Clinical analysis demonstrates that ID4 is amplified and overexpressed at a higher frequency in BRCA1-mutant BLBC compared with sporadic BLBC, providing genetic evidence for an interaction between ID4 and DNA damage repair pathways. These data link the interactions of ID4 with MDC1 to DNA damage repair in the aetiology of BLBC and HGSOV.

cancer biology↗

bioRxiv2017

Targeting stromal remodeling and cancer stem cell plasticity to overcome chemoresistance in triple negative breast cancer

The cellular and molecular basis of stromal cell recruitment, activation and crosstalk in carcinomas is poorly understood, limiting the development of targeted anti-stromal therapies. In mouse models of triple negative breast cancer (TNBC), Hh ligand produced by neoplastic cells reprogrammed cancer-associated fibroblast (CAF) gene expression, driving tumor growth and metastasis. Hh-activated CAFs upregulated expression of FGF5 and production of fibrillar collagen, leading to FGFR and FAK activation in adjacent neoplastic cells, which then acquired a stem-like, drug-resistant phenotype. Treatment with smoothened inhibitors (SMOi) reversed these phenotypes. Stromal treatment of TNBC patient-derived xenograft (PDX) models with SMOi downregulated the expression of cancer stem cell markers and sensitized tumors to docetaxel, leading to markedly improved survival and reduced metastatic burden. In the phase I clinical trial EDALINE, 3 of 12 patients with metastatic TNBC derived clinical benefit from combination therapy with the SMOi Sonidegib and docetaxel chemotherapy, with one patient experiencing a complete response. Markers of pathway activity correlated with response. These studies identify Hh signaling to CAFs as a novel mediator of cancer stem cell plasticity and an exciting new therapeutic target in TNBC.\n\nSIGNIFICANCECompared to other breast cancer subtypes, TNBCs are associated with significantly worse patient outcomes. Standard of care systemic treatment for patients with non-BRCA1/2 positive TNBC is cytotoxic chemotherapy. However, the failure of 70% of treated TNBCs to attain complete pathological response reflects the relative chemoresistance of these tumors. New therapeutic strategies are needed to improve patient survival and quality of life. Here, we provide new insights into the dynamic interactions between heterotypic cells within a tumor. Specifically, we establish the mechanisms by which CAFs define cancer cell phenotype and demonstrate that the bidirectional CAF-cancer cell crosstalk can be successfully targeted in mice and humans using anti-stromal therapy.

cancer biology↗