Intestinal crypts are test tube-like structures lined with an epithelial monolayer. Under homeostasis, mitotic forces drive epithelial cells to migrate up the crypt, from the stem cell niche. As the cells migrate up the crypt, they differentiate into specialised cells. This process is regulated by morphogen gradients established by distinct populations of subepithelial fibroblasts, and recent studies suggest fibroblasts and epithelial cells have co-evolved to maintain crypt structure and function via complementary morphogen expression. We present a mathematical model of fibroblast–epithelial cross-talk, in which fibroblast and epithelial phenotypes emerge from morphogen binding to cell surface receptors. The model predicts the formation of distinct zones of mutually supporting phenotypes at different crypt heights. These findings support the idea that fibroblast and epithelial cell phenotypes are an emergent property of the crypt microenvironment. We use the model to investigate how mutations in the fibroblasts may disrupt these phenotypic zones. Our results suggest that such mutations may lead to uncontrolled epithelial cell growth and, as such, indicate how dysfunctional fibroblasts may contribute to the emergence of colorectal cancer.

Abstract In colorectal cancer (CRC), tumours classified 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 polyinosinic:polycytidylic acid, 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 poor-prognostic tumours, by reprogramming stem cell states and activation of an innate-adaptive anti-metastatic response.

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 (PGE 2 ) is a dominant regulator of CRC cell-fate plasticity. Stromal PGE 2 converts DACH1 + epithelia from a chemosensitive proliferative colonic stem cell (proCSC) fate into a chemorefractory and prometastatic revival colonic stem cell (revCSC) fate. PGE 2 -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.

The colorectal epithelium is rapidly renewing, with remarkable capacity to regenerate following injury. In colorectal cancer (CRC), this regenerative capacity can be co-opted to drive epithelial plasticity. Although oncogenic MAPK signalling in CRC is common, with frequent mutations of both KRAS (40-50%) and BRAF (10%)1, inhibition of this pathway typically drives resistance clinically. Here, given the development of KRAS inhibitors and licensing of BRAF inhibitor combinations2-4, we have interrogated key mechanisms of resistance to these agents in advanced preclinical CRC models. We show that oncogenic MAPK signalling induces epithelial-state changes in vivo, driving adoption of a regenerative/revival stem-like population, whereas inhibition leads to rapid transcriptional remodelling of both Kras-mutant and Braf-mutant tumours, favouring a WNT-associated, canonical stem phenotype. This drives acute therapeutic resistance in Kras-driven and delayed resistance in Braf-driven models. Where plasticity is restrained, such as in early metastatic disease, or through targeting ligand-dependent WNT pathway Rnf43 mutations, marked therapeutic responses are observed. This explains the super response to BRAF + EGFR-targeted therapies previously observed in a BRAF-RNF43 co-mutant patient population, highlighting the criticality of cellular plasticity in therapeutic response. Together, our data provide clear insight into the mechanisms underpinning resistance to MAPK-targeted therapies in CRC. Moreover, strategies that aim to corral stem cell fate, restrict epithelial plasticity or intervene when tumours lack heterogeneity may improve therapeutic efficacy of these agents.

Knockdown of Cdx1, Cdx2 and Hnf4a fails to influence organoid morphology or expression of Btnl genes

The ConfoundR resource enables stromal influence estimation in cancer tissue. A, Schematic overview of the cohorts and analyses available within the ConfoundR app, accessible via https://confoundr.qub.ac.uk/. B, Expression Boxplots analysis module of ConfoundR enabling the expression of a single gene to be compared between stroma and epithelium samples in each of the ConfoundR datasets. C, Expression Heatmap analysis module of ConfoundR enabling the expression of multiple genes to be visually compared between stroma and epithelium samples in each of the ConfoundR datasets. D, GSEA analysis module of ConfoundR allowing GSEA of existing gene sets from established gene set collections or custom user defined gene sets to be performed comparing stroma with epithelium in each of the ConfoundR datasets.

Impact of Btnl1 and Btnl6 expression on Vgamma7+ cells and the tumor microenvironment

Platelets are anucleate blood cells vital for hemostasis and immunity. During cell death and aberrant mitosis, nucleated cells release DNA, resulting in "cell-free" DNA in plasma (cfDNA). An excess of cfDNA is deleterious. Given their ability to internalize pathogen-derived nucleic acids, we hypothesized that platelets may also clear endogenous cfDNA. We found that, despite lacking a nucleus, platelets contained a repertoire of DNA fragments mapping across the nuclear genome. We detected fetal DNA in maternal platelets and cancer-derived DNA in platelets from patients with premalignant and cancerous lesions. As current liquid biopsy approaches utilize platelet-depleted plasma, important genetic information contained within platelets is being missed. This study establishes a physiological role for platelets that has not previously been highlighted, with broad translational relevance.

BACKGROUND:The risk of developing advanced neoplasia (AN; colorectal cancer and/or high-grade dysplasia) in ulcerative colitis (UC) patients with a low-grade dysplasia (LGD) lesion is variable and difficult to predict. This is a major challenge for effective clinical management. OBJECTIVE:We aimed to provide accurate AN risk stratification in UC patients with LGD. We hypothesised that the pattern and burden of somatic genomic copy number alterations (CNAs) in LGD lesions could predict future AN risk. DESIGN:We performed a retrospective multicentre validated case-control study using n=270 LGD samples from n=122 patients with UC. Patients were designated progressors (n=40) if they had a diagnosis of AN in the ~5 years following LGD diagnosis or non-progressors (n=82) if they remained AN-free during follow-up. DNA was extracted from the baseline LGD lesion, low-coverage whole genome sequencing performed and data processed to detect CNAs. Survival analysis was used to evaluate CNAs as predictors of future AN risk. RESULTS:CNA burden was significantly higher in progressors than non-progressors (p=2×10-6 in discovery cohort) and was a very significant predictor of AN risk in univariate analysis (OR=36; p=9×10-7), outperforming existing clinical risk factors such as lesion size, shape and focality. Optimal risk prediction was achieved with a multivariate model combining CNA burden with the known clinical risk factor of incomplete LGD resection. Within-LGD lesion genetic heterogeneity did not confound risk prediction. CONCLUSION:Measurement of CNAs in LGD is an accurate predictor of AN risk in inflammatory bowel disease and is likely to support clinical management.

Tumour content plays a pivotal role in directing the bioinformatic analysis of molecular profiles such as copy number variation (CNV). In clinical application, tumour purity estimation (TPE) is achieved either through visual pathological review [conventional pathology (CP)] or the deconvolution of molecular data. While CP provides a direct measurement, it demonstrates modest reproducibility and lacks standardisation. Conversely, deconvolution methods offer an indirect assessment with uncertain accuracy, underscoring the necessity for innovative approaches. SoftCTM is an open-source, multiorgan deep-learning (DL) model for the detection of tumour and non-tumour cells in H&E-stained slides, developed within the Overlapped Cell on Tissue Dataset for Histopathology (OCELOT) Challenge 2023. Here, using three large multicentre colorectal cancer (CRC) cohorts (N = 1,097 patients) with digital pathology and multi-omic data, we compare the utility and accuracy of TPE with SoftCTM versus CP and bioinformatic deconvolution methods (RNA expression, DNA methylation) for downstream molecular analysis, including CNV profiling. SoftCTM showed technical repeatability when applied twice on the same slide (r = 1.0) and excellent correlations in paired H&E slides (r > 0.9). TPEs profiled by SoftCTM correlated highly with RNA expression (r = 0.59) and DNA methylation (r = 0.40), while TPEs by CP showed a lower correlation with RNA expression (r = 0.41) and DNA methylation (r = 0.29). We show that CP and deconvolution methods respectively underestimate and overestimate tumour content compared to SoftCTM, resulting in 6-13% differing CNV calls. In summary, TPE with SoftCTM enables reproducibility, automation, and standardisation at single-cell resolution. SoftCTM estimates (M = 58.9%, SD +/- 16.3%) reconcile the overestimation by molecular data extrapolation (RNA expression: M = 79.2%, SD +/- 10.5, DNA methylation: M = 62.7%, SD +/- 11.8%) and underestimation by CP (M = 35.9%, SD +/- 13.1%), providing a more reliable middle ground. A fully integrated computational pathology solution could therefore be used to improve downstream molecular analyses for research and clinics. (c) 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Clinicopathological characteristics of colon cancer patient cohorts.

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 use a disease-positioned mouse and human tissue to explore the consequences of pathological BMP signalling dysregulation on epithelial-mesenchymal interaction. Aberrant pan-epithelial expression of the secreted BMP antagonist Grem1 results in ectopic crypt formation, with lineage tracing demonstrating the presence of Lgr5(-) stem/progenitor cells. Isolated epithelial cell Grem1 expression has no effect on individual cell fate, indicating an intercompartmental impact of mucosal-wide BMP antagonism. Treatment with an anti-Grem1 antibody abrogates the polyposis phenotype, and triangulation of specific pathway inhibitors defines a pathological sequence of events, with Wnt-ligand-dependent ectopic stem cell niches forming 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.

Deletion of Apc in organoids increases WNT target genes

Disruption of WNT gradient in normal intestinal villi reduces gamma delta T cells

Identification of a WNT responsive element (WRE) in the HNF4G genomic locus.

Mammalian tissue architecture is central to biological function, and its disruption is a hallmark of disease. Medical imaging techniques can generate large point cloud datasets that capture changes in the cellular composition of such tissues with disease progression. However, regions of interest (ROIs) are usually defined by quadrat-based methods that ignore intrinsic structure and risk fragmenting meaningful features. Here, we introduce TopROI, a topology-informed, network-based method for partitioning point clouds into ROIs that preserves both local geometry and higher-order architecture. TopROI integrates geometry-informed networks with persistent homology, combining cell neighbourhoods and multiscale cycles to guide community detection. Applied to synthetic point clouds that mimic glandular structure, TopROI outperforms quadrat-based and purely geometric partitions by maintaining biologically plausible ROI geometry and better preserving ground-truth structures. Applied to cellular point clouds obtained from human colorectal cancer biopsies, TopROI generates ROIs that preserve crypt-like structures and enable persistent homology analysis of individual regions. This study reveals a continuum of architectural changes from healthy mucosa to carcinoma, reflecting progressive disorganisation in tissue structure. TopROI thus provides a principled and flexible framework for defining biologically meaningful ROIs in large point clouds, enabling more accurate quantification of tissue organization and new insights into structural changes associated with disease progression.

Correlation between gut-specific transcription factors, gamma delta T cell density and BTNL genes in human tumors