Biology subjects

Dunne, P. D.

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

At least 19 recordsLinked to original sources

bioRxiv2026

Stromal Prostaglandin is a Dominant Spatial Regulator of Cell-fate Plasticity in Colorectal Cancer

Colorectal cancer (CRC) tumours with high stromal content have a worse outcome, but the mechanisms governing this are unclear. Using high-throughput single-cell perturbation analysis of CRC patient-derived organoids (PDOs) and cancer-associated fibroblasts (CAFs) we find that epithelial cells with high stromal-communication potential are marked by the transcriptional co-repressor DACH1. To define the causal regulators of stromal-epithelial signalling, we developed a novel CRISPR screening platform to perturb the CAF secretome and measure epithelial stem cell responses at single-cell resolution. Intercellular CRISPR screening and full factorial ligand analysis revealed that stromal Prostaglandin E2 (PGE2) is a dominant regulator of CRC cell-fate plasticity. Stromal PGE2 converts DACH1+ epithelia from a chemosensitive proliferative colonic stem cell (proCSC) fate into a chemorefractory and prometastatic revival colonic stem cell (revCSC) fate. PGE2-driven epithelial transdetermination is rapid and reversible, providing an acute mechanism for stromal-driven plasticity in CRC tumours. Genetic and pharmacological inhibition of stromal COX2 inhibits epithelial plasticity, trapping CRC epithelia in an anti-metastatic and chemosensitive proCSC fate. PTGS2+ CAFs support a spatially resolved revCSC to proCSC plasticity gradient in human CRC tumours marked by increasing DACH1 expression. These results reveal that stromal prostaglandin is a dominant spatial regulator of poor-prognosis cell-fates and may explain the benefit of anti-COX therapies in both preventing and treating CRC.

cancer biology↗

bioRxiv2026

Phenotype-dependent subtyping exposes high MYC activity as a targetable dependency in LuAd

C-MYC (MYC) occupies a critical nexus of oncogenic signalling and deregulated expression of MYC is widespread across most human cancer types, suggesting that MYC should be an attractive target for therapeutic intervention. Although 30-40% of human Non-Small Cell lung cancers show low level amplification of c-MYC and genetic evidence has shown that c-Myc is a key downstream effector of KRas-driven lung tumourigenesis in mouse models, the functional contribution of MYC to human lung cancer remains unclear. We applied a phenotype-based classifier to the TCGA Lung Adenocarcinoma (LuAd) cohort and found that high MYC transcriptional activity identifies a subset of LuAd with significantly reduced survival. Application of the same methodology to a panel of genetically engineered mouse models identified multiple genotypes that give rise to the high MYC activity phenotype, disease positioning such models as reflective of a distinct subset of human LuAd. We show that high MYC activity predicts sensitivity to a small molecule dual-inhibitor of the MYC co-factors, EZH2 and G9A, HKMTi-1-005, and that treatment with HKMTi-1-005 strongly reduced MYC protein expression, induced B cell-mediated immune surveillance and suppressed growth of autochthonous KRasG12D-driven lung tumours. Statement of significanceThis work establishes the principle of indirectly targeting MYC in LuAd, via inhibition of associated enzymatic cofactors, EZH2 and G9A, and identifies a large subset of aggressive human LuAd with a high MYC activity signature that may benefit from this approach.

cancer biology↗

bioRxiv2025

Stromal cells modulate innate immune cell phenotype and function in colorectal cancer via the Sialic acid/Siglec axis

BackgroundThe immunosuppressive tumour microenvironment (TME) reduces immune response effectiveness in stromal-rich tumours, including CMS4 colorectal cancer (CRC). Mesenchymal stromal cells (MSCs), precursors to cancer-associated fibroblasts (CAFs), promote cancer progression by suppressing anti-tumour immune responses. Hypersialylation of glycans on tumours engages Siglec receptors on immune cells, driving immune dysfunction, but its role in stromal-mediated immunosuppression remains unclear. MethodsSialic acids and Siglec ligands were measured on CRC tissue, primary human CAFs, and tumour-conditioned-mesenchymal stromal cells (MSCTCS) and CAF using immunohistochemistry and flow cytometry. The effect of stromal cell sialylation on macrophages and NK cells was assessed in ex vivo primary stromal and immune cell co-cultures and expression of Siglec-10 and immune cell phenotype markers and function were measured by flow cytometry. Using an immunocompetent Balb/c CT26 mouse model, we induced tumours with/without conditioned stromal cells, with/without pre-treatment of stromal cells with sialyltransferase inhibitor (3FAX) or sialidase (E610). We assessed the effect of stromal cell sialylation on macrophages and NK cells in the tumour and secondary lymphoid tissues by flow cytometry. ResultsStromal cells, including CAFs, in CRC tumours are highly sialylated compared to epithelial cancer cells and are associated with high expression of ST6GalNAC6. Genetic knockdown of ST6GalNAC6 reduced the expression of stromal cell Siglec-10 ligands in MSCs. CAFs and MSCTCS induced Siglec-10 on macrophages and NK cells and impaired NK cell cytotoxicity. Sialidase treatment reduced Siglec-10 expression, restoring NK cell function. In vivo, desialylation of stromal cells increased macrophage activation (CD11b+CD80+) and reduced immunosuppressive marker expression (CD206, PD-L1, Siglec-G) in lymphoid tissues, indicating sustained systemic anti-tumour immunity. Intratumoural NK cells exhibited high Siglec-G expression and impaired cytotoxicity, and granzyme B expression significantly increased with sialidase treatment of stromal cells. In an inflammatory tumour model, inflammatory tumour-conditioned (iTCS) MSCs promoted metastasis and Siglec-G induction on NK cells and macrophages, both reversed by sialyltransferase inhibition, underscoring the effects of stromal modulation of innate immune cell function in inflammatory tumours. ConclusionStromal cell sialylation modulates innate immune suppression in CRC via the sialic acid/Siglec axis. Targeting stromal sialylation restores NK cytotoxicity and macrophage activation, offering a novel therapeutic strategy for immunosuppressive stromal-rich tumours. What is already known on this topicO_LIThe tumour microenvironment of consensus molecular subtype 4 (CMS4) colorectal cancer (CRC) is associated with high stromal burden, poor immune infiltration, poor response to anti-cancer therapies and thus poor patient prognosis. Immune checkpoint inhibitors (ICIs) have limited impact on stromal-rich CRC tumours, therefore highlighting the need to discover and target novel mechanisms of tumour immune evasion. C_LIO_LIEmerging studies have highlighted that stromal cells in CRC and pancreatic ductal adenocarcinoma (PDAC) are highly sialylated, expressing even higher levels of sialic acid on their cell surface than epithelial cancer cells. Targeting stromal cell sialylation has unveiled promising data in restoring the anti-tumour activity of T cells and macrophages. There is a need to explore the effects of targeting stromal cell sialylation on other immune cells of the TME and to evaluate the Siglec/sialic acid axis of stromal and immune cells in resistant CRC tumours. C_LI What this study addsO_LIWe reveal ST6GalNAC6 as a sialyltransferase enzyme that regulates the production of Siglec-10 ligands in CRC stromal cells. Overexpression of ST6GalNAC6 and Siglec-10 correlated with poor survival in CRC and mesenchymal CRC tumours. C_LIO_LIWe show for the first time an induction of Siglec-10 expression on macrophages and NK cells in stromal-immune co-culture experimental models with hypersialylated MSCs and CAFs in vitro and ex vivo. Targeting stromal cell sialylation increased NK cell cytotoxicity of CRC cells, indicating a direct functional role for stromal cell sialylation in immunosuppression. C_LIO_LIAn immunogenic mouse model of CRC was used to evaluate the potential therapeutic efficacy of targeting stromal cell sialylation in overcoming stromal cell-mediated immunosuppression in CRC. Sialic acid-targeting of stroma slowed tumour growth and reduced inflammation-driven metastasis. This was associated with greater infiltration and activation of macrophages and NK cells with stromal cell sialic acid depletion, highlighting stromal cell sialylation as a mechanism of innate immune cell suppression in stromal-rich CRC. C_LI How this study might affect Research, Practice or PolicyO_LIOur research provides insight into a novel mechanism of stromal cell-mediated immunosuppression of innate immune cells in CRC and may open up new avenues of research for targeting stromal cells in stromal-rich TMEs such as pancreatic, breast and ovarian cancers. C_LIO_LIOur research identifies a stromal cell effect of enhancing Siglec expression on tumour infiltrating innate immune cells as a novel immune checkpoint, which may be useful in identifying potential novel immunotherapeutic combinations in future. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/642985v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@cbafc3org.highwire.dtl.DTLVardef@80474forg.highwire.dtl.DTLVardef@1c9d050org.highwire.dtl.DTLVardef@8b6551_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Viral mimicry redirects immunosuppressed colorectal tumour landscapes towards a proinflammatory and CMS1-like regenerative state

In colorectal cancer (CRC), tumours classifier as consensus molecular subtype 4 (CMS4) have the worst prognosis and derive negligible benefit from chemotherapy. We previously described how repressed interferon-related signalling is associated with increased relapse in CMS4 tumours. Although the viral mimetic poly(I:C) can reduce liver metastasis in vivo, the initial phenotypic changes that underpin its anti-metastatic response remain poorly described, particularly in the immunosuppressed CMS4 tumour microenvironment. Here we characterise lineage-specific anti-metastatic responses induced by poly(I:C), including acute macrophage polarisation and a novel CMS1-like regenerative stem cell state, which drive pro-inflammatory microenvironmental changes in CRC. These insights enabled the development of tractable biomarkers that identify an "immune-warm" patient subset most likely to respond to poly(I:C), enriched for mismatch-repair proficient (pMMR), anti-inflammatory macrophages and CMS4-like features. The viral mimetic poly(I:C) offers a tailored treatment option for CMS4 tumours, by reprogramming stem cell states and activation of an innate-adaptive anti-metastatic response.

cancer biology↗

bioRxiv2024

Mitochondrial metabolism is a key determinant of chemotherapy sensitivity in Colorectal Cancer

Therapy resistance is attributed to over 80% of cancer deaths per year emphasizing the urgent need to overcome this challenge for improved patient outcomes. Despite its widespread use in colorectal cancer (CRC) treatment, resistance to 5-fluorouracil (5FU) remains poorly understood. Here, we investigate the transcriptional responses of CRC cells to 5FU treatment, revealing significant metabolic reprogramming towards heightened mitochondrial activity. Utilizing CRC models, we demonstrate sustained enhancement of mitochondrial biogenesis and function following 5FU treatment, leading to resistance in both in vitro and in vivo settings. Furthermore, we show that targeting mitochondrial metabolism, specifically by inhibiting Complex I (CI), sensitizes CRC cells to 5FU, resulting in delayed tumour growth and prolonged survival in preclinical models. Additionally, our analysis of patient data suggests that oxidative metabolism signatures may predict responses to 5FU-based chemotherapy. These findings shed light on mechanisms underlying 5FU resistance and propose a rational strategy for combination therapy in CRC, emphasizing the potential clinical benefit of targeting mitochondrial metabolism to overcome resistance and enhance patient outcomes.

cancer biology↗

bioRxiv2024

Library size can undermine accurate molecular and phenotypic subtyping in spatial transcriptomics data.

In an era where transcriptomics-based subtyping, phenotyping and mechanistic understanding is increasingly being driven by state-of-the-art spatially resolved transcriptomic (ST) technologies, it is imperative that researchers, journals, and funders do all they can to ensure that as a community we are interpreting these exciting data as accurately as possible with awareness of their limitations. In this short report, we highlight one potential bias in ST data that could undermine accurate interpretation of transcriptional signatures, providing the field with an opportunity to identify and avoid this issue prior to release of new mechanistic findings. This issue is particularly relevant for platforms that produce some of the most granular and high-resolution spatial information at single cell (and sub-cellular) resolution, with the compromise of a reduced transcriptome panel of genes (Figure 1A). O_FIG O_LINKSMALLFIG WIDTH=141 HEIGHT=200 SRC="FIGDIR/small/602370v1_fig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1219fb3org.highwire.dtl.DTLVardef@7bd4a4org.highwire.dtl.DTLVardef@1c55c42org.highwire.dtl.DTLVardef@2bf924_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Visualisation of the iCMS3 up signature A: Schematic overview of enrichment using a full gene panel versus a reduced panel. B: Overlap between the genes represented on the CosMx and Xenium gene panels. C: Correlation between the single sample scores of the full iCMS3 up signature (74 genes) and the 15 genes from the signature represented on the CosMx platform. Two samples are highlighted which have a similar iCMS3 up enrichment for the full signature (CRC-JSC-S06: 4535.746; SMC16: 4265.263) but extreme enrichments for the signature composed only of the genes present on the CosMx array (CRC-JSC-S06: 301.0396; SMC16: 11430.593). Median (4047.994) shown by red line. D: Visualisation of the rank of each sample across for the full (CRC-JSC-S06: position 25872/44458; SMC16: position 23907/44458) and CosMx (CRC-JSC-S06: position 513/44458; SMC16: position 44241/44458) signature enrichment. E: Heatmap showing the relative enrichment of each gene with the iCMS3 up signature for each sample, with the genes present on the CosMx array in red. F: Subset of samples (n=628) +/-1% of the median (4007.514 - 4088.474), with the top 100 and bottom 100 samples for CosMx enrichment in red. G: Heatmap of the top 100 and bottom 100 samples (shown in red in F) for CosMx enrichment with a full iCMS3 up enrichment around the median. The samples are arranged by the rank of each sample for CosMx signature enrichment, with the sum of the genes within the iCMS3 up signature present on the array (CosMx [n=16]) and those not present on the array (Non CosMx [n=58]) overlaid as barplots. C_FIG

cancer biology↗

bioRxiv2024

Evaluation of Gene Set Enrichment Analysis (GSEA) tools highlights the value of single sample approaches over pairwise for robust biological discovery.

BackgroundGene set enrichment analysis (GSEA) tools can be used to identify biological insights from transcriptional datasets and have become an integral analysis within gene expression-based cancer studies. Over the years, additional methods of GSEA-based tools have been developed, providing the field with an ever-expanding range of options to choose from. Although several studies have compared the statistical performance of these tools, the downstream biological implications that arise when choosing between the range of pairwise or single sample forms of GSEA methods remain understudied. MethodsIn this study, we compare the statistical and biological interpretation of results obtained when using a variety of pre-ranking methods and options for pairwise GSEA and fast GSEA (fGSEA), alongside single sample GSEA (ssGSEA) and gene set variation analysis (GSVA). These analyses are applied to a well-established cohort of n=215 colon tumour samples, using the clinical feature of cancer recurrence status, non-relapse (NR) and relapse (R), as an initial exemplar, in conjunction with the Molecular Signatures Database "Hallmark" gene sets. ResultsDespite minor fluctuations in statistical performance, pairwise analysis revealed remarkably similar results when deployed using a range of gene pre-ranking methods or across a range of choices of GSEA versus fGSEA, with the same well-established prognostic signatures being consistently returned as significantly associated with relapse status. In contrast, when the same statistically significant signatures, such as Interferon Gamma Response, were assessed using ssGSEA and GSVA approaches, there was a complete absence of biological distinction between these groups (NR and R). ConclusionsData presented here highlights how pairwise methods can overgeneralise biological enrichment within a group, assigning strong statistical significance to gene sets that may be inadvertently interpreted as equating to distinct biology. Importantly, single sample approaches allow users to clearly visualise and interpret statistical significance alongside biological distinction between samples within groups-of-interest; thus, providing a more robust and reliable basis for discovery research.

cancer biology↗

bioRxiv2024

A targetable PREX2/RAC1/PI3Kβ signalling axis confers resistance to clinically relevant therapeutic approaches in melanoma

Metastatic melanoma remains a major clinical challenge. Large-scale genomic sequencing of melanoma has identified bona fide activating mutations in RAC1, with mutations of its upstream regulator, the RAC-GEF PREX2, also commonly detected. Crucially, RAC1 mutations are associated with resistance to BRAF-targeting therapies. Despite the role of its homologue PREX1 in melanomagenesis, and evidence that some truncating PREX2 mutations drive increased RAC1 activity, no hotspot mutations have been identified, and the impact of PREX2 mutation remains contentious. Here, we use genetically engineered mouse models and patient-derived BRAFV600E-driven melanoma cell lines to dissect the role of PREX2 in melanomagenesis and response to therapy. We show that while PREX2 is dispensable for the initiation and progression of melanoma, its loss confers sensitivity to clinically relevant therapeutics. Importantly, genetic and pharmacological targeting of the RAC1 effector kinase PI3K{beta} phenocopies PREX2 loss, sensitizing our model systems to therapy. Our data reveal a druggable PREX2/RAC1/PI3K{beta} signalling axis in BRAF-mutant melanoma that could be exploited clinically. Statement of SignificanceMetastatic melanoma remains both a clinical problem, and an opportunity for therapeutic benefit. Co-targeting of the MAPK pathway and the PREX2/RAC1/PI3K{beta} has remarkable efficacy and outperforms monotherapy MAPK targeting in vivo.

cancer biology↗

bioRxiv2022

Impaired Function in Diabetic Patient iPSCs-derived Blood Vessel Organoids Stem from a Subpopulation of Vascular Cells

The presence of both endothelial cells (ECs) and mural cells are central to the proper function of blood vessels in health and pathological changes in diseases including diabetes. Although iPSCs-derived vascular organoids (VOs) provide an appealing in vitro disease model and platform for drug screening, whether these organoids recapitulate human disease remains debatable. Here, we show human diabetic (DB)-VOs represent impaired vascular function including enhanced ROS activity, with higher mitochondrial content and activity, increased pro-inflammatory cytokines, and less regenerative potential in vivo. Using single-cell RNA sequencing, we identify all specialized types of vascular cells (artery, capillary, vein, lymphatic and tip cells, as well as pericytes and vSMCs) within vascular organoids, while demonstrating the dichotomy landscape of ECs and mural cells. Furthermore, we reveal basal heterogeneity within vascular organoids and demonstrate differences between diabetic and non-diabetic VOs. Of note, a subpopulation of ECs significantly enrich for ROS and oxidative phosphorylation hallmarks in DB-VOs, may represent early signs of aberrant angiogenesis in diabetes. This study helps to identify key biomarkers for diabetic disease progression and find signalling molecules amenable to drug intervention.

cell biology↗

bioRxiv2022

β-catenin obstructs γδ T cell immunosurveillance in colon cancer through loss of BTNL expression

WNT/{beta}-catenin signaling endows cancer cells with proliferative capacity and immune-evasive functions that impair anti-cancer immunosurveillance by conventional, cytoxtoic T cells. However, the impact of dysregulated WNT signalling on unconventional, tissue-resident T cells, specifically in colon cancer is unknown. Here, we show that cancer cells in Apc-mutant mouse models escape immunosurveillance from gut-resident intraepithelial lymphocytes (IELs) expressing {gamma}{delta} T cell receptors ({gamma}{delta}TCRs). Analysis of late-stage tumors from mice and humans revealed that {gamma}{delta}IELs are largely absent from the tumor microenvironment, and that butyrophilin-like (BTNL) molecules, which can critically regulate {gamma}{delta}IEL through direct {gamma}{delta}TCR-interactions, are also downregulated. We could attribute this to {beta}-catenin stabilization, which rapidly decreased expression of the transcription factors, HNF4A and HNF4G, that we found to bind promoter regions of Btnl genes, thereby driving their expression in normal gut epithelial cells. Indeed, inhibition of {beta}-catenin signaling restored Btnl1 gene expression and {gamma}{delta} T cell infiltration into tumors. These observations highlight an immune-evasion mechanism specific to WNT-driven colon cancer cells that disrupts {gamma}{delta}IEL immunosurveillance and furthers cancer progression.

cancer biology↗

bioRxiv2022

Molecular phenotyping of colorectal neoplasia shows dynamic and adaptive cancer stem cell population admixture

Intestinal homeostasis is underpinned by LGR5+ve crypt-base columnar stem cells (CBCs), but following injury, dedifferentiation results in the emergence of LGR5-ve regenerative stem cell populations (RSCs), characterised by fetal transcriptional profiles. Neoplasia hijacks regenerative signalling, so we assessed the distribution of CBCs and RSCs in mouse and human intestinal tumors. Using combined molecular-morphological analysis we demonstrate variable expression of stem cell markers across a range of lesions. The degree of CBC-RSC admixture was associated with both epithelial mutation and microenvironmental signalling disruption, and could be mapped across disease molecular subtypes. The CBC-RSC equilibrium was adaptive, with a dynamic response to acute selective pressure, and adaptability was associated with chemoresistance. We propose a fitness landscape model where individual tumors have equilibrated stem cell population distributions along a CBC-RSC phenotypic axis. Cellular plasticity is represented by position shift along this axis, and is influenced by cell-intrinsic, extrinsic and therapeutic selective pressures.

cancer biology↗

bioRxiv2022

Biological misinterpretation of transcriptional signatures in tumour samples can unknowingly undermine mechanistic understanding and faithful alignment with preclinical data

Precise mechanism-based gene expression signatures (GESs) have been developed in appropriate in vitro and in vivo model systems, to identify important cancer-related signalling processes. However, some GESs originally developed to represent specific disease processes, primarily with an epithelial cell focus, are being applied to heterogeneous tumour samples where the expression of the genes in the signature may no longer be epithelial-specific. Therefore, unknowingly, even small changes in tumour stroma percentage can directly influence GESs, undermining the intended mechanistic signalling. Using colorectal cancer as an exemplar, we deployed numerous orthogonal profiling methodologies, including laser capture microdissection, flow cytometry, bulk and multiregional biopsy clinical samples, single cell RNAseq and finally spatial transcriptomics, to perform a comprehensive assessment of the potential for the most widely-used GESs to be influenced, or confounded, by stromal content in tumour tissue. To complement this work, we generated a freely-available resource, ConfoundR; https://confoundr.qub.ac.uk/, that enables users to test the extent of stromal influence on an unlimited number of the genes/signatures simultaneously across colorectal, breast, pancreatic, ovarian and prostate cancer datasets. Findings presented here demonstrate the clear potential for misinterpretation of the meaning of GESs, due to widespread stromal influences, which in-turn can undermine faithful alignment between clinical samples and preclinical data/models, particularly cell lines and organoids, or tumour models not fully recapitulating the stromal and immune microenvironment. As such, efforts to faithfully align preclinical models of disease using phenotypically-designed GESs must ensure that the signatures themselves remain representative of the same biology when applied to clinical samples.

cancer biology↗

bioRxiv2021

The Molecular Subtyping Resource (MouSR): a user-friendly tool for rapid biological discovery from human or mouse transcriptional data

Generation of transcriptional data has dramatically increased in the last decade, driving the development of analytical algorithms that enable interrogation of the biology underpinning the profiled samples. However, these resources require users to have expertise in data wrangling and analytics, reducing opportunities for biological discovery by "wet-lab" users with a limited programming skillset. Although commercial solutions exist, costs for software access can be prohibitive for academic research groups. To address these challenges, we have developed an open source and user-friendly data analysis platform for on-the-fly bioinformatic interrogation of transcriptional data derived from human or mouse tissue, called "MouSR". This internet-accessible analytical tool, https://mousr.qub.ac.uk/, enables users to easily interrogate their data using an intuitive "point and click" interface, which includes a suite of molecular characterisation options including QC, differential gene expression, gene set enrichment and microenvironmental cell population analyses from RNA-Seq. Users are provided with adjustable options for analysis parameters to generate results that can be saved as publication-quality images. To highlight its ability to perform high quality data analysis, we utilise the MouSR tool to interrogate our recently published tumour dataset, derived from genetically engineered mouse models and matched organoids, where we rapidly reproduced the key transcriptional findings. The MouSR online tool provides a unique freely-available option for users to perform rapid transcriptomic analyses and comprehensive interrogation of the signalling underpinning transcriptional datasets, which alleviates a major bottleneck for biological discovery.

bioinformatics↗

bioRxiv2021

Stromal cell sialylation suppresses T cells in inflammatory tumour microenvironments: A new tumour stromal cell immune checkpoint?

Immunosuppressive tumour microenvironments (TME) reduce the effectiveness of immune responses in cancer. Non-haematopoietic mesenchymal stromal cells (MSC), the precursor to cancer associated fibroblasts (CAFs), dictate tumour progression by enhancing immune cell suppression. Hyper-sialylation of glycans promotes immune evasion in cancer, but the role of sialyation in stromal cell-mediated immunosuppression is unknown. Here we study changes in sialyltransferase (ST) enzymes and associated surface expressed sialic acid in stromal cells following inflammatory and tumour secretome conditioning. We show that tumour conditioned stromal cells have increased levels of sialyltransferases, 2,3/6 linked sialic acid and siglec ligands. In tumour models of solid (colorectal cancer) and haematological (multiple myeloma) stromal rich tumours, stromal cell sialylation is associated with enhanced immunosuppression. Using datasets and patient samples, we confirm that targeting sialylation in tumour stromal cells reverses immune cell exhaustion. Targeting stromal cell sialylation may represent a novel immune checkpoint to reactivate anti-tumour immunity.

immunology↗

bioRxiv2021

Development of a semi-automated method for tumor budding assessment in colorectal cancer and comparison with manual methods

Tumor budding is an established prognostic feature in multiple cancers but routine assessment has not yet been incorporated into clinical pathology practice. Recent efforts to standardize and automate assessment have shifted away from haematoxylin and eosin (H&E)-stained images towards cytokeratin (CK) immunohistochemistry. In this study, we compare established manual H&E and cytokeratin budding assessment methods with a new, semi-automated approach built within the QuPath open-source software. We applied our method to tissue cores from the advancing tumor edge in a cohort of stage II/III colon cancers (n=186). The total number of buds detected by each method, over the 186 TMA cores, were as follows; manual H&E (n=503), manual CK (n=2290) and semi-automated (n=5138). More than four times the number of buds were detected using CK compared to H&E. A total of 1734 individual buds were identified both using manual assessment and semi-automated detection on CK images, representing 75.7% of the total buds identified manually (n=2290) and 33.7% of the total buds detected using our proposed semi-automated method (n=5138). Higher bud scores by the semi-automated method were due to any discrete area of CK immunopositivity within an accepted area range being identified as a bud, regardless of shape or crispness of definition, and to inclusion of tumor cell clusters within glandular lumina ("luminal pseudobuds"). Although absolute numbers differed, semi-automated and manual bud counts were strongly correlated across cores ({rho}=0.81, p<0.0001). Despite the random, rather than "hotspot", nature of tumor core sampling, all methods of budding assessment demonstrated poorer survival associated with higher budding scores. In conclusion, we present a new QuPath-based approach to tumor budding assessment, which compares favorably to current established methods and offers a freely-available, rapid and transparent tool that is also applicable to whole slide images.

pathology↗

bioRxiv2020

Clinical positioning of the IAP antagonist tolinapant (ASTX660) in colorectal cancer

Cancer cells frequently express elevated levels of Inhibitor of Apoptosis Proteins (IAPs): cIAPI, cIAP2 and XIAP. Elevated expression of cIAP1 and cIAP2 (but not XIAP) significantly correlated with poor prognosis in microsatellite stable (MSS) stage-III colorectal cancer (CRC) patients treated with adjuvant chemotherapy, suggesting their involvement in promoting resistance. Preclinical analysis of the IAP inhibitor tolinapant in CRC cell lines demonstrated robust on-target effects and caspase-8-dependent apoptosis that was inhibited by the caspase-8 paralogs FLIP and, unexpectedly, caspase-10. Importantly, tolinipant-induced apoptosis was augmented by standard-of-care chemotherapy (FOLFOX) in CRC disease models, due (at least in part) to FOLFOX-induced downregulation of Class-I histone deacetylases, leading to acetylation of the FLIP-binding partner Ku70 and downregulation of FLIP. Moreover, this effect could be phenocopied using a Class-I HDAC inhibitor. Further analyses revealed that caspase-8-knockout RIPK3-positive CRC models were sensitive to tolinostat-induced necroptosis, an effect that could be exploited with the FDA-approved caspase inhibitor emricasan. Our study provides evidence for immediate clinical exploration of tolinapant in combination with FOLFOX chemotherapy in poor prognosis MSS CRC with elevated cIAP1/2 expression.

cancer biology↗

bioRxiv2019

FLIP(L) determines p53 induced life or death

How p53 differentially activates cell cycle arrest versus cell death remains poorly understood. Here, we demonstrate that upregulation of canonical pro-apoptotic p53 target genes in colon cancer cells imposes a critical dependence on the long splice form of the caspase-8 regulator FLIP (FLIP(L)), which we identify as a direct p53 transcriptional target. Inhibiting FLIP(L) expression with siRNA or Class-I HDAC inhibitors promotes apoptosis in response to p53 activation by the MDM2 inhibitor Nutlin-3A, which otherwise predominantly induces cell-cycle arrest. When FLIP(L) upregulation is inhibited, apoptosis is induced in response to p53 activation via a novel ligand-independent TRAIL-R2/caspase-8 complex, which, by activating BID, induces mitochondrial-mediated apoptosis. Notably, FLIP(L) depletion inhibits p53-induced expression of the cell cycle regulator p21 and enhances p53-mediated upregulation of PUMA, with the latter activating mitochondrial-mediated apoptosis in FLIP(L)-depleted, Nutlin-3A-treated cells lacking TRAIL-R2/caspase-8. Thus, we report two previously undescribed, novel FLIP(L)-dependent mechanisms that determine cell fate following p53 activation.

cancer biology↗

bioRxiv2018

ACE: A Workbench using Evolutionary Genetic Algorithms for analysing association in TCGA Data

Modern methods in generating molecular data have dramatically scaled in recent years, allowing researchers to efficiently acquire large volumes of information. However, this has increased the challenge of recognising interesting patterns within the data. Atlas Correlation Explorer (ACE) is a user-friendly workbench for seeking associations between attributes in the cancer genome atlas (TCGA) database. It allows any combination of clinical and genomic data streams to be selected for searching, and highlights significant correlations within the chosen data. It is based on an evolutionary algorithm which is capable of producing results for very large searches in a short time.

bioinformatics↗