Stromal R-spondin orchestrates gastric epithelial stem cells and gland homeostasis

Wilhelm F., Simon E., Böger C., Behrens H., Krüger S., Röcken C.

Abstract Recently, it was shown that leucine-rich repeat-containing receptor 5 (LGR5)–expressing stem cells are the cellular origin of intestinal-type gastric cancer. The aim of our study was to uncover regulatory mechanisms of LGR5 expression in gastric mucosa and their implications for cancer development. Reporter assays identified an LGR5 promoter fragment, which is highly relevant for active LGR5 expression. Chromatin immunoprecipitation verified that SP1 is bound within this region, and reporter activity increased in SP1 transfected cells. Subsequently, the expression of R-spondins (RSPO1 and RSPO2), ligands of LGR5, was explored in neoplastic and nonneoplastic gastric tissue and gastric cancer cell lines. Using IHC, distinct spatial expression patterns of LGR5, RSPO1, and RSPO2 were found in nonneoplastic stomach mucosa and gastric cancer. RSPO expression was lower in gastric cancer compared with nonneoplastic mucosa on both the transcriptional (P = 0.003 for RSPO1 and P = 0.000 for RSPO2; n = 50) and the translational level. Methylation-specific PCR showed higher methylation levels of RSPO1/2 and reexpression of RSPOs in the gastric cancer cell lines MKN45 and MKN74 were induced by demethylating 5-aza-C treatment. Finally, expression patterns of LGR5 and RSPO were similar in gastric cancer. Implications: This report identifies a regulatory mechanism of LGR5 expression in gastric carcinogenesis, with SP1 as an important component of the transcriptional complex and LGR5 activity, which is modulated by its ligands RSPO1 and RSPO2, whose expression is modulated by methylation. Visual Overview: http://mcr.aacrjournals.org/content/15/6/776/F1.large.jpg. Mol Cancer Res; 15(6); 776–85. ©2017 AACR.

Matsuo J., Kimura S., Yamamura A., Koh C.P., Hossain M.Z., Heng D.L., Kohu K., Voon D.C., Hiai H., Unno M., So J.B., Zhu F., Srivastava S., Teh M., Yeoh K.G., et. al.

Little is known about the mechanisms of gastric carcinogenesis, partly because it has been a challenge to identify characterize gastric stem cells. Runx genes regulate development and their products are transcription factors associated with cancer development. A Runx1 enhancer element, eR1, is a marker of hematopoietic stem cells. We studied expression from eR1 in the stomach and the roles of gastric stem cells in gastric carcinogenesis in transgenic mice.We used in situ hybridization and immunofluorescence analyses to study expression of Runx1 in gastric tissues from C57BL/6 (control) mice. We then created mice that expressed enhanced green fluorescent protein (EGFP) or CreERT2 under the control of eR1 (eR1-CreERT2;Rosa-Lox-Stop-Lox [LSL]-tdTomato, eR1-CreERT2;Rosa-LSL-EYFP mice). Gastric tissues were collected and lineage-tracing experiments were performed. Gastric organoids were cultured from eR1-CreERT2(5-2);Rosa-LSL-tdTomato mice and immunofluorescence analyses were performed. We investigated the effects of expressing oncogenic mutations in stem cells under control of eR1 using eR1-CreERT2;LSL-KrasG12D/+ mice; gastric tissues were collected and analyzed by histology and immunofluorescence.Most proliferation occurred in the isthmus; 86% of proliferating cells were RUNX1-positive and 76% were MUC5AC-positive. In eR1-EGFP mice, EGFP signals were detected mainly in the upper part of the gastric unit, and 83% of EGFP-positive cells were located in the isthmus/pit region. We found that eR1 marked undifferentiated stem cells in the isthmus and a smaller number of terminally differentiated chief cells at the base. eR1 also marked cells in the pyloric gland in the antrum. Lineage-tracing experiments demonstrated that stem cells in the isthmus and antrum continuously gave rise to mature cells to maintain the gastric unit. eR1-positive cells in the isthmus and pyloric gland generated organoid cultures in vitro. In eR1-CreERT2;LSL-Kras G12D/+ mice, MUC5AC-positive cells rapidly differentiated from stem cells in the isthmus, resulting in distinct metaplastic lesions similar to that observed in human gastric atrophy.Using lineage-tracing experiments in mice, we found that a Runx1 enhancer element, eR1, promotes its expression in the isthmus stem cells of stomach corpus as well as pyloric gland in the antrum. We were able to use eR1 to express oncogenic mutations in gastric stem cells, proving a new model for studies of gastric carcinogenesis.

Sigal M., Rothenberg M.E., Logan C.Y., Lee J.Y., Honaker R.W., Cooper R.L., Passarelli B., Camorlinga M., Bouley D.M., Alvarez G., Nusse R., Torres J., Amieva M.R.

Background & Aims Helicobacter pylori infection is the main risk factor for gastric cancer. We characterized the interactions of H pylori with gastric epithelial progenitor and stem cells in humans and mice and investigated how these interactions contribute to H pylori–induced pathology. Methods We used quantitative confocal microscopy and 3-dimensional reconstruction of entire gastric glands to determine the localizations of H pylori in stomach tissues from humans and infected mice. Using lineage tracing to mark cells derived from leucine-rich repeat-containing G-protein coupled receptor 5–positive (Lgr5+) stem cells (Lgr5-eGFP-IRES-CreERT2/Rosa26-TdTomato mice) and in situ hybridization, we analyzed gastric stem cell responses to infection. Isogenic H pylori mutants were used to determine the role of specific virulence factors in stem cell activation and pathology. Results H pylori grow as distinct bacterial microcolonies deep in the stomach glands and interact directly with gastric progenitor and stem cells in tissues from mice and humans. These gland-associated bacteria activate stem cells, increasing the number of stem cells, accelerating Lgr5+ stem cell proliferation, and up-regulating expression of stem cell–related genes. Mutant bacteria with defects in chemotaxis that are able to colonize the stomach surface but not the antral glands in mice do not activate stem cells. In addition, bacteria that are unable to inject the contact-dependent virulence factor CagA into the epithelium colonized stomach glands in mice, but did not activate stem cells or produce hyperplasia to the same extent as wild-type H pylori. Conclusions H pylori colonize and manipulate the progenitor and stem cell compartments, which alters turnover kinetics and glandular hyperplasia. Bacterial ability to alter the stem cells has important implications for gastrointestinal stem cell biology and H pylori–induced gastric pathology.

Tomasetti C., Vogelstein B.

Crunching the numbers to explain cancer Why do some tissues give rise to cancer in humans a million times more frequently than others? Tomasetti and Vogelstein conclude that these differences can be explained by the number of stem cell divisions. By plotting the lifetime incidence of various cancers against the estimated number of normal stem cell divisions in the corresponding tissues over a lifetime, they found a strong correlation extending over five orders of magnitude. This suggests that random errors occurring during DNA replication in normal stem cells are a major contributing factor in cancer development. Remarkably, this “bad luck” component explains a far greater number of cancers than do hereditary and environmental factors. Science , this issue p. 78

Schlaermann P., Toelle B., Berger H., Schmidt S.C., Glanemann M., Ordemann J., Bartfeld S., Mollenkopf H.J., Meyer T.F.

Background and aims Helicobacter pylori is the causative agent of gastric diseases and the main risk factor in the development of gastric adenocarcinoma. In vitro studies with this bacterial pathogen largely rely on the use of transformed cell lines as infection model. However, this approach is intrinsically artificial and especially inappropriate when it comes to investigating the mechanisms of cancerogenesis. Moreover, common cell lines are often defective in crucial signalling pathways relevant to infection and cancer. A long-lived primary cell system would be preferable in order to better approximate the human in vivo situation.Methods Gastric glands were isolated from healthy human stomach tissue and grown in Matrigel containing media supplemented with various growth factors, developmental regulators and apoptosis inhibitors to generate long-lasting normal epithelial cell cultures.Results Culture conditions were developed which support the formation and quasi-indefinite growth of three dimensional (3D) spheroids derived from various sites of the human stomach. Spheroids could be differentiated to gastric organoids after withdrawal of Wnt3A and R-spondin1 from the medium. The 3D cultures exhibit typical morphological features of human stomach tissue. Transfer of sheared spheroids into 2D culture led to the formation of dense planar cultures of polarised epithelial cells serving as a suitable in vitro model of H. pylori infection.Conclusions A robust and quasi-immortal 3D organoid model has been established, which is considered instrumental for future research aimed to understand the underlying mechanisms of infection, mucosal immunity and cancer of the human stomach.

Clevers H., Loh K.M., Nusse R.

Controlling stem cells and their niches Adult organs such as the intestines and skin continually renew themselves every few days or weeks. In several mammalian tissues, this renewal relies on Wnt signaling. Clevers et al. review this crucial role in stem cell self renewal. Wnt plays a pivotal role in tissue regeneration even in the earliest animals. Wnt proteins function mainly as short-range signals between adjacent cells. The short-range, spatially-constrained nature of Wnt signals underpins mammalian stem cell niche architecture and tissue self-organization. Science , this issue 10.1126/science.1248012

Herring B., Hoggatt A.M., Burlak C., Offermanns S.

Hayakawa Y., Jin G., Wang H., Chen X., Westphalen C.B., Asfaha S., Renz B.W., Ariyama H., Dubeykovskaya Z.A., Takemoto Y., Lee Y., Muley A., Tailor Y., Chen D., Muthupalani S., et. al.

Objective Progastrin is the incompletely cleaved precursor of gastrin that is secreted by G-cells in the gastric antrum. Both gastrin and progastrin bind to the CCK2 receptor (Cckbr or CCK2R) expressed on a subset of gastric epithelial cells. Little is known about how gastrin peptides and CCK2R regulate gastric stem cells and carcinogenesis. Interconversion among progenitors in the intestine is documented, but the mechanisms by which this occurs are poorly defined.Design We generated CCK2R-CreERT mice and performed inducible lineage tracing experiments. CCK2R+ antral cells and Lgr5+ antral stem cells were cultured in a three-dimensional in vitro system. We crossed progastrin-overexpressing mice with Lgr5-GFP-CreERT mice and examined the role of progastrin and CCK2R in Lgr5+ stem cells during MNU-induced carcinogenesis.Results Through lineage tracing experiments, we found that CCK2R defines antral stem cells at position +4, which overlapped with an Lgr5neg or low cell population but was distinct from typical antral Lgr5high stem cells. Treatment with progastrin interconverts Lgr5neg or low CCK2R+ cells into Lgr5high cells, increases CCK2R+ cell numbers and promotes gland fission and carcinogenesis in response to the chemical carcinogen MNU. Pharmacological inhibition or genetic ablation of CCK2R attenuated progastrin-dependent stem cell expansion and carcinogenesis.Conclusions CCK2R labels +4 antral stem cells that can be activated and expanded by progastrin, thus identifying one hormonal trigger for gastric stem cell interconversion and a potential target for gastric cancer chemoprevention and therapy.

Leushacke M., Ng A., Galle J., Loeffler M., Barker N.

Summary The pyloric epithelium continuously self-renews throughout life, driven by limited reservoirs of resident Lgr5+ adult stem cells. Here, we characterize the population dynamics of these stem cells during epithelial homeostasis. Using a clonal fate-mapping strategy, we demonstrate that multiple Lgr5+ cells routinely contribute to epithelial renewal in the pyloric gland and, similar to what was previously observed in the intestine, a balanced homeostasis of the glandular epithelium and stem cell pools is predominantly achieved via neutral competition between symmetrically dividing Lgr5+ stem cells. Additionally, we document a lateral expansion of stem cell clones via gland fission under nondamage conditions. These findings represent a major advance in our basic understanding of tissue homeostasis in the stomach and form the foundation for identifying altered stem cell behavior during gastric disease.

Stange D., Koo B., Huch M., Sibbel G., Basak O., Lyubimova A., Kujala P., Bartfeld S., Koster J., Geahlen J., Peters P., van Es J., van de Wetering M., Mills J., Clevers H.

Proliferation of the self-renewing epithelium of the gastric corpus occurs almost exclusively in the isthmus of the glands, from where cells migrate bidirectionally toward pit and base. The isthmus is therefore generally viewed as the stem cell zone. We find that the stem cell marker Troy is expressed at the gland base by a small subpopulation of fully differentiated chief cells. By lineage tracing with a Troy-eGFP-ires-CreERT2 allele, single marked chief cells are shown to generate entirely labeled gastric units over periods of months. This phenomenon accelerates upon tissue damage. Troy(+) chief cells can be cultured to generate long-lived gastric organoids. Troy marks a specific subset of chief cells that display plasticity in that they are capable of replenishing entire gastric units, essentially serving as quiescent "reserve" stem cells. These observations challenge the notion that stem cell hierarchies represent a "one-way street."

van Amerongen R., Bowman A., Nusse R.

The mammary epithelium undergoes extensive growth and remodeling during pregnancy, suggesting a role for stem cells. Yet their origin, identity, and behavior in the intact tissue remain unknown. Using an Axin2(CreERT2) allele, we labeled and traced Wnt/β-catenin-responsive cells throughout mammary gland development. This reveals a switch in Wnt/β-catenin signaling around birth and shows that, depending on the developmental stage, Axin2(+) cells contribute differently to basal and luminal epithelial cell lineages of the mammary epithelium. Moreover, an important difference exists between the developmental potential tested in transplantation assays and that displayed by the same cell population in situ. Finally, Axin2(+) cells in the adult build alveolar structures during multiple pregnancies, demonstrating the existence of a Wnt/β-catenin-responsive adult stem cell. Our study uncovers dynamic changes in Wnt/β-catenin signaling in the mammary epithelium and offers insights into the developmental fate of mammary gland stem and progenitor cells.

Seshagiri S., Stawiski E.W., Durinck S., Modrusan Z., Storm E.E., Conboy C.B., Chaudhuri S., Guan Y., Janakiraman V., Jaiswal B.S., Guillory J., Ha C., Dijkgraaf G.J., Stinson J., Gnad F., et. al.

Exomes, transcriptomes and copy-number alterations in a sample of more than 70 primary human colonic tumours were analysed in an attempt to characterize the genomic landscape; in addition to finding alterations in genes associated with commonly mutated signalling pathways, recurrent gene fusions involving R-spondin family members were also found to occur in approximately 10% of colonic tumours, revealing a potential new therapeutic target. An analysis of exomes, transcriptomes and copy-number alterations in more than 70 primary human colon tumours and matched normal controls has identified more than 35,000 protein-altering somatic mutations, most of which have been validated. In addition to alterations in genes involved in the Wnt pathway, chromatin remodelling and receptor-tyrosine-kinase signalling, the authors identify recurrent gene fusions involving R-spondin family members that collectively occur in 10% of colon tumors; as such they may provide a potential therapeutic target. There is evidence to suggest that these fusions may have a role in the activation of Wnt signalling and tumorigenesis. Identifying and understanding changes in cancer genomes is essential for the development of targeted therapeutics1. Here we analyse systematically more than 70 pairs of primary human colon tumours by applying next-generation sequencing to characterize their exomes, transcriptomes and copy-number alterations. We have identified 36,303 protein-altering somatic changes that include several new recurrent mutations in the Wnt pathway gene TCF7L2, chromatin-remodelling genes such as TET2 and TET3 and receptor tyrosine kinases including ERBB3. Our analysis for significantly mutated cancer genes identified 23 candidates, including the cell cycle checkpoint kinase ATM. Copy-number and RNA-seq data analysis identified amplifications and corresponding overexpression of IGF2 in a subset of colon tumours. Furthermore, using RNA-seq data we identified multiple fusion transcripts including recurrent gene fusions involving R-spondin family members RSPO2 and RSPO3 that together occur in 10% of colon tumours. The RSPO fusions were mutually exclusive with APC mutations, indicating that they probably have a role in the activation of Wnt signalling and tumorigenesis. Consistent with this we show that the RSPO fusion proteins were capable of potentiating Wnt signalling. The R-spondin gene fusions and several other gene mutations identified in this study provide new potential opportunities for therapeutic intervention in colon cancer.

Koo B., Spit M., Jordens I., Low T.Y., Stange D.E., van de Wetering M., van Es J.H., Mohammed S., Heck A.J., Maurice M.M., Clevers H.

In vivo and in vitro studies show that the stem-cell E3 ubiquitin ligases RNF43 and ZNRF3 act as tumour suppressors in colorectal cancer models, and are involved in the negative regulation of the cancer-associated Wnt signalling pathway through limiting the cell-surface expression of Wnt receptors. Wnt signalling is critical for the function of intestinal stem cells; it also drives colorectal tumorigenesis. Bon-Kyoung Koo et al. find that two targets of Wnt signalling, the E3 ligases RNF43 and ZNFR3, are also important negative-feedback regulators of Wnt signalling. They act by limiting the cell-surface expression of Wnt receptors. Deletion of both genes in the mouse intestine leads to expansion of LGR5+ intestinal stem cells and the development of adenomas. Furthermore, in human colon cancer cells, the expression of RNF43 reduces Wnt signalling. Mutated RNF43 has been found in human colorectal cancers, indicating that Wnt-pathway inhibitors that act at the level of Wnt secretion or Wnt-receptor activation may have therapeutic potential. LGR5+ stem cells reside at crypt bottoms, intermingled with Paneth cells that provide Wnt, Notch and epidermal growth factor signals1. Here we find that the related RNF43 and ZNRF3 transmembrane E3 ubiquitin ligases are uniquely expressed in LGR5+ stem cells. Simultaneous deletion of the two genes encoding these proteins in the intestinal epithelium of mice induces rapidly growing adenomas containing high numbers of Paneth and LGR5+ stem cells. In vitro, growth of organoids derived from these adenomas is arrested when Wnt secretion is inhibited, indicating a dependence of the adenoma stem cells on Wnt produced by adenoma Paneth cells. In the HEK293T human cancer cell line, expression of RNF43 blocks Wnt responses and targets surface-expressed frizzled receptors to lysosomes. In the RNF43-mutant colorectal cancer cell line HCT116, reconstitution of RNF43 expression removes its response to exogenous Wnt. We conclude that RNF43 and ZNRF3 reduce Wnt signals by selectively ubiquitinating frizzled receptors, thereby targeting these Wnt receptors for degradation.

Neufeld S., Rosin J.M., Ambasta A., Hui K., Shaneman V., Crowder R., Vickerman L., Cobb J.

Summary: R-spondins are secreted ligands that bind cell surface receptors and activate Wnt/β-catenin signaling. Human mutations and gene inactivation studies in mice have revealed a role for these four proteins (RSPO1-4) in diverse developmental processes ranging from sex determination to limb development. Among the genes coding for R-spondins, only inactivation of Rspo3 shows early embryonic lethality (E10.5 in mice). Therefore, a conditional allele of this gene is necessary to understand the function of R-spondins throughout murine development. To address this need, we have produced an allele in which loxP sites flank exons 2–4 of Rspo3, allowing tissue-specific deletion of these exons in the presence of Cre recombinase. We used these mice to investigate the role of Rspo3 during limb development and found that limbs ultimately developed normally in the absence of Rspo3 function. However, severe hindlimb truncations resulted when Rspo3 and Rspo2 mutations were combined, demonstrating redundant function of these genes. genesis 50:741–749, 2012. © 2012 Wiley Periodicals, Inc.

Muñoz J., Stange D.E., Schepers A.G., van de Wetering M., Koo B., Itzkovitz S., Volckmann R., Kung K.S., Koster J., Radulescu S., Myant K., Versteeg R., Sansom O.J., van Es J.H., Barker N., et. al.

Two types of stem cells are currently defined in small intestinal crypts: cycling crypt base columnar (CBC) cells and quiescent '+4' cells. Here, we combine transcriptomics with proteomics to define a definitive molecular signature for Lgr5(+) CBC cells. Transcriptional profiling of FACS-sorted Lgr5(+) stem cells and their daughters using two microarray platforms revealed an mRNA stem cell signature of 384 unique genes. Quantitative mass spectrometry on the same cell populations identified 278 proteins enriched in intestinal stem cells. The mRNA and protein data sets showed a high level of correlation and a combined signature of 510 stem cell-enriched genes was defined. Spatial expression patterns were further characterized by mRNA in-situ hybridization, revealing that approximately half of the genes were expressed in a gradient with highest levels at the crypt bottom, while the other half was expressed uniquely in Lgr5(+)stem cells. Lineage tracing using a newly established knock-in mouse for one of the signature genes, Smoc2, confirmed its stem cell specificity. Using this resource, we find-and confirm by independent approaches-that the proposed quiescent/'+4' stem cell markers Bmi1, Tert, Hopx and Lrig1 are robustly expressed in CBC cells.

Yoshihiro T., Yamaguchi K., Ariyama H., Koreishi S., Uehara K., Ohmura H., Ito M., Tsuchihashi K., Isobe T., Shindo K., Ohuchida K., Nakamura M., Nagao Y., Oda Y., Akashi K., et. al.

Kemp L.J., Monster J.L., Wood C.S., Moers M., Vliem M.J., Khalil A.A., Jamieson N.B., Brosens L.A., Kodach L.L., van Dieren J.M., Bisseling T.M., van der Post R.S., Gloerich M.

BackgroundDevelopment of diffuse-type gastric cancer (DGC) starts with intramucosal lesions that are primarily composed of differentiated, non-proliferative signet ring cells (SRCs). These indolent lesions can advance into highly proliferative and metastatic tumours, which requires suppression of DGC cell differentiation.ObjectiveOur goal was to identify molecular changes contributing to the progression of indolent to aggressive DGC lesions.DesignWe conducted spatial transcriptomic analysis of patient tumours at different stages of hereditary DGC, comparing transcriptional differences in tumour cell populations and tumour-associated cells. We performed functional analysis of identified changes in a human gastric (CDH1KO) organoid model recapitulating DGC initiation.ResultsOur analysis reveals that distinct DGC cell populations exhibit varying levels of Wnt-signalling activity, and high levels of Wnt signalling prevent differentiation into SRCs. We identify multiple adaptations during DGC progression that converge on Wnt signalling, allowing tumour cells to remain in an undifferentiated state as they disseminate away from the gastric stem cell niche. First, DGC cells establish a cell-autonomous source for Wnt-pathway activation through upregulated expression of Wnt-ligands and ‘secreted frizzled-related protein 2’ (SFRP2) that potentiates ligand-induced Wnt signalling. Second, early tumour development is marked by extracellular matrix remodelling, including increased deposition of collagen I whose interactions with DGC cells suppress their differentiation in the absence of exogenous Wnt ligands.ConclusionsOur findings demonstrate that tumour cell-derived ligand expression and extracellular matrix remodelling sustain Wnt signalling during DGC progression. These complementary mechanisms promote niche independence enabling expansion of undifferentiated DGC cells needed for the development of advanced tumours.

Cherubini A., Pistoni C., Iachini M.C., Mei C., Rusconi F., Peli V., Barilani M., Tace D., Elia N., Lepore F., Caporale V., Piemonti L., Lazzari L.

Wizenty J., Sigal M.

Wong G.P., Hartmann S., Nonn O., Cannon P., Nguyen T., Kandel M., de Alwis N., Murphy C.N., Pritchard N., Dechend R., Hannan N.J., Tong S., Simmons D.G., Kaitu’u-Lino T.J.

Leucine-rich repeat-containing G protein-coupled receptors 5/4 (LGR5/LGR4) are critical stem cell markers in epithelial tissues including intestine. They agonise wingless-related integration site (WNT) signalling. Until now, LGR5/LGR4 were uncharacterised in placenta, where analogous functions may exist. We characterised LGR5/LGR4, their ligands/targets in human placenta, with further assessments on dysregulation in preeclampsia/fetal growth restriction (FGR). LGR5 mRNA was unaltered in first trimester (n = 11), preterm (n = 9) and term (n = 11) placental lysate. LGR5 was enriched in human trophoblast stem cells (hTSCs) and downregulated with differentiation to extravillous trophoblasts (p < 0.0215) and syncytiotrophoblasts (p < 0.0350). In situ hybridisation localised LGR5 to unique, proliferative MKI67 + mononuclear trophoblasts underlying syncytium which concurred with proposed progenitor identities in single-cell transcriptomics. LGR5 expression was significantly reduced in placentas from early-onset preeclampsia (p < 0.0001, n = 81 versus n = 19 controls), late-onset preeclampsia (p = 0.0046, n = 20 versus n = 33 controls) and FGR (p = 0.0031, n = 34 versus n = 17 controls). LGR4 was elevated in first trimester versus preterm and term placentas (p = 0.0412), in placentas with early-onset preeclampsia (p = 0.0148) and in FGR (p = 0.0417). Transcriptomic analysis and in vitro hTSC differentiation to both trophoblast lineages suggested LGR4 increases with differentiation. Single-nucleus RNA sequencing of placental villous samples supported LGR5 and LGR4 localisation findings. Hypoxia/proinflammatory cytokine treatment modelling elements experienced by the placenta in placental insufficiency pathogenesis did not significantly alter LGR5/LGR4. Ligands R-spondins 1/3/4, and neutralising targets ring finger protein 43 (RNF43) and zinc and ring finger 3 (ZNRF3) were also reduced in placentas from preeclamptic pregnancies. This study is the first to describe LGR5/LGR4 and their signalling partner expression in human placenta. Their dysregulations in the preeclamptic placenta allude to disruptions to integral trophoblast stem cell function/differentiation that may occur during placental development related to WNT signalling.

Liu C., He P., Lu H., Sun Y., Zhang Z., Dong K.

Abstract Background Medical microbiology represents a fundamental core curriculum for medical students. In order to adapt to the cultivation of high-level medical talents under the guidance of the concept of holistic education, the focus of this course is on the development of scientific thinking and professionalism. This paper took the example of Helicobacter pylori, an important pathogen in the digestive system, addressing the teaching design of this chapter in medical microbiology. Teaching design The design of this chapter includes learning objectives, teaching content, teaching model and assessment. The learning objectives were designed under the outcome-based education philosophy, integrating the three dimensions of attitude, skill and knowledge. The content of this chapter is designed to integrate a number of key concepts, including systematic thinking, critical thinking, the One Health perspective, and the mission and responsibility of infectious disease prevention and control. Question-oriented online-and-offline blended teaching model was adopted, and a multi-objective assessment system was applied. Implementation A series of questions were designed before, during, and after the class. These questions served as the primary focus of learning, guiding students through various teaching activities, including online self-study, offline discussion and analysis, and online extended learning. Concurrently, the evaluation is integrated throughout the teaching process, with particular emphasis on formative and process evaluations. Conclusions This teaching design facilitated the achievement of learning objectives and optimized the teaching effect. The students’ self-assessment of their overall improvement in knowledge, skills and attitude, as well as their high level of satisfaction with the classroom environment, is presented herewith. The teaching design of this chapter has a particular demonstrative effect.

Valenzuela-Bezanilla D., Mardones M.D., Galassi M., Arredondo S.B., Santibanez S.H., Gutierrez-Jimenez S., Merino-Véliz N., Bustos F.J., Varela-Nallar L.

Abstract In the dentate gyrus of the adult hippocampus, neurogenesis from neural stem cells (NSCs) is regulated by Wnt signals from the local microenvironment. The Wnt/β-catenin pathway is active in NSCs, where it regulates proliferation and fate commitment, and subsequently its activity is strongly attenuated. The mechanisms controlling Wnt activity are poorly understood. In stem cells from adult peripheral tissues, secreted R-spondin proteins (RSPO1-4) interact with LGR4-6 receptors and control Wnt signaling strength. Here, we found that RSPO1-3 and LGR4-6 are expressed in the adult dentate gyrus and in cultured NSCs isolated from the adult mouse hippocampus. LGR4-5 expression decreased in cultured NSCs upon differentiation, concomitantly with the reported decrease in Wnt activity. Treatment with RSPO1-3 increased NSC proliferation and the expression of Cyclin D1, but did not induce the expression of Axin2 or RNF43, two well-described Wnt target genes. However, RSPOs enhanced the effect of Wnt3a on Axin2 and RNF43 expression, as well as on Wnt/β-catenin reporter activity, indicating that they can potentiate Wnt activity in NSCs. Moreover, RSPO1-3 were found to be expressed by cultured dentate gyrus astrocytes, a crucial component of the neurogenic niche. In co-culture experiments, the astrocyte-induced proliferation of NSCs was prevented by RSPO2 knockdown in astrocytes and LGR5 knockdown in hippocampal NSCs. Additionally, RSPO2 knockdown in the adult mouse dentate gyrus reduced proliferation of neural stem and progenitor cells in vivo. Altogether, our results indicate that RSPO/LGR signaling is present in the dentate gyrus and plays a crucial role in regulating neural precursor cell proliferation.

Mokhtarpour K., Akbarzadehmoallemkolaei M., Rezaei N.

Traditionally, the role of the gut microbiome has been evaluated in relation to diseases and pathologies affecting the gut. However, recent clinical trials have highlighted the significant impact of the microbiome on extraintestinal diseases. Similarly, studies have transitioned from exploring the impacts of the microbiome and the efficacy of methods like fecal microbiota transplantation (FMT) on intestinal diseases to actively modulating the microbiome to influence non-intestinal conditions. Therefore, it is not far-fetched that in this chapter we will examine the impact of the microbiome on the second cause of death, cancer, and in this way put the most important and most up-to-date microbiome modification technique, FMT, under the microscope. Therefore, we will address questions such as how this technique affects the tumor microenvironment (TME), the current position of microbiome elements on cancer, and how to manipulate the gut microbiome to enhance therapeutic responses for different cancer types. In addition, we also collect recent clinical trials and relevant information and discuss safety and side effects to provide a foundation for the development of personalized medicine in further studies.

Mulè P., Fernandez-Perez D., Amato S., Manganaro D., Oldani P., Brandini S., Diaferia G., Cuomo A., Recordati C., Soriani C., Dondi A., Zanotti M., Rustichelli S., Bisso A., Pece S., et. al.

WNT signaling is central to spatial tissue arrangement and regulating stem cell activity, and it represents the hallmark of gastrointestinal cancers. Although its role in driving intestinal tumors is well characterized, WNT's role in gastric tumorigenesis remains elusive.

Cao C., Yue S., Lu A., Liang C.

The critical role of the gut microbiome in gastrointestinal cancers is becoming increasingly clear. Imbalances in the gut microbial community, referred to as dysbiosis, are linked to increased risks for various forms of gastrointestinal cancers. Pathogens like Fusobacterium and Helicobacter pylori relate to the onset of esophageal and gastric cancers, respectively, while microbes such as Porphyromonas gingivalis and Clostridium species have been associated with a higher risk of pancreatic cancer. In colorectal cancer, bacteria such as Fusobacterium nucleatum are known to stimulate the growth of tumor cells and trigger cancer-promoting pathways. On the other hand, beneficial microbes like Bifidobacteria offer a protective effect, potentially inhibiting the development of gastrointestinal cancers. The potential for therapeutic interventions that manipulate the gut microbiome is substantial, including strategies to engineer anti-tumor metabolites and employ microbiota-based treatments. Despite the progress in understanding the influence of the microbiome on gastrointestinal cancers, significant challenges remain in identifying and understanding the precise contributions of specific microbial species and their metabolic products. This knowledge is essential for leveraging the role of the gut microbiome in the development of precise diagnostics and targeted therapies for gastrointestinal cancers.

Lin M., Tu R., Wu S., Zhong Q., Weng K., Wu Y., Lin G., Wang J., Zheng C., Xie J., Lin J., Chen Q., Huang C., Cao L., Li P.

AbstractHelicobacter pylori (HP) infection initiates and promotes gastric carcinogenesis. ONECUT2 shows promise for tumor diagnosis, prognosis, and treatment. This study explored ONECUT2’s role and the specific mechanism underlying HP infection-associated gastric carcinogenesis to suggest a basis for targeting ONECUT2 as a therapeutic strategy for gastric cancer (GC). Multidimensional data supported an association between ONECUT2, HP infection, and GC pathogenesis. HP infection upregulated ONECUT2 transcriptional activity via NFκB. In vitro and in vivo experiments demonstrated that ONECUT2 increased the stemness of GC cells. ONECUT2 was also shown to inhibit PPP2R4 transcription, resulting in reduced PP2A activity, which in turn increased AKT/β-catenin phosphorylation. AKT/β-catenin phosphorylation facilitates β-catenin translocation to the nucleus, initiating transcription of downstream stemness-associated genes in GC cells. HP infection upregulated the reduction of AKT and β-catenin phosphorylation triggered by ONECUT2 downregulation via ONECUT2 induction. Clinical survival analysis indicated that high ONECUT2 expression may indicate poor prognosis in GC. This study highlights a critical role played by ONECUT2 in promoting HP infection-associated GC by enhancing cell stemness through the PPP2R4/AKT/β-catenin signaling pathway. These findings suggest promising therapeutic strategies and potential targets for GC treatment.

Kakiailatu N.J., Kuil L.E., Windster J.D., Bindels E., Zink J.T., Vermeulen M., de Graaf B.M., Sahadew D., van den Bosch T.P., Huijgen D., Sloots C.E., Wijnen R.M., Hofstra R.M., Melotte V., Alves M.M.

AbstractHirschsprung disease (HSCR) is a congenital disorder characterized by the absence of an enteric nervous system (ENS) in the distal gut, causing obstruction and constipation. Despite the known importance of the ENS for normal gut function due to its interaction with other intestinal cells, the impact of ENS loss on intestinal homeostasis remains largely unexplored. In this manuscript, we investigate changes in intestinal composition associated with loss of an ENS, by performing single cell RNA sequencing on zebrafish intestines collected from 5 days post-fertilization wildtype zebrafish and aretmutant HSCR model. Significant findings were validated through immunofluorescence and fluorescencein situhybridization in zebrafish and human tissues. Notable shifts included a reduction in enterocytes and enterochromaffin cells, alongside an increase in immune and endothelial cells, as well as BEST4+ enterocytes, in the HSCR model. These discoveries elucidate significant changes in the intestinal cellular composition in HSCR, highlighting potential pathways to secondary complications and offering insights into new therapeutic possibilities aimed at enhancing patient outcomes.

Hill A.B., Murphy Y.M., Polkoff K.M., Edwards L., Walker D.M., Moatti A., Greenbaum A., Piedrahita J.A.

Recent advancements in genome editing techniques, notably CRISPR-Cas9 and TALENs, have marked a transformative era in biomedical research, significantly enhancing our understanding of disease mechanisms and helping develop novel therapies. These technologies have been instrumental in creating precise animal models for use in stem cell research and regenerative medicine. For instance, we have developed a transgenic pig model to enable the investigation of LGR5-expressing cells. The model was designed to induce the expression of H2B-GFP under the regulatory control of the LGR5 promoter via CRISPR/Cas9-mediated gene knock-in. Notably, advancements in stem cell research have identified distinct subpopulations of LGR5-expressing cells within adult human, mouse, and pig tissues. LGR5, a leucine-rich repeat-containing G protein-coupled receptor, enhances WNT signaling and these LGR5+ subpopulations demonstrate varied roles and anatomical distributions, underscoring the necessity for suitable translational models. This transgenic pig model facilitates the tracking of LGR5-expressing cells and has provided valuable insights into the roles of these cells across different tissues and species. For instance, in pulmonary tissue, Lgr5+ cells in mice are predominantly located in alveolar compartments, driving alveolar differentiation of epithelial progenitors via Wnt pathway activation. In contrast, in pigs and humans, these cells are situated in a unique sub-basal position adjacent to the airway epithelium. In fetal stages a pattern of LGR5 expression during lung bud tip formation is evident in humans and pigs but is lacking in mice. Species differences with respect to LGR5 expression have also been observed in the skin, intestines, and cochlea further reinforcing the need for careful selection of appropriate translational animal models. This paper discusses the potential utility of the LGR5+ pig model in exploring the role of LGR5+ cells in tissue development and regeneration with the goal of translating these findings into human and animal clinical applications.

Kumar N., Prakash P.G., Wentland C., Kurian S.M., Jethva G., Brinkmann V., Mollenkopf H., Krammer T., Toussaint C., Saliba A., Biebl M., Jürgensen C., Wiedenmann B., Meyer T.F., Gurumurthy R.K., et. al.

AbstractThe gastroesophageal squamocolumnar junction (GE-SCJ) is a critical tissue interface between the esophagus and stomach, with significant relevance in the pathophysiology of gastrointestinal diseases. Despite this, the molecular mechanisms underlying GE-SCJ development remain unclear. Using single-cell transcriptomics, organoids, and spatial analysis, we examine the cellular heterogeneity and spatiotemporal dynamics of GE-SCJ development from embryonic to adult mice. We identify distinct transcriptional states and signaling pathways in the epithelial and mesenchymal compartments of the esophagus and stomach during development. Fibroblast-epithelial interactions are mediated by various signaling pathways, including WNT, BMP, TGF-β, FGF, EGF, and PDGF. Our results suggest that fibroblasts predominantly send FGF and TGF-β signals to the epithelia, while epithelial cells mainly send PDGF and EGF signals to fibroblasts. We observe differences in the ligands and receptors involved in cell-cell communication between the esophagus and stomach. Our findings provide insights into the molecular mechanisms underlying GE-SCJ development and fibroblast-epithelial crosstalk involved, paving the way to elucidate mechanisms during adaptive metaplasia development and carcinogenesis.

Rao X., Zhang Z., Pu Y., Han G., Gong H., Hu H., Ji Q., Liu N.

AbstractBackgroundHelicobacter pylori (H. pylori) accounts for the majority of gastric cancer (GC) cases globally. The present study found that H. pylori promoted GC stem cell (CSC)‐like properties, therefore, the regulatory mechanism of how H. pylori promotes GC stemness was explored.MethodsSpheroid‐formation experiments were performed to explore the self‐renewal capacity of GC cells. The expression of R‐spondin 3 (RSPO3), Nanog homeobox, organic cation/carnitine transporter‐4 (OCT‐4), SRY‐box transcription factor 2 (SOX‐2), CD44, Akt, glycogen synthase kinase‐3β (GSK‐3β), p‐Akt, p‐GSK‐3β, β‐catenin, and G protein subunit gamma 7 (GNG7) were detected by RT‐qPCR, western blotting, immunohistochemistry (IHC), and immunofluorescence. Co‐immunoprecipitation (CoIP) and liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) were performed to identify proteins interacting with RSPO3. Lentivirus‐based RNA interference constructed short hairpin (sh)‐RSPO3 GC cells. Small interfering RNA transfection was performed to inhibit GNG7. The in vivo mechanism was verified using a tumor peritoneal seeding model in nude mice.ResultsH. pylori extracts promoted a CSC‐like phenotype in GC cells and elevated the expression of RSPO3. RSPO3 knockdown significantly reduced the CSC‐like properties induced by H. pylori. Previous studies have demonstrated that RSPO3 potentiates the Wnt/β‐catenin signaling pathway, but the inhibitor of Wnt cannot diminish the RSPO3‐induced activation of β‐catenin. CoIP and LC–MS/MS revealed that GNG7 is one of the transmembrane proteins interacting with RSPO3, and it was confirmed that RSPO3 directly interacted with GNG7. Recombinant RSPO3 protein increased the phosphorylation level of Akt and GSK‐3β, and the expression of β‐catenin in GC cells, but this regulatory effect of RSPO3 could be blocked by GNG7 knockdown. Of note, GNG7 suppression could diminish the promoting effect of RSPO3 to CSC‐like properties. In addition, RSPO3 suppression inhibited MKN45 tumor peritoneal seeding in vivo. IHC staining also showed that RSPO3, CD44, OCT‐4, and SOX‐2 were elevated in H. pylori GC tissues.ConclusionRSPO3 enhanced the stemness of H. pylori extracts‐infected GC cells through the GNG7/β‐catenin signaling pathway.