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The stem/progenitor cells of the developing intestine are biologically distinct from their adult counterparts. Here, we examine the microenvironmental cues that regulate the embryonic stem/progenitor population, focusing on the role of Notch pathway factor delta-like protein-1 (DLK1). mRNA-seq analyses of intestinal mesenchymal cells (IMCs) collected from embryonic day 14.5 (E14.5) or adult IMCs and a novel coculture system with E14.5 intestinal epithelial organoids were used. Following addition of recombinant DLK1 (rDLK) or

Modelling human colon cancer has long been the ambition of researchers and oncologists with the aim to better replicate disease progression and treatment response. Advances in our understanding of genetics, stem cell biology, tumour microenvironment and immunology have prepared the groundwork for recent major advances. In the last two years the field has seen the progression of: using patient derived organoids (alone and in co-culture) as predictors of treatment response; molecular stratification of tumours that predict outcome and treatment response; mouse models of metastatic disease; and transplant models that can be used to de-risk clinical trials. We will discuss these advances in this review.

Bone morphogenetic protein (BMP) signaling is required for early forebrain development and cortical formation. How the endogenous modulators of BMP signaling regulate the structural and functional maturation of the developing brain remains unclear. Here, we show that expression of the BMP antagonist Grem1 marks committed layer V and VI glutamatergic neurons in the embryonic mouse brain. Lineage tracing of Grem1-expressing cells in the embryonic brain was examined by administration of tamoxifen to pregnant Grem1creERT; Rosa26LSLTdtomato mice at 13.5 days post coitum (dpc), followed by collection of embryos later in gestation. In addition, at 14.5 dpc, bulk mRNA-seq analysis of differentially expressed transcripts between FACS-sorted Grem1-positive and -negative cells was performed. We also generated Emx1-cre-mediated Grem1 conditional knockout mice (Emx1-Cre;Grem1flox/flox) in which the Grem1 gene was deleted specifically in the dorsal telencephalon. Grem1Emx1cKO animals had reduced cortical thickness, especially layers V and VI, and impaired motor balance and fear sensitivity compared with littermate controls. This study has revealed new roles for Grem1 in the structural and functional maturation of the developing cortex., Summary: The BMP antagonist Grem1 is expressed by committed deep-layer glutamatergic neurons in the embryonic mouse cortex. Grem1 conditional knockout mice display cortical and behavioral abnormalities.

The intestinal stroma provides an important microenvironment for immune cell activation. The perturbation of this tightly regulated process can lead to excessive inflammation. We know that upregulated Toll-like receptor 4 (TLR4) in the intestinal epithelium plays a key role in the inflammatory condition of preterm infants, such as necrotizing enterocolitis (NEC). However, the surrounding stromal contribution to excessive inflammation in the pre-term setting awaits careful dissection. Ex vivo co-culture of embryonic day 14.5 (E14.5) or adult murine intestinal stromal cells with exogenous monocytes was undertaken. We also performed mRNAseq analysis of embryonic and adult stromal cells treated with vehicle control or lipopolysaccharide (LPS), followed by pathway and network analyses of differentially regulated transcripts. Cell characteristics were compared using flow cytometry and pHrodo red phagocytic stain, candidate gene analysis was performed via siRNA knockdown and gene expression measured by qPCR and ELISA. Embryonic stromal cells promote the differentiation of co-cultured monocytes to CD11bhighCD11chigh mononuclear phagocytes, that in turn express decreased levels of CD103. Global mRNAseq analysis of stromal cells following LPS stimulation identified TLR signaling components as the most differentially expressed transcripts in the immature compared to adult setting. We show that CD14 expressed by CD11b+CD45+ embryonic stromal cells is a key inducer of TLR mediated inflammatory cytokine production and phagocytic activity of monocyte derived cells. We utilise transcriptomic analyses and functional ex vivo modelling to improve our understanding of unique molecular cues provided by the immature intestinal stroma.

Evasion of apoptosis is a hallmark of cancer, which is frequently mediated by upregulation of the antiapoptotic BCL-2 family proteins. In colorectal cancer (CRC), previous work has highlighted differential antiapoptotic protein dependencies determined by the stage of the disease. While intestinal stem cells (ISCs) require BCL-2 for adenoma outgrowth and survival during transformation, ISC-specific MCL1 deletion results in disturbed intestinal homeostasis, eventually contributing to tumorigenesis. Colon cancer stem cells (CSCs), however, no longer require BCL-2 and depend mainly on BCL-XL for their survival. We therefore hypothesized that a shift in antiapoptotic protein reliance occurs in ISCs as the disease progresses from normal to adenoma to carcinoma. By targeting antiapoptotic proteins with specific BH3 mimetics in organoid models of CRC progression, we found that BCL-2 is essential only during ISC transformation while MCL1 inhibition did not affect adenoma outgrowth. BCL-XL, on the other hand, was crucial for stem cell survival throughout the adenoma-to-carcinoma sequence. Furthermore, we identified that the limited window of BCL-2 reliance is a result of its downregulation by miR-17-5p, a microRNA that is upregulated upon APC-mutation driven transformation. Here we show that BCL-XL inhibition effectively impairs adenoma outgrowth in vivo and enhances the efficacy of chemotherapy. In line with this dependency, expression of BCL-XL, but not BCL-2 or MCL1, directly correlated to the outcome of chemotherapy-treated CRC patients. Our results provide insights to enable the rational use of BH3 mimetics in CRC management, particularly underlining the therapeutic potential of BCL-XL targeting mimetics in both early and late-stage disease.

Bone morphogenetic protein (BMP) signaling is required for early forebrain development and cortical formation. How the endogenous modulators of BMP signaling regulate the structural and functional maturation of the developing brain remains unclear. Here we show that expression of the BMP antagonist,Grem1, marks a neuroprogenitor that gives rise to layer V and VI glutamatergic neurons in the embryonic mouse brain. Lineage tracing ofGrem1-expressing cells in the embryonic brain was examined by administration of tamoxifen to pregnantGrem1creERT Rosa26LSLTdtomatomice at 13.5 days post coitum (dpc), followed by collection of embryos later in gestation. In addition, at 14.5 dpc, bulk mRNA seq analysis of differentially expressed transcripts between FACS sortedGrem1positive and negative cells was performed. We also generatedEmx1-cremediatedGrem1conditional knockout mice (Emx1-Cre;Grem1flox/flox) in which theGrem1gene was deleted specifically in the dorsal telencephalon.Grem1Emx1cKOanimals had reduced cortical thickness, especially layers V and VI and impaired motor balance and fear sensitivity compared to littermate controls. This study has revealed new roles for Grem1 in the structural and functional maturation of the developing cortex.Summary statementThe BMP antagonist,Grem1, marks neuroprogenitors that give rise to deep layer glutamatergic neurons in the embryonic mouse brain.Grem1conditional knockout mice display cortical and behavioural abnormalities.

Background & Aims Cancer-associated fibroblasts (CAFs), key constituents of the tumor microenvironment, either promote or restrain tumor growth. Attempts to therapeutically target CAFs have been hampered by our incomplete understanding of these functionally heterogeneous cells. Key growth factors in the intestinal epithelial niche, bone morphogenetic proteins (BMPs), also play a critical role in colorectal cancer (CRC) progression. However, the crucial proteins regulating stromal BMP balance and the potential application of BMP signaling to manage CRC remain largely unexplored. Methods Using human CRC RNA expression data, we identified CAF-specific factors involved in BMP signaling, then verified and characterized their expression in the CRC stroma by in situ hybridization. CRC tumoroids and a mouse model of CRC hepatic metastasis were used to test approaches to modify BMP signaling and treat CRC. Results We identified Grem1 and Islr as CAF-specific genes involved in BMP signaling. Functionally, GREM1 and ISLR acted to inhibit and promote BMP signaling, respectively. Grem1 and Islr marked distinct fibroblast subpopulations and were differentially regulated by transforming growth factor β and FOXL1, providing an underlying mechanism to explain fibroblast biological dichotomy. In patients with CRC, high GREM1 and ISLR expression levels were associated with poor and favorable survival, respectively. A GREM1-neutralizing antibody or fibroblast Islr overexpression reduced CRC tumoroid growth and promoted Lgr5+ intestinal stem cell differentiation. Finally, adeno-associated virus 8 (AAV8)–mediated delivery of Islr to hepatocytes increased BMP signaling and improved survival in our mouse model of hepatic metastasis. Conclusions Stromal BMP signaling predicts and modifies CRC progression and survival, and it can be therapeutically targeted by novel AAV-directed gene delivery to the liver.

Colorectal cancer is a major cause of cancer death and approximately 20% arises within serrated polyps, which are under-recognized and poorly understood. Human serrated colorectal polyps frequently exhibit both oncogenic BRAF mutation and widespread DNA methylation changes, which are important in silencing genes restraining neoplastic progression. Here, we investigated whether in vivo induction of mutant Braf is sufficient to result in coordinated promoter methylation changes for multiple cancer-related genes. The BrafV637E mutation was induced in murine intestine on an FVB;C57BL/6J background and assessed for morphological and DNA methylation changes at multiple time points from 10 days to 14 months. Extensive intestinal hyperplasia developed by 10 days post-induction of the mutation. By 8 months, most mice had murine serrated adenomas with dysplasia and invasive cancer developed in 40% of mice by 14 months. From 5 months onwards, Braf mutant mice showed extensive, gene-specific increases in DNA methylation even in hyperplastic mucosa without lesions. This demonstrates that persistent oncogenic Braf signaling is sufficient to induce widespread DNA methylation changes. This occurs over an extended period of time, mimicking the long latency followed by rapid progression of human serrated neoplasia. This study establishes for the first time that DNA methylation arises slowly in direct response to prolonged oncogenic Braf signaling in serrated polyps; this finding has implications both for chemoprevention and for understanding the origin of DNA hypermethylation in cancer generally.

International audience; Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is an autosomal-dominant cancer-predisposition syndrome with a significant risk of gastric, but not colorectal, adenocarcinoma. We mapped the gene to 5q22 and found loss of the wild-type allele on 5q in fundic gland polyps from affected individuals. Whole-exome and -genome sequencing failed to find causal mutations but, through Sanger sequencing, we identified point mutations in APC promoter 1B that co-segregated with disease in all six families. The mutations reduced binding of the YY1 transcription factor and impaired activity of the APC promoter 1B in luciferase assays. Analysis of blood and saliva from carriers showed allelic imbalance of APC, suggesting that these mutations lead to decreased allele-specific expression in vivo. Similar mutations in APC promoter 1B occur in rare families with familial adenomatous polyposis (FAP). Promoter 1A is methylated in GAPPS and sporadic FGPs and in normal stomach, which suggests that 1B transcripts are more important than 1A in gastric mucosa. This might explain why all known GAPPS-affected families carry promoter 1B point mutations but only rare FAP-affected families carry similar mutations, the colonic cells usually being protected by the expression of the 1A isoform. Gastric polyposis and cancer have been previously described in some FAP-affected individuals with large deletions around promoter 1B. Our finding that GAPPS is caused by point mutations in the same promoter suggests that families with mutations affecting the promoter 1B are at risk of gastric adenocarcinoma, regardless of whether or not colorectal polyps are present.

Introduction Cancer-associated fibroblasts (CAFs), a heterogeneous component of the tumor microenvironment, substantially influence tumor progression. Bone morphogenetic proteins (BMP) play a critical part in defining the intestinal epithelial niche and either promote or retard cancer progression in a context-dependent manner. However, the role of BMP signaling in the colorectal cancer (CRC) stroma remains to be fully elucidated. This study investigated the significance of mesenchymal BMP signaling as a potential therapeutic target in CRC progression. Design Using CRC expression array data, we identified two CAF-specific factors involved in BMP signaling, then verified their upregulation in the human CRC stroma by in-situ hybridization (ISH). We took advantage of a preclinical mouse model of CRC hepatic metastasis to test approaches targeting the BMP signaling pathway. Results CRC microarray data identified GREM1 and ISLR as CAF-specific genes involved in BMP signaling. In colonic myofibroblasts, Grem1-overexpression inhibited BMP signaling whereas BMP7 signaling was augmented by Islr overexpression, suggesting opposing roles for GREM1 and ISLR in the regulation of BMP signaling. ISH using human rectal cancer samples revealed that GREM1 and ISLR were expressed in distinct CAF subpopulations and that GREM1 and ISLR expression predicted poor and favorable survival, respectively. Notably, Grem1 and Islr expression was differentially regulated by Foxl1, an intestinal mesenchyme-lineage transcription factor, and TGF-b, indicating a mechanism for generating fibroblast heterogeneity. Finally, adeno-associated virus 8-mediated in-vivo overexpression of Islr in hepatocytes retarded growth and generated more differentiated histology in CRC hepatic metastases. Conclusion These data suggest that increased stromal BMP signaling may ameliorate CRC progression and provide a rationale for targeting stromal BMP signaling to inhibit CRC progression and metastasis. Citation Format: Hiroki Kobayashi, Krystyna A. Gieniec, Tongtong Wang, Josephine A. Wright, Nobumi Suzuki, Tamsin RM Lannagan, Yoku Hayakawa, Simon J. Leedham, Nicholas Arpaia, Siddhartha Mukherjee, Timothy C. Wang, Atsushi Enomoto, Masahide Takahashi, Daniel L. Worthley, Susan L. Woods. Stromal BMP signaling imbalance mediated by GREM1 and ISLR regulates colorectal cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3977.

The mechanisms that regulate the induction of term or preterm delivery (PTD) are not fully understood. Infection is known to play a role in the induction of pro-inflammatory cascades in uteroplacental tissues associated with preterm pathological parturition. Similar but not identical cascades are evident in term labour. In the current study, we used a mouse model to evaluate the role of prokineticins in term and preterm parturition. Prokineticins are multi-functioning secreted proteins that signal through G-protein-coupled receptors to induce gene expression, including genes important in inflammatory responses. Expression of prokineticins (Prok1andProk2) was quantified in murine uteroplacental tissues by QPCR in the days preceding labour (days 16–19).Prok1mRNA expression increased significantly on D18 in fetal membranes (compared with D16) but not in uterus or placenta. Intrauterine injection of PROK1 on D17 induced fetal membrane mRNA expression of the pro-inflammatory mediatorsIl6,Il1b,Tnf,Cxcl2andCxcl5, which are not normally up-regulated until D19 of pregnancy. However, intrauterine injection of PROK1 did not result in PTD. As expected, injection of lipopolysaccharide (LPS) induced PTD, but this was not associated with changes in expression ofProk1or its receptor (Prokr1) in fetal membranes. These results suggest that althoughProk1exhibits dynamic mRNA regulation in fetal membranes preceding labour and induces a pro-inflammatory response when injected into the uterus on D17, it is insufficient to induce PTD. Additionally, prokineticin up-regulation appears not to be part of the LPS-induced inflammatory response in mouse fetal membranes.

All developing and adult organs are supported by connective tissues. We recently demonstrated that Gremlin 1 expressing cells in the bone (osteochondroreticular stem cells) and the bowel (intestinal reticular stem cells) are connective tissue stem cells, during development and healing. The contribution of these stem cells to the desmoplasia surrounding gastrointestinal and skeletal cancers, however, is unknown. In this study we first established that typical markers of bone marrow skeletal stem cells, also identify colony forming unit-fibroblasts (CFU-Fs) in the tumour microenvironment (identified by CD45-Ter-119-CD31-CD1040a+CD105+). Next we tested the local origins of alpha-smooth muscle actin (Acta2) expressing cancer-associated fibroblasts in orthotopic (colonoscopically delivered MC38 and carcinogenesis AOM/DSS) mouse models of colorectal cancer. Using transgenic mouse models to lineage trace and report the connective tissues in the bone and bowel, including Grem1-creERT;R26-LSL-ZsGreen; Acta2-RFP and our Acta2-CreERT line, we found that normal Grem1-expressing and Acta2-expressing cells each contribute to some, but not all, of the reactive cancer-associated fibroblasts surrounding our mouse models of cancer. Whilst, neoplastic cells appear to make a significant contribution to cancer-associated fibroblasts in some other cancers, using an epithelial specific Cre (K19-cre) there was no contribution of epithelium to Acta2-expressing cells in our AOM-DSS colorectal cancer models. We investigated Grem1 and Acta2 derived cells from the tumor microenvironment and found that these cells were clonagenic. Finally, we compared their capacity to support the in vitro growth of colorectal normal and neoplastic organoids compared to other colonic mesenchymal cell types. We are currently examining the role of these cells on expanding intestinal stem cells in normal and neoplastic gastrointestinal glands and examining the secreted factors from these cells that are relevant to tumor initiation and spread. Citation Format: Tamsin Lannagan, Susan Woods, Laura Vrbanac, Miao Yang, Jia Ng, Tongtong Wang, Yagnesh Tailor, Samuel Asfaha, Timothy Wang, Daniel L. Worthley. Desmoplasia stem and progenitor cells within the tumor microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1721.

The onset of labour is associated with inflammatory cascades in the uteroplacental unit, and these are prematurely activated in preterm labour. Infection can induce an inflammatory response, which results in premature onset of the pro-inflammatory parturient cascades and preterm delivery. We have become interested in two proteins called prokineticins and the role they may play in labour and its premature onset. Prokineticin 1 and 2 bind to two G-protein coupled receptors, called prokineticin receptor 1 and 2. Expression of the prokineticins and their receptors is elevated in the uteroplacental unit during labour and they can induce expression of a host of genes known to be important in initiating the inflammatory and contractile events of labour. Prokineticins have also been shown to directly induce contractility of smooth muscles. Analysing the promoter regions of the prokineticins and their receptors highlights their potential regulation by pathways activated by infectious agents. Hence, we propose that infection can result in premature activation of prokineticin expression and signalling in the uteroplacental unit and this initiates a premature inflammatory and contractile cascade leading to preterm birth. Antagonism of prokineticin action may provide a suitable therapy for preterm labour that targets both inflammation and contractile pathways.