Biology subjects

Cook, D. P.

Publications and source records attributed to Cook, D. P..

15 recordsLinked to original sources

bioRxiv2026

Tumour architecture shapes polarized epithelial states that predict survival in high-grade serous ovarian cancer

Epithelial heterogeneity defines high-grade serous ovarian carcinoma (HGSC), yet principles that generate this diversity within and across tumours remain unclear. Integrating single-cell RNA sequencing (scRNA-seq) data from 13 studies (1,980,703 cells, 371 samples), we resolve a dominant axis of secretory cell polarization spanning proliferative, progenitor-like SecA cells and quiescent SecB cells expressing a mucosal injury response program. Targeted spatial transcriptomics across 8 whole HGSC tissues and a 97-patient tissue microarray shows this axis is spatially deterministic: tumour architecture shapes a hypoxic gradient along which SecB cells localize to avascular, luminal regions, where HIF/NF-{kappa}B-driven survival and glycolysis displace the mitogenic signalling of SecA. Within this niche, SecB cells rewire adhesion, ECM-remodelling, and immune-regulatory programs. Transcriptionally reprogrammed macrophages are enriched in this niche, while lymphocytes are excluded or dysfunctional. These cells assemble a coordinated multicellular niche poised for dissemination. SecB cells are enriched both in ascites and after chemotherapy, and are progressively lost in patient-derived organoids, only partially restored by IFN{gamma}, suggesting SecB is environmentally programmed rather than clonally fixed. SecB proportion independently predicts worse overall (HR = 1.31, p = 0.023) and progression-free survival (HR = 1.28, p = 0.011). Tumour architecture is thus a primary axis of malignant identity in HGSC, coupling microenvironment, cell state, and immune niche to clinical outcomes.

cancer biology↗

bioRxiv2024

LINE1 promotes nuclear compartmentalization to repress the 8-cell state in embryonic stem cells

The family of LINE1 transposable elements underwent a massive expansion in mammalian genomes. While traditionally viewed as a mutagenic selfish element, recent studies point to roles for LINE1 in early mouse development, T cell quiescence and neurogenesis. Here we show that human LINE1 RNA is essential for self-renewal and identity of human embryonic stem cells (hESCs). Silencing of LINE1 using either antisense oligonucleotides or CRISPR interference in naive hESCs leads to a strong induction of 8C-like cells (8CLCs). We found that genes derepressed upon LINE1 KD are not uniformly distributed across the genome, with an enrichment for chromosome 19, which includes key markers of the 8C state such as TPRX1. Silencing of TPRX1, but not other putative 8C regulators p53 or H3.XY, suppresses the induction of the 8C program in LINE1 KD hESCs. We found that LINE1 RNA is preferentially localized to the lamina and periphery of the nucleolus in hESCs. Sequencing of Lamina-Associated Domains (LADs) and Nucleolus-Associated Domains (NADs) reveals a preferential association of chromosome 19 with NADs in hESCs. However, 8CLCs have a distinct nucleolar morphology and a lower association of chromosome 19 and TPRX1 loci with the nucleolus relative to naive and primed hESCs, suggesting a role for nucleolar dynamics in the 8CLC-hESC transition. In agreement, LINE1 KD leads to disruption of nucleolar architecture with signs of nucleolar stress. Independent perturbations of the nucleolus induce the 8C program in hESCs. Genes induced by LINE1 KD are enriched for targets of Polycomb Repressive Complex (PRC2), and inhibition of PRC2 leads to a strong induction of 8C genes. Our results indicate that LINE1 coordinates nuclear compartmentalization and chromatin-mediated gene repression to prevent developmental reversion of hESCs. HighlightsO_LIKnockdown of LINE1 induces TPRX1-dependent emergence of 8C-like cells in hESCs. C_LIO_LIGenes de-repressed upon LINE1 KD are enriched for Chr 19 and PRC2 targets. C_LIO_LI8CLCs display dissociation of Chromosome 19 from the nucleolus. C_LIO_LIDisruption of the nucleolus or inhibition of PRC2 strongly induce the 8C program. C_LI

developmental biology↗

bioRxiv2024

Preventing surgery induced immune suppression and metastases by inhibiting PI3K-gamma signalling in Myeloid-Derived Suppressor Cells.

Myeloid derived suppressor cells (MDSCs) have a dominating presence in the postoperative period and mediate the suppression of Natural Killer (NK) cells and promotion of cancer metastases after surgery. However, their functional characteristics and effect on cellular immunity after surgery have not been comprehensively investigated. Here, we characterize the expansion of surgery-induced (sx) MDSCs via multi-colour flow cytometry, single-cell RNA sequencing, and functional ex vivo NK cell suppression assays. We then screened a small molecule library using our sx-MDSC:NK cell suppression assay to identify compounds that could inhibit sx-MDSCs. These studies provide evidence that PI3K-{gamma} signalling is upregulated in sx-MDSCs and blockade with PI3K-{gamma} specific inhibitors attenuates NK cell suppression in humans and mice and reduces postoperative metastases in murine models. Upregulated PI3K-{gamma} in sx-MDSCs is a potential pathway amenable to therapeutic targeting in the postoperative period. One Sentence SummaryThe suppressive mechanisms of surgery-induced myeloid derived suppressor cells use PI3K signalling and are amenable to PI3K-gamma specific inhibitors.

immunology↗

bioRxiv2023

A Comparative Analysis of Imaging-Based Spatial Transcriptomics Platforms

Spatial transcriptomics is a rapidly evolving field, overwhelmed by a multitude of technologies. This study aims to offer a comparative analysis of datasets generated from leading in situ imaging platforms. We have generated spatial transcriptomics data from serial sections of prostate adenocarcinoma using the 10x Genomics Xenium and NanoString CosMx SMI platforms. Additionally, orthogonal single-nucleus RNA sequencing (snRNA-seq) was performed on the same FFPE tissue to establish a reference for the tumors transcriptional profiles. We assessed various technical aspects, such as reproducibility, sensitivity, dynamic range, cell segmentation, cell type annotation, and congruence with single-cell profiling. The practicality of assessing cellular organization and biomarker localization was evaluated. Although fewer genes are measured (CosMx: 960, Xenium: 377, with an overlap of 125), Xenium consistently demonstrates higher sensitivity, a broader dynamic range, and better alignment with single-cell reference profiles. Conversely, CosMxs out-of-the-box segmentation outperformed Xeniums, resulting in noticeable transcript misassignment in Xenium within certain tissue areas. However, the impact of this on the cells transcriptional profile was minimal. Together, this comprehensive comparison of two leading commercial platforms for spatial transcriptomics provides essential metrics for assessing their performance, offering invaluable insights for future research and technological advancements in this dynamic field.

genomics↗

bioRxiv2023

Emergence of disease specific endothelial and stromal cell populations involved in arterial remodeling during development of pulmonary arterial hypertension

AbstractPulmonary arterial hypertension (PAH) is a severe and lethal pulmonary vascular disease characterized by arteriolar pruning and occlusive vascular remodeling leading to increased pulmonary vascular resistance and eventually right heart failure. While endothelial cell (EC) injury and apoptosis are known triggers for this disease, the mechanisms by which they lead to complex arterial remodeling remain obscure. We employed multiplexed single-cell RNA sequencing (scRNA-seq) at multiple timepoints during the onset and progression of disease in a model of severe PAH to identify mechanisms involved in the development of occlusive arterial lesions. There was significant loss of arterial volume as early as 1-week by microCT, preceding any evidence of occlusive arteriopathy, consistent with early arteriolar dropout. Maximal arterial pruning was seen by 5 to 8 weeks, with signs of progressive occlusive remodeling. Analysis of the scRNA-seq data resolved 44 lung cell populations, with widespread early transcriptomic changes at 1 week affecting endothelial, stromal and immune cell populations. Notably, this included emergence of a relatively dedifferentiated (dD) EC population that was enriched for Cd74 expression compared to general capillary (gCap) ECs which were primed to undergo endothelial-mesenchymal transition, as evidenced by RNA velocity analysis. However, at late timepoints (5 and 8 weeks), activated arterial ECs (aAECs) were the only cell population exhibiting persistent differential gene expression. This was characterized by a growth regulated state, including high expression of Tm4sf1, a gene implicated in cancer cell growth, which was also expressed by a smooth muscle (SM)-like pericyte cluster. Both these populations were localized to regions of arterial remodeling in the rat model and PAH patients, with aAECs contributing to intimal occlusive lesions and SM-like pericytes forming bands of medial muscularization. Together these findings implicate disease-specific vascular cells in PAH progression and suggest that TM4SF1 may be a novel therapeutic target for arterial remodeling.

molecular biology↗

bioRxiv2023

Comparative analysis of syngeneic mouse models of high-grade serous ovarian cancer

Ovarian cancers often exhibit high rates of recurrence and poor treatment response. Preclinical models that recapitulate the heterogeneity of human disease are critical to develop new therapeutic approaches. While patient-derived models are a powerful tool for testing various therapeutics, their dependence on immune-compromised mice is severely limiting. Syngeneic mouse models, however, allow for the generation of tumours comprising the full repertoire of non-malignant cell types. Here we have performed a comparative analysis of diverse models of high-grade serous ovarian cancer based on transcriptomic profiling of 22 cell line models, and intrabursal and intraperitoneal tumours from 12 models. Among cell lines, we identify distinct features in signalling activity, such as elevated inflammatory signalling in STOSE and OVE16 models, and MAPK/ERK signalling in ID8 and OVE4 models; metabolic features, such as predicted reduction in glycolysis associated with subsets of engineered ID8 subclones; and relevant functional properties, including differences in EMT activation, PD-L1 and MHC class I expression, and predicted chemosensitivity. Finally, we evaluate variability in properties of the tumour microenvironment among models. We anticipate that this work will serve as a valuable resource, providing new insight to help in the selection of models for specific experimental objectives.

cancer biology↗

bioRxiv2022

Alterations in neural stem cell quiescence and activation in the 3xTG-AD model of Alzheimers Disease

Alzheimers Disease (AD) is the most common form of dementia with progressive cognitive deficits and mood disorders (Knopman et al., 2021). Recent studies have associated AD pathology with the impairment of adult neurogenesis, as indicated by impaired neural stem cell (NSCs) homeostasis (Bond et al., 2015). Recent work has further associated AD progression with a decline in the number and maturation of adult-born neurons in the SGZ, distinct from typical age-related decline (Moreno-Jimenez et al., 2019). In 3xTG-AD mice, a well-established mouse model of AD, our and other groups have demonstrated impairments to NSC pool and neural progenitor proliferation, as well as adult-born neurons, before the onset of A{beta} plaques and NFTs (Hamilton et al., 2010, 2015; Rodriguez et al., 2008, 2009). However, the regulatory mechanisms underlying the functional impairment of adult NSCs remain to be resolved. Here, we employ single-cell RNA-Seq to establish population-specific defects in the 3xTG-AD mouse model during adult SGZ neurogenesis. Relative to control mice, we observe a dramatic AD-induced decrease in the primed and activated NSC population, which results in a progressive loss of cells committed to neurogenesis. Transcriptome measurements suggest that 3xTG-AD NSCs and their progeny represent enhanced ribosomal and mitochondrial biogenesis, and disturbed Notch signaling pathway. RNA velocity analysis reveals reduced NSC activation as evidenced by a large fraction of Ascl1-postive cells, instead of entering cell cycle, returning to the primed and quiescent state. This is further supported by reduced numbers of Lpar1-expressing cells, a marker of neural progenitor cells, in the SGZ. Our work explores, at a stage-specific resolution, changes in the regulatory networks guiding adult neurogenesis, and identifies niche disturbances in the regulation of NSC quiescence and activation. These NSC deficits underlying impaired neurogenesis identified in AD mice, may be key contributors underlying the compromised hippocampal function in AD.

neuroscience↗

bioRxiv2021

Newly Emergent Apelin Expressing Endothelial Stem-like Cells Orchestrate Lung Microvascular Repair

QuestionWe sought to define the mechanism underlying lung microvascular regeneration in a severe acute lung injury (ALI) model induced by selective lung endothelial cell ablation. MethodsChanges in lung cell populations and gene expression profiles were determined in transgenic mice expressing human diphtheria toxin (DT) receptor targeted to ECs using single-cell RNA sequencing at baseline (day 0) and days 3, 5 and 7 after lung EC ablation. ResultsEight distinct endothelial clusters were resolved, including alveolar aerocytes (aCap) ECs expressing apelin at baseline, and general capillary (gCap) ECs expressing the apelin receptor. Intratracheal instillation of DT resulted in ablation of >70% of lung ECs, producing severe ALI with near complete resolution by 7 days. At 3 days post injury, a novel gCap population emerged characterized by de novo expression of apelin, together with the stem cell marker, protein C receptor. These stem-like cells transitioned to proliferative ECs, expressing apelin receptor together with the pro-proliferative transcription factor, FoxM1. This progenitor-like cell population was responsible for the rapid replenishment of all depleted EC populations by 7 days post injury, including aerocytes which play a critical role in re-establishment of the air-blood barrier. Treatment with an apelin receptor antagonist prevented recovery and resulted in excessive mortality, consistent with a central role for apelin signaling in EC regeneration and microvascular repair. ConclusionThe lung has a remarkable capacity for microvasculature EC regeneration which is orchestrated by signaling between newly emergent apelin-expressing gCap endothelial stem-like cells and highly proliferative, apelin receptor positive endothelial progenitors. Take-Home messageUsing sublethal lung endothelial cell (EC) ablation, we show for the first that EC regeneration and resolution of acute lung injury is orchestrated by novel apelin-expressing, gCap endothelial stem-like cells by a mechanism requiring apelin signaling. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY A schematic representation of EC populations contributing to microvascular repair. At baseline (Day 0), there are two main alveolar groups of capillary ECs: larger apelin positive aCap ECs, termed aerocytes, that play a key structural role in forming the air-blood barrier; and smaller apelin receptor (Aplnr) expressing gCap ECs, which are found in the thicker regions at the corners of the alveoli. After DT-induced EC ablation, there is a marked depletion of both EC populations and the appearance of novel transitional and transient populations. At Day 3, there is the appearance of stem-like gCap ECs that paradoxically express apelin, but not its receptor, and are characterized by various stem and progenitor cell markers but show no evidence of proliferation. By Day 5, these transition to ECs expressing Aplnr which have a strong proliferative phenotype, as evidenced by FoxM1 and Ki67 expression, and then rapidly replenish depleted EC pools, including aCap ECs, by Day 7. This transition is orchestrated by the interaction of apelin with its receptor as a critical mechanism in lung microvascular regeneration after EC injury. AT1 = alveolar type -1 epithelial cell; AT2 = alveolar type-2 epithelial cell; APLNR = apelin receptor; ANGPT2 = angiopoietin 2; EPCR = Endothelial protein C receptor.

cell biology↗

bioRxiv2021

Single-cell RNA sequencing-based characterization of resident lung mesenchymal stromal cells in bronchopulmonary dysplasia

Late lung development is a period of alveolar and microvascular formation, which is pivotal in ensuring sufficient and effective gas exchange. Defects in late lung development manifest in premature infants as a chronic lung disease named bronchopulmonary dysplasia (BPD). Numerous studies demonstrated the therapeutic properties of exogenous bone marrow and umbilical cord-derived mesenchymal stromal cells (MSCs) in experimental BPD. However, very little is known regarding the regenerative capacity of resident lung MSCs (L-MSCs) during normal development and in BPD. In this study we aimed to characterize the L-MSC population in homeostasis and upon injury. We used single-cell RNA sequencing (scRNA-seq) to profile in situ Ly6a+ L-MSCs in the lungs of normal and O2-exposed neonatal mice (a well-established model to mimic BPD) at three developmental timepoints (postnatal days 3, 7 and 14). Hyperoxia exposure increased the number, and altered the expression profile of L-MSCs, particularly by increasing the expression of multiple pro-inflammatory, pro-fibrotic, and anti-angiogenic genes. In order to identify potential changes induced in the L-MSCs transcriptome by storage and culture, we profiled 15,000 Ly6a+ L-MSCs after in vitro culture. We observed great differences in expression profiles of in situ and cultured L-MSCs, particularly those derived from healthy lungs. Additionally, we have identified the location of L-MSCs in the developing lung and propose Serpinf1 as a novel, culture-stable marker of L-MSCs. Finally, cell communication analysis suggests inflammatory signals from immune and endothelial cells as main drivers of hyperoxia-induced changes in L-MSCs transcriptome.

cell biology↗

bioRxiv2021

Transcriptional census of epithelial-mesenchymal plasticity in cancer

Epithelial-mesenchymal plasticity (EMP) contributes to tumour progression, promoting therapy resistance and immune cell evasion. Definitive molecular features of this plasticity have largely remained elusive due to the limited scale of most studies. Leveraging scRNA-seq data from 160 tumours spanning 8 different cancer types, we identify expression patterns associated with intratumoural EMP. Integrative analysis of these programs confirmed a high degree of diversity among tumours. These diverse programs are associated with combinations of various common regulatory mechanisms initiated from cues within the tumour microenvironment. We highlight that inferring regulatory features can inform effective therapeutics to restrict EMP.

cancer biology↗

bioRxiv2020

Rac guanine nucleotide exchange factors promoting Lgl1 phosphorylation

The protein Lgl1 has key roles in the regulation of cell polarity. We have shown that Lgl1 is inactivated by hyperphosphorylation in glioblastoma as a consequence of PTEN loss and aberrant activation of the PI 3-kinase pathway; this contributes to glioblastoma pathogenesis both by promoting invasion and repressing glioblastoma cell differentiation. Lgl1 is phosphorylated by atypical protein kinase C in a complex with Par6 and activated Rac. Here we have investigated the role of specific Rac guanine nucleotide exchange factors in Lgl1 hyperphosphorylation in glioblastoma. We used CRISPR/Cas9 to knockout PREX1, a PI 3-kinase pathway-responsive Rac guanine nucleotide exchange factor, in patient-derived glioblastoma cells. Knockout cells had reduced Lgl1 phosphorylation which could be reversed by re-expressing PREX1. PREX1 knockout cells showed reduced motility and an altered phenotype suggestive of partial neuronal differentiation; consistent with this, RNA-seq analyses identified sets of PREX1-regulated genes associated with changes in cell motility and neuronal differentiation. PREX1 knockout in glioblastoma cells from a second patient did not affect Lgl1 phosphorylation. These cells overexpressed a short isoform of the Rac guanine nucleotide exchange factor TIAM1; knockdown of TIAM1 in PREX1-knockout cells from this patient reduced Lgl1 phosphorylation. These data show that PREX1 links aberrant PI 3-kinase signaling to Lgl1 phosphorylation in glioblastoma, but that TIAM1 can also promote Lgl phosphorylation in a subset of patients. While this shows redundant mechanisms for Lgl1 phosphorylation, PREX1 appears to have a non-redundant role in glioblastoma cell motility, as this was impaired in PREX1 knockout cells from both patients.

biochemistry↗

bioRxiv2020

Transcriptional heterogeneity of stemness phenotypes in the ovarian epithelium

The ovarian surface epithelium (OSE) is a monolayer of epithelial cells covering the surface of the ovary. During ovulation, the OSE is ruptured to allow release of the oocyte. This wound is quickly repaired, but mechanisms of this repair are poorly understood. The contribution of tissue-resident stem cells in the homeostasis of several epithelial tissues is widely accepted, such as the intestinal epithelium, airway epithelium, and skin, but their involvement in OSE maintenance is unclear. While putative stem cell populations in the OSE have been described, how they are regulated is poorly defined. We show that traits associated with stem cells (stemness) can be increased in OSE following exposure to the cytokine TGFB1, overexpression of the transcription factor Snai1, or deletion of Brca1. By assessing the gene expression profiles of these populations, we show that stemness is often linked to mesenchymal-associated gene expression and higher activation of ERK signalling, but it is not consistently dependent on their activation. Expression profiles of these populations are extremely context specific, suggesting that stemness may not correspond to a single, distinct population, but rather is a heterogenous state that can possibly emerge from diverse environmental cues. Together, these findings support that the OSE may not require distinct stem cell populations for long-term maintenance, and may achieve this through transient dedifferentiation into a stem-like state.

cell biology↗

bioRxiv2020

Cre-mediated deletion of SMARCA5 disrupts pluripotency in mouse embryonic stem cells

In embryonic stem cells (ESCs), the SWI/SNF, CHD, and INO80 families of ATP-dependent chromatin remodellers have been implicated in maintaining pluripotency-associated gene expression. At the time of this study, the importance of ISWI family remodellers had yet to be defined, and we had sought to assess their involvement. During this time, Barisic et al. (Nature, 2019) elegantly demonstrated that the ISWI homologue SNF2H (Smarca5) is important for nucleosomal periodicity, the binding of select transcription factors, and proper differentiation of mouse ESCs. While we do not dispute their findings to any extent, our experiments have led to slightly different conclusions, and we have chosen to use this platform to share our results. Here, we explore the importance of SNF2H by deriving a conditional knockout mouse ESC line and observing the consequences of SNF2H depletion on the pluripotent state. Cre-mediated deletion of Snf2h disrupts hallmark characteristics of pluripotency, resulting in distinct morphological changes; reduced expression of the master transcription factors Oct4, Sox2, and Nanog; and reduced alkaline phosphatase activity. To understand the mechanisms of SNF2H-mediated regulation, we mapped SNF2H-bound nucleosomes genome-wide. SNF2H is broadly distributed across the genome but is preferentially enriched at active regulatory regions and transcription factor binding sites.

cell biology↗

bioRxiv2019

Multiplexed single-cell transcriptomic analysis of normal and impaired lung development in the mouse

During late lung development alveolar and microvascular development is finalized to enable sufficient gas exchange. Impaired late lung development manifests as bronchopulmonary dysplasia (BPD) in preterm infants. Single-cell RNA sequencing (scRNA-seq) allows for assessment of complex cellular dynamics during biological processes, such as development. Here, we use MULTI-seq to generate scRNA-seq profiles of over 66,000 cells from 36 mice during normal or impaired lung development secondary to hyperoxia. We observed dynamic populations of cells, including several rare cell types and putative progenitors. Hyperoxia exposure, which mimics the BPD phenotype, alters the composition of all cellular compartments, particularly alveolar epithelium, capillary endothelium and macrophage populations. We identified several BPD-associated signatures, including Pdgfra in fibroblasts, Activin A in capillary endothelial cells, and Csf1-Csf1r and Ccl2-Ccr2 signaling in macrophages and neutrophils. Our data provides a novel single-cell view of cellular changes associated with late lung development in health and in disease.

cell biology↗

bioRxiv2019

Comparing transcriptional dynamics of the epithelial-mesenchymal transition

Epithelial-mesenchymal (E/M) heterogeneity is ubiquitous within all epithelial tissues and the reversible transition between these two states provides cells with plasticity that contributes to organogenesis in the developing embryo, tissue homeostasis in adults, and tumour progression1. While the epithelial-mesenchymal transition (EMT) has been extensively studied, no common, EMT-defining gene expression program has been identified2. Here, we leverage highly multiplexed single-cell RNA sequencing (scRNA-seq) to compare the transcriptional program associated with the EMT across a variety of contexts, assessing 103,999 cells from 960 samples, comprising 12 EMT time course experiments and 16 independent kinase inhibitor screens. We demonstrate that the EMT is not simply a linear transition between E/M states, and transcriptional dynamics are widely variable across contexts, regardless of the cell type and cytokine used to induce the transition. While many canonical EMT genes were poor markers of the transition in our models, we identified 86 conserved mesenchymal-associated genes also coexpressed in a variety of mouse and human epithelial and carcinoma tissues. Despite the heterogeneous transcriptional responses, we identified a core set of largely conserved transcription factors coordinating these dynamics, including RELB and SOX4. Finally, we found that the EMT is associated with a broad increase in expression of secreted factors. Kinase inhibitor screens revealed multiple paracrine dependencies of the EMT, including a novel association between TGFB1 and the TNF-associated kinase RIPK1. Together, these results comprehensively highlight the complexity and diversity of the EMT, but also reveal dynamics conserved across contexts. This work will provide the foundation for understanding the nature of E/M heterogeneity and its functional consequences, which could elucidate various physiological processes and be leveraged for cancer treatments.

cancer biology↗