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

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

Clinicopathological characteristics of colon cancer patient cohorts.

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.

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

WNT expression is independent of BTN3A1 and BTN2A1

In adult tissues, stem cells (SCs) reside in specialized niches, where they remain relatively stationary state until activated by injury. While migration is essential for regeneration, mechanisms guiding SCs towards injury sites remain poorly understood due to challenges in tracking them in vivo. Here, we present an experimental framework to monitor intestinal SC (ISC) movement in real time during early gut regeneration. We identify the Drosophila PDGF-VEGF-related receptor, Pvr, as a critical regulator of this process, with ISC-specific Pvr depletion strongly impairing migration and regeneration. The ligand, Pvf1, produced by gut-associated trachea after damage, serves as a guidance cue directing ISCs towards injury sites. Our work highlights a critical role of gut-trachea crosstalk in guiding ISC migration during regeneration. As neovascularization of injury sites is a key feature of tissue repair in both flies and mammals, these findings could have broader implications for regenerative processes across diverse adult tissues.

AbstractSickness-induced sleep is a behavior conserved across species that promotes recovery from illness, yet the underlying mechanisms are poorly understood. Here, we show that interleukin-6-like cytokine signaling from theDrosophilagut to brain glial cells regulates sleep. Under healthy conditions, this pathway promotes wakefulness. However, elevated gut cytokine signaling in response to oxidative stress – triggered by immune and inflammatory responses in the intestine – induces sleep. The cytokines Unpaired 2 and -3 are upregulated by oxidative stress in enteroendocrine cells and activate JAK-STAT signaling in glial cells, including those of the blood-brain barrier (BBB). This activity maintains elevated sleep during oxidative-stress-induced intestinal disturbances, suggesting that the JAK-STAT pathway in glia inhibits wake-promoting signaling to facilitate sleep-dependent restoration under these conditions. We find that the enteric peptide Allatostatin A (AstA) enhances wakefulness, and during intestinal oxidative stress, gut-derived Unpaired 2/3 inhibits AstA receptor expression in BBB glia, thereby sustaining an elevated sleep state during gut inflammation or illness. Taken together, our work identifies a gut-to-glial communication pathway that couples sleep with intestinal homeostasis and disease, enhancing sleep during intestinal sickness, and contributes to our understanding of how sleep disturbances arise from gastrointestinal disturbances.

While the role of the Tumor necrosis factor-α (referred to as TNF here) as a key mediator of pro-inflammatory and immune responses is well-established, non-pathological functions of the TNF-TNF receptor (TNFR) signaling are less explored. In this issue of The EMBO Journal, Esteban-Collado et al, (2024) describe a TNF independent, pro-survival role of the Drosophila TNFR Wengen during damage-induced tissue regeneration. Recent study identifies the Drosophila TNF receptor Wengen as a TNF-independent mediator of tissue regeneration in response to apoptosis.

Objectives Adenomas are known precursors to colorectal cancer (CRC). Current UK post-polypectomy surveillance guidelines use polyp size, numbers, and histology to stratify the risk of patients developing metachronous polyps or CRC. However, these risk guidelines suffer from poor predictive value, often leading to under/over surveillance. Design Adenomas removed from 1257 patients at bowel screening colonoscopy were retrospectively identified to investigate mutational profile and protein expression trends associated with the detection of metachronous polyps or CRC. The presence or absence of metachronous polyps or CRC was recorded 6 months to 6 years after index polypectomy. Results APC and KRAS were the most mutated genes in these patients (87% and 34% respectively), and both were significantly co-occurring with the 6th most mutated gene SOX9 (17% co-occurring with APC , p=0.047; 23% co-occurring with KRAS , p=0.012). High SOX9 cytoplasmic expression was significantly associated with the detection of metachronous polyps or CRC (HR 1.543, p=0.001) and improved high risk stratification when combined with BSG2020 guidelines versus guidelines alone (HR 2.626, p<0.0001). High cytoplasmic SOX9 alone and in combination with current guidelines was an independent predictor of metachronous polyps or CRC according to various regression models. This was validated in an independent test dataset, where high cytoplasmic expression was significantly associated with the detection of metachronous polyps or CRC (HR 1.654, p=0.012) and enhanced risk stratification when combined with BSG2020 guidelines versus guidelines alone (HR 2.473, p=0.0018). Conclusion High cytoplasmic SOX9 expression within adenomas is associated with shorter time to detection of metachronous polyps or CRC.   ### Competing Interest Statement DA was employed by BioClavis LTD at the time the work for this manuscript was carried out. All other authors declare no conflicts of interest. ### Funding Statement This work was funded by Cancer Research UK (Grant ID 105858), Innovate UK (Grants 105858 and 10054829), Medical Research Scotland (Grant OHD-50246-2020), and Cancer Research UK Scottish Cancer Centre (CTRQQR-2021/100006). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The West of Scotland Research Ethics Committee gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Mutational data used in this study are available at Datacite DOI: 10.5525/gla.researchdata.1223, Datacite DOI: 10.5525/gla.researchdata.1307, Datacite DOI: 10.5525/gla.researchdata.1498, and Datacite DOI: 10.5525/gla.researchdata.1602.Data used in this study is available upon reasonable request under condition of collaboration.

ABSTRACT Purpose Carbonic anhydrase IX (CAIX) is a well‐established prognostic marker in breast cancer (BC). Nevertheless, this prognostic value is yet to be confirmed in BC subtypes. This study aims to investigate the prognostic effects of CAIX in oestrogen receptor (ER)‐negative (ER−) BCs and to establish pathways related to cytoplasmic CAIX expression in ER− and lymph node‐negative BCs. Methods Immunohistochemistry was performed to identify the prognostic role of CAIX protein expression in ER− tissue microarrays (TMAs) (n = 191). CAIX‐positive samples (n = 37) were transcriptionally profiled by TempO‐Seq and analysed by STRING. Real‐time quantitative PCR (RT‐qPCR) analysis was used to validate differentially expressed genes. Results Overexpression of cytoplasmic CAIX was an independent predictor of recurrence free survival, disease‐free survival and overall survival in ER− cohort. RNA transcriptomic analysis identified 10 significant genes in ER− cohort and 3 genes in the node‐negative group. The STRING database demonstrated a significant interaction between MUCL1 and GALNT6, which were linked with extracellular matrix organisation, degradation of the extracellular matrix and disease of glycosylation pathways. In the node‐negative group, SPNS2 is mainly involved in the sphingolipid de novo biosynthesis pathway. A significant correlation between cytoplasmic SphK1 and cytoplasmic hypoxia‐inducible factor‐1α was observed. Among the 10 genes, 7 genes (SERHL2, GALNT6, MUCL1, MMP7, PITX2, CEACAM6 and SPNS2) were selected, and their expression was quantitatively assessed by RT‐qPCR. The PCR data of these genes showed that SERHL2, GALNT6, MUCL1, PITX2, and SPNS2 mRNA levels were expressed in MDA‐MB‐231 BC cell lines at variable levels of hypoxic exposure. Conclusion Cytoplasmic CAIX was independently associated with poor prognosis in ER− BC. Gene expression profiles shed light on the pathways and genes associated with hypoxia in ER− BC. In node‐negative patients, SPNS2 was of particular interest.

Colorectal cancer (CRC) is a heterogenous malignancy underpinned by dysregulation of cellular signaling pathways. Previous literature has implicated aberrant JAK/STAT3 signal transduction in the development and progression of solid tumors. In this study we investigate the effectiveness of inhibiting JAK/STAT3 in diverse CRC models, establish in which contexts high pathway expression is prognostic and perform in depth analysis underlying phenotypes. In this study we investigated the use of JAK inhibitors for anti-cancer activity in CRC cell lines, mouse model organoids and patient-derived organoids. Immunohistochemical staining of the TransSCOT clinical trial cohort, and 2 independent large retrospective CRC patient cohorts was performed to assess the prognostic value of JAK/STAT3 expression. We performed mutational profiling, bulk RNASeq and NanoString GeoMx® spatial transcriptomics to unravel the underlying biology of aberrant signaling. Inhibition of signal transduction with JAK1/2 but not JAK2/3 inhibitors reduced cell viability in CRC cell lines, mouse, and patient derived organoids (PDOs). In PDOs, reduced Ki67 expression was observed post-treatment. A highly significant association between high JAK/STAT3 expression within tumor cells and reduced cancer-specific survival in patients with high stromal invasion (TSP high ) was identified across 3 independent CRC patient cohorts, including the TrasnSCOT clinical trial cohort. Patients with high phosphorylated STAT3 (pSTAT3) within the TSP high group had higher influx of CD66b + cells and higher tumoral expression of PDL1. Bulk RNAseq of full section tumors showed enrichment of NFκB signaling and hypoxia in these cases. Spatial deconvolution through GeoMx® demonstrated higher expression of checkpoint and hypoxia-associated genes in the tumor (pan-cytokeratin positive) regions, and reduced lymphocyte receptor signaling in the TME (pan-cytokeratin- and αSMA-) and αSMA (pan-cytokeratin- and αSMA +) areas. Non-classical fibroblast signatures were detected across αSMA + regions in cases with high pSTAT3. Therefore, in this study we have shown that inhibition of JAK/STAT3 represents a promising therapeutic strategy for patients with stromal-rich CRC tumors. High expression of JAK/STAT3 proteins within both tumor and stromal cells predicts poor outcomes in CRC, and aberrant signaling is associated with distinct spatially-dependant differential gene expression.

Rapidly renewable tissues adapt different strategies to cope with environmental insults. While tissue repair is associated with increased intestinal stem cell (ISC) proliferation and accelerated tissue turnover rates, reduced calorie intake triggers a homeostasis-breaking process causing adaptive resizing of the gut. Here we show that activins are key drivers of both adaptive and regenerative growth. Activin-β (Actβ) is produced by stem and progenitor cells in response to intestinal infections and stimulates ISC proliferation and turnover rates to promote tissue repair. Dawdle (Daw), a divergent Drosophila activin, signals through its receptor, Baboon, in progenitor cells to promote their maturation into enterocytes (ECs). Daw is dynamically regulated during starvation-refeeding cycles, where it couples nutrient intake with progenitor maturation and adaptive resizing of the gut. Our results highlight an activin-dependent mechanism coupling nutrient intake with progenitor-to-EC maturation to promote adaptive resizing of the gut and further establish activins as key regulators of adult tissue plasticity.

AbstractColorectal cancer (CRC) is a heterogenous malignancy and research is focused on identifying novel ways to subtype patients. In this study, a novel classification system, tumour microenvironment score (TMS), was devised based on Klintrup–Mäkinen grade (KMG), tumour stroma percentage (TSP), and tumour budding. TMS was performed using a haematoxylin and eosin (H&E)‐stained section from retrospective CRC discovery and validation cohorts (n = 1,030, n = 787). TMS0 patients had high KMG, TMS1 were low for KMG, TSP, and budding, TMS2 were high for budding, or TSP and TMS3 were high for TSP and budding. Scores were assessed for association with survival and clinicopathological characteristics. Mutational landscaping and Templated Oligo‐Sequencing (TempO‐Seq) profiling were performed to establish differences in the underlying biology of TMS. TMS was independently prognostic in both cohorts (p < 0.001, p < 0.001), with TMS3 predictive of the shortest survival times. TMS3 was associated with adverse clinical features including sidedness, local and distant recurrence, higher T stage, higher N stage, and presence of margin involvement. Gene set enrichment analysis of TempO‐Seq data showed higher expression of genes associated with hallmarks of cancer pathways including epithelial to mesenchymal transition (p < 0.001), IL2 STAT5 signalling (p = 0.007), and angiogenesis (p = 0.017) in TMS3. Additionally, enrichment of immunosuppressive immune signatures was associated with TMS3 classification. In conclusion, TMS represents a novel and clinically relevant method for subtyping CRC patients from a single H&E‐stained tumour section.

Inhibition of β-catenin transcriptional activity increases expression of HNF4A, HNF4G, and butyrophilin-like molecules. A, Vγ7+ cell viability in cocultures with CT26 or CT26-B1/6 cells. Each dot represents one paired biological replicate (n = 4). B, CD25 expression by Vγ7+ cells in cocultures with CT26 or CT26-B1/6 cells. Each dot represents one paired biological replicate (n = 4). C, CT26 and CT26-B1/6 cancer cell death using flow cytometry after coculture with Vγ7+ cell as indicated. Each dot represents one biological replicate (n = 4). D, Kaplan–Meier survival analysis of doxycycline-treated CT26 and CT26-B1/6 tumor-bearing mice (n = 4 CT26, 5 CT26-B1/6) using the log-rank test. E, γδ T-cell numbers in tumors from doxycycline-treated CT26 and CT26-B1/6 tumor-bearing mice. Each dot represents one mouse (n = 4 CT26, 5 CT26-B1/6). F and H, Images taken from serially stained sections of indicated protein in tumors from indicated mouse models (n = 3–4; scale bar, 500 μm. G and I, Images of Trdc expression in tumors from indicated mouse models; scale bar, 500 μm. γδ T-cell numbers in tumors. Each dot represents one mouse. Data are presented as mean ± SD. *, P < 0.05; **, P < 0.01 (paired t test or unpaired t test or one-way ANOVA followed by Tukey post hoc test).

Activation of β-catenin decreases butyrophilin-like molecule expression. A and B, Correlation between indicated genes as determined by TempO-seq and γδ T-cell density determined by IHC in the Scotland cohort. Units on axes are normalized read counts × 103. Each dot represents one tumor (n = 77 left and 82 right). P and r values determined by Pearson correlation. C, Correlation between CTNNB1 or SOX9 expression and BTNL3 or BTNL8 expression in the Marisa cohort (28). Units on axes are normalized counts × 103. Each dot represents one tumor (n = 258). P and r values determined by Pearson correlation. D, Images of organoids derived from indicated mouse models taken 4 days after tamoxifen treatment. E, Fold change in expression levels of indicated genes in organoids from various genotypes measured at indicated days after tamoxifen treatment. Each dot represents one organoid derived from one mouse (n = 3). F, Fold change in expression levels of indicated genes in WT organoids treated with 3 or 10 μmol/L CHIR-99021 for indicated days. Each dot represents one organoid derived from one mouse (n = 3). Data are presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-way ANOVA followed by Dunnett post hoc test).

232 Background: Following colorectal polypectomy, 20-50% of patients develop metachronous polyps, and a proportion have increased colorectal cancer (CRC) risk. However, there are currently no molecular biomarkers for predicting metachronous polyp risk. Constitutive activation of the Wnt signalling cascade is a hallmark of polyp development and carcinogenesis, therefore biomarkers from this pathway were investigated for association with detection of metachronous polyps. Methods: A retrospective study was performed on a tissue microarray (TMA) of left sided colonic polyps from 279 patients undergoing screening polypectomy (May’09-Dec’16) followed by surveillance colonoscopy (up to 6 years). Mutational analysis was performed using the GPOL cancer plus panel, protein expression by Immunohistochemistry and RNA-sequencing by TempO-Seq (BioClavis). Log rank statistics determined associations with time to detection of metachronous polyp. Index polyp number, histology and size were compared to metachronous lesion outcome using χ 2 /ANOVA and multivariate polynomial regression identified independent predictors of advanced future lesions. Results: APC (91%), KRAS (30%) and SOX9 (23%) were the most frequently mutated genes and APC and SOX9 are both implicated in Wnt signalling. Mutations in APC and co-mutations in SOX9 with ARID1 were associated with a shorter time to detection of metachronous polyps. Neither variant classification nor location of mutations were associated with metachronous polyp detection. Luminal epithelial cells with high expression of E-Cadherin or SOX9 were significantly associated with earlier detection of metachronous polyps or CRC (HR 2.3, 95%CI 1.6-3.3: p<0.001 and: HR 2.0, 95%CI 1.3-3.0; p=0.001 respectively). By χ 2 /ANOVA analysis, E-Cadherin and SOX9 expression was associated with detection of metachronous polyp (p>0.001, p=0.037 respectively). On multivariate regression, number of polyps (HR 1.2, 95%CI 1.1-1.3; p<0.001), and E-Cadherin expression (HR 2.1, 95%CI 1.5-3; p<0.001), independently predicted metachronous lesions. Gene set enrichment analysis (GSEA) was used to explore differential biology between high and low E-Cadherin or SOX9 expression. For E-Cadherin, all seven immune-annotated MSigDB hallmarks had negative enrichment, however Wnt--catenin signaling was positively enriched (NES=1.50, p-adj<0.1). The top signature enriched for SOX9 was ‘NFB signaling via TNF (NES=1.70, p-adj<0.001). The LGR5 stemness signature was enriched in both high E-Cadherin (NES=1.97 p-adj<0.0001) and high SOX9 (NES=1.48, p-adj<0.0001) groups. Conclusions: Wnt signalling-associated protein expression within index polyp tissue is valuable for predicting metachronous polyp risk. In addition, expression of E-Cadherin and SOX9 in colonic polyps are linked to immune and stemness signatures, suggesting immune and stemness biology may be an underlying driver of metachronous polyp development.