Biology subjects

Leedham, S. J.

Publications and source records attributed to Leedham, S. J..

10 recordsLinked to original sources

bioRxiv2024

MuSpAn: A Toolbox for Multiscale Spatial Analysis

Advances in multiplex imaging and spatial omics have revolutionised spatial data generation in biology, revealing complex tissue organisation across multiple scales. However, methods for analysing these data have lagged behind, with fragmented, study-specific pipelines and limited guidance for tool selection. To address this, we introduce MuSpAn, a Multiscale Spatial Analysis package offering intuitive, flexible access to a wide range of mathematical tools - including spatial statistics, topological data analysis, geometry, and networks - within a unified framework. MuSpAn supports efficient data querying, is agnostic to imaging modality, and provides extensive documentation and community support. It enables users to create custom pipelines or conduct unbiased exploratory analyses. We demonstrate MuSpAns capacity to interrogate cross-compartmental cell interactions at multiple length scales in both normal and neoplastic tissue using mouse intestinal spatial transcriptomic datasets. Applied to a CMS4-like murine intestinal cancer model, MuSpAn identifies a continuum of fibroblastic functional phenotypes associated with discrete and coordinated fibroblast-immune interactions, highlighting its utility as a discovery tool across diverse biological contexts.

cell biology↗

bioRxiv2024

Integrating diverse statistical methods to analyse stage-discriminatory cell interactions in colorectal neoplasia

Spatial biology has the potential to unlock information about the disrupted cellular ecosystems that define human disease. Quantitative analysis of spatially-resolved cell interactions allows mapping of tissue self-organisation and assessment of why cells interact differently in physiological and pathological contexts. However, the complexity of mammalian tissues, that occur across a spectrum of length scales, presents significant challenges for spatial analysis, increasing the gap between our capacity to generate and biologically interpret these datasets. Here, we have adapted a range of mathematical tools to develop a suite of spatial descriptors, and deployed them to determine how cell interactions change as colorectal cancer progresses from benign precursors. We demonstrate that combining mathematical analyses permits insightful examination of tissue organisational structures and identifies variable cell-interaction pathways that underpin disease progression. Mathematical tool triangulation can cross-corroborate spatial biology findings, facilitating development of analysis pipelines that are robust to individual method limitations.

cancer biology↗

bioRxiv2024

GREMLIN1 disrupts intestinal epithelial-mesenchymal crosstalk to induce a wnt-dependent ectopic stem cell niche via stromal remodelling

In homeostasis, counterbalanced morphogen signalling gradients along the vertical axis of the intestinal mucosa regulate the fate and function of epithelial and stromal cell compartments. Here, we used a disease-positioned mouse, and human tissue, to explore the consequences of pathological Bone Morphogenetic Protein (BMP) signalling dysregulation on epithelial- mesenchymal interaction. Aberrant pan-epithelial expression of the secreted BMP antagonist GREM1, resulted in ectopic crypt formation with lineage tracing demonstrating the presence of Lgr5(-) stem/progenitor cells. Isolated epithelial cell Grem1 expression had no effect on individual cell fate, indicating an intercompartmental impact of mucosal-wide BMP antagonism. Treatment with a novel anti-Grem1 antibody abrogated the polyposis phenotype, and triangulation of specific pathway inhibitors defined a pathological sequence of events, with wnt-ligand dependent ectopic stem cell niches formed through stromal remodelling following BMP disruption. These data support an emerging co-evolutionary model of intestinal cell compartmentalisation based on bidirectional regulation of epithelial-mesenchymal cell fate and function. One Sentence SummaryPathological epithelial GREM1 expression induces therapeutically reversible ectopic stem cell niches through stromal remodelling

cancer biology↗

bioRxiv2023

Extended correlation functions for spatial analysis of multiplex imaging data

Imaging platforms for generating highly multiplexed histological images are being continually developed and improved. Significant improvements have also been made in the accuracy of methods for automated cell segmentation and classification. However, less attention has focussed on the quantification and analysis of the resulting point clouds which describe the spatial coordinates of individual cells. We focus here on a particular spatial statistical method, the cross-pair correlation function (cross-PCF), which can identify positive and negative spatial correlation between cells across a range of length scales. However, limitations of the cross-PCF hinder its widespread application to multiplexed histology. For example, it can only consider relations between pairs of cells, and cells must be classified using discrete categorical labels (rather than labelling continuous labels such as stain intensity). In this paper, we present three extensions to the cross-PCF which address these limitations and permit more detailed analysis of multiplex images: Topographical Correlation Maps (TCMs) can visualise local clustering and exclusion between cells; Neighbourhood Correlation Functions (NCFs) can identify colocalisation of two or more cell types; and weighted-PCFs (wPCFs) describe spatial correlation between points with continuous (rather than discrete) labels. We apply the extended PCFs to synthetic and biological datasets in order to demonstrate the insight that they can generate. Impact statementThis paper introduces three methods for performing spatial analysis on multiplex digital pathology images. We apply the methods to synthetic datasets and regions of interest from a murine colorectal carcinoma, in order to illustrate their relative strengths and weaknesses. We note that these methods have wider application to marked point pattern data from other sources.

pathology↗

bioRxiv2023

Innate immune receptor C5aR1 regulates cancer cell fate and can be targeted to improve radiotherapy in tumours with immunosuppressive microenvironments

An immunosuppressive microenvironment causes poor tumour T-cell infiltration and is associated with reduced patient overall survival in colorectal cancer. How to improve treatment responses in these tumours is still a challenge. Using an integrated screening approach to identify cancer-specific vulnerabilities, we identify complement receptor C5aR1 as a druggable target which when inhibited improves radiotherapy even in tumours displaying immunosuppressive features and poor CD8+ T-cell infiltration. While C5aR1 is well-known for its role in the immune compartment, we find that C5aR1 is also robustly expressed on malignant epithelial cells, highlighting potential tumour-cell specific functions. C5aR1 targeting results in increased NF-{kappa}B-dependent apoptosis specifically in tumours and not normal tissues; indicating that in malignant cells, C5aR1 primarily regulates cell fate. Collectively, these data reveal that increased complement gene expression is part of the stress response mounted by irradiated tumours and that targeting C5aR1 can improve radiotherapy even in tumours displaying immunosuppressive features.

cancer 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↗

bioRxiv2021

Reconstruction of Contemporary Human Stem Cell Dynamics with Oscillatory Molecular Clocks

Molecular clocks record cellular ancestry. However, currently used clocks tick too slowly to measure the short-timescale dynamics of cellular renewal in adult tissues. Here we develop rapidly oscillating DNA methylation clocks where ongoing (de)methylation causes the clock to tick-tock back-and-forth between methylated and unmethylated states like a pendulum. We identify oscillators using standard methylation arrays and develop a mathematical modelling framework to quantitatively measure human adult stem cell dynamics from these data. Small intestinal crypts were inferred to contain slightly more stem cells than colon (6.5 {+/-} 1.0 vs 5.8 {+/-} 1.7 stem cells/crypt) with slower stem cell replacement in small intestine (0.79 {+/-} 0.5 vs 1.1 {+/-} 0.8 replacements/stem cell/year). Germline APC mutation increased the number of replacements per crypt (13.0 {+/-} 2.4 replacements/crypt/year vs 6.9 {+/-} 4.6 for healthy colon). In blood, we measure rapid expansion of acute leukaemia and slower growth of chronic disease. Rapidly oscillating molecular clocks are a new methodology to quantitatively measure human somatic cell dynamics.

bioinformatics↗

bioRxiv2021

BMP pathway antagonism by Grem1 regulates epithelial cell fate in intestinal regeneration

In the intestine, the homeostatic effect of Bone Morphogenetic Protein (BMP) on cell fate has predominantly been inferred through pathway inactivation. Here, we assessed the impact of autocrine Bmp4 expression on secretory cell fate. Ligand exposure reduced proliferation, expedited terminal differentiation, abrogated long-term secretory cell survival and prevented dedifferentiation. As stem cell plasticity is required for regenerative adaptive reprogramming, we spatiotemporally mapped and functionally explored BMPs role in epithelial restitution. Following ulceration, physiological attenuation of BMP signalling arose through upregulation of the secreted antagonist, Grem1, from topographically distinct populations of stromal cells. Concomitant expression supported functional compensation, following Grem1 deletion from tissue-resident fibroblasts. BMP pathway manipulation showed that antagonist-mediated BMP attenuation was obligatory, but functionally sub-maximal, as regeneration was impaired or enhanced by epithelial overexpression of Bmp4 or Grem1 respectively. Mechanistically, Bmp4 abrogated regenerative stem cell reprogramming, despite a convergent impact of YAP/TAZ on cell fate in remodelled wounds.

cell biology↗

bioRxiv2019

Image-based consensus molecular subtype classification (imCMS) of colorectal cancer using deep learning

Image analysis is a cost-effective tool to associate complex features of tissue organisation with molecular and outcome data. Here we predict consensus molecular subtypes (CMS) of colorectal cancer (CRC) from standard H&E sections using deep learning. Domain adversarial training of a neural classification network was performed using 1,553 tissue sections with comprehensive multi- omic data from three independent datasets. Image-based consensus molecular subtyping (imCMS) accurately classified CRC whole-slide images and preoperative biopsies, spatially resolved intratumoural heterogeneity and provided accurate secondary calls with higher discriminatory power than bioinformatic prediction. In all three cohorts imCMS established sensible classification in CMS unclassified samples, reproduced expected correlations with (epi)genomic alterations and effectively stratified patients into prognostic subgroups. Leveraging artificial intelligence for the development of novel biomarkers extracted from histological slides with molecular and biological interpretability has remarkable potential for clinical translation.

cancer biology↗