Identifying and characterising gastric stem cells and their role in H. pylori-induced stomach cancer

Project Overview

There is increasing evidence that bacterial infections are promoters or co-factors of human cancers. Strong epidemiological associations have been reported especially for  Helicobacter pylori that is considered to be responsible of 70% of the gastric malignancies (Boccellato and Meyer 2015). To figure causal relationships between an infection and cancer onset is difficult, mainly because of the long time span between the two events. Stronger connections to cancers might be delineated by understanding the regenerative capacity of gastric stem cells upon infection. If cancer arises at the site of infection, the cancer cell should derive from an infected cell with regenerative capacity. Epithelial cells are the primary target of H.pylori infection and the bacterium has an extraordinary capacity to disrupt epithelial barrier integrity by cleaving cellular junctions (Backert, Bernegger et al. 2018) and subverting apical basal polarity of epithelial cells (Bagnoli, Buti et al. 2005). As a consequence of the damage caused by the infection the epithelium undergo an intense regenerative program fuelled by stem cells (Sigal, Rothenberg et al. 2015). However, how these stem cells are activated and if they play a role in disease progression into cancer remain poorly understood.

 

We have recently developed a marker that enables stem cells to be differentiated from non-stem cell populations and a fluorescent reporter designed to enable the isolation of live stem cells from dissociated tissues or organoids. Moreover we have also harnessed air-liquid interface (ALI) methodology to establish functional, long-lived, epithelial polarised monolayers from healthy human stomachs, which fully recapitulate the different cell lineages found in situ, and accumulate mucins on the apical side. This ‘mucosoid’ culture system recapitulates the in vivo gastric epithelial layer and permits advanced studies of the bacterial sensing mechanisms and epithelial inflammation (Boccellato, Woelffling et al. 2018).

 

 In this project, we will use a combination of human and mouse tissue as well as organoid and mucosoid cultures to detect and characterise gastric stem cell populations and the impact of H. pylori infection on their activation, capacity to regenerate the gastric epithelium, and their contribution to cancer progression.

Training Opportunities

The project will provide training in a wide range of advanced 3D culture systems, flow cytometry and cell sorting as well as real-time live cell imaging, and will also provide opportunities for single cell analysis of gastric stem cells. All students at the Ludwig Institute are provided with training in all aspects of scientific development and are integrated into a highly interdisciplinary and collaborative environment.

Theme

Genetics & Genomics

Admissions

Project reference number: 999

Funding and admissions information

Supervisors

Name Department Institution Country Email
Dr Francesco Boccellato Oxford Ludwig Institute Oxford University, Old Road Campus Research Building GBR francesco.boccellato@ludwig.ox.ac.uk
Professor Colin R Goding Oxford Ludwig Institute Oxford University, Old Road Campus Research Building GBR colin.goding@ludwig.ox.ac.uk

Sigal M, Rothenberg ME, Logan CY, Lee JY, Honaker RW, Cooper RL, Passarelli B, Camorlinga M, Bouley DM, Alvarez G, Nusse R, Torres J, Amieva MR. 2015. Helicobacter pylori Activates and Expands Lgr5(+) Stem Cells Through Direct Colonization of the Gastric Glands. Gastroenterology, 148 (7), pp. 1392-404.e21. Read abstract | Read more

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. Hide abstract

Boccellato F, Woelffling S, Imai-Matsushima A, Sanchez G, Goosmann C, Schmid M, Berger H, Morey P, Denecke C, Ordemann J, Meyer TF. 2018. Polarised epithelial monolayers of the gastric mucosa reveal insights into mucosal homeostasis and defence against infection. Gut, Read abstract | Read more

OBJECTIVE: causes life-long colonisation of the gastric mucosa, leading to chronic inflammation with increased risk of gastric cancer. Research on the pathogenesis of this infection would strongly benefit from an authentic human in vitro model. DESIGN: Antrum-derived gastric glands from surgery specimens served to establish polarised epithelial monolayers via a transient air-liquid interface culture stage to study cross-talk with and the adjacent stroma. RESULTS: The resulting 'mucosoid cultures', so named because they recapitulate key characteristics of the gastric mucosa, represent normal stem cell-driven cultures that can be passaged for months. These highly polarised columnar epithelial layers encompass the various gastric antral cell types and secrete mucus at the apical surface. By default, they differentiate towards a foveolar, MUC5AC-producing phenotype, whereas Wnt signalling stimulates proliferation of MUC6-producing cells and preserves stemness-reminiscent of the gland base. Stromal cells from the lamina propria secrete Wnt inhibitors, antagonising stem-cell niche signalling and inducing differentiation. On infection with , a strong inflammatory response is induced preferentially in the undifferentiated basal cell phenotype. Infection of cultures for several weeks produces foci of viable bacteria and a persistent inflammatory condition, while the secreted mucus establishes a barrier that only few bacteria manage to overcome. CONCLUSION: Gastric mucosoid cultures faithfully reproduce the features of normal human gastric epithelium, enabling new approaches for investigating the interaction of with the epithelial surface and the cross-talk with the basolateral stromal compartment. Our observations provide striking insights in the regulatory circuits of inflammation and defence. Hide abstract

Boccellato F, Meyer TF. 2015. Bacteria Moving into Focus of Human Cancer. Cell Host Microbe, 17 (6), pp. 728-30. Read abstract | Read more

Although bacteria have long been associated with human cancer, drawing causal relationships has been difficult. In this issue of Cell Host & Microbe, Scanu et al. (2015) provide evidence for a transforming activity of Salmonella Typhimurium on predisposed host cells, which can subsequently form tumors in a xenograft model. Hide abstract

Bagnoli F, Buti L, Tompkins L, Covacci A, Amieva MR. 2005. Helicobacter pylori CagA induces a transition from polarized to invasive phenotypes in MDCK cells. Proc. Natl. Acad. Sci. U.S.A., 102 (45), pp. 16339-44. Read abstract | Read more

CagA is a bacterial effector protein of Helicobacter pylori that is translocated via a type IV secretion system into gastric epithelial cells. We previously described that H. pylori require CagA to disrupt the organization and assembly of apical junctions in polarized epithelial cells. In this study, we provide evidence that CagA expression is not only sufficient to disrupt the apical junctions but also perturbs epithelial differentiation. CagA-expressing cells lose apicobasal polarity and cell-cell adhesion, extend migratory pseudopodia, and degrade basement membranes, acquiring an invasive phenotype. Expression of the CagA C-terminal domain, which contains the tyrosine phosphorylated EPIYA motifs, induces pseudopodial activity but is not sufficient to induce cell migration. Conversely, the N terminus targets CagA to the cell-cell junctions. Neither domain is sufficient to disrupt cell adhesion or cell polarity, but coexpressed in trans, the N terminus determines the localization of both polypeptides. We show that CagA induces a morphogenetic program in polarized Madin-Darby canine kidney cells resembling an epithelial-to-mesenchymal transition. We propose that altered cell-cell and cell matrix interactions may serve as an early event in H. pylori-induced carcinogenesis. Hide abstract

Backert S, Bernegger S, Skórko-Glonek J, Wessler S. 2018. Extracellular HtrA serine proteases: An emerging new strategy in bacterial pathogenesis. Cell. Microbiol., 20 (6), pp. e12845. Read abstract | Read more

The HtrA family of chaperones and serine proteases is important for regulating stress responses and controlling protein quality in the periplasm of bacteria. HtrA is also associated with infectious diseases since inactivation of htrA genes results in significantly reduced virulence properties by various bacterial pathogens. These virulence features of HtrA can be attributed to reduced fitness of the bacteria, higher susceptibility to environmental stress and/or diminished secretion of virulence factors. In some Gram-negative and Gram-positive pathogens, HtrA itself can be exposed to the extracellular environment promoting bacterial colonisation and invasion of host tissues. Most of our knowledge on the function of exported HtrAs stems from research on Helicobacter pylori, Campylobacter jejuni, Borrelia burgdorferi, Bacillus anthracis, and Chlamydia species. Here, we discuss recent progress showing that extracellular HtrAs are able to cleave cell-to-cell junction factors including E-cadherin, occludin, and claudin-8, as well as extracellular matrix proteins such as fibronectin, aggrecan, and proteoglycans, disrupting the epithelial barrier and producing substantial host cell damage. We propose that the export of HtrAs is a newly discovered strategy, also applied by additional bacterial pathogens. Consequently, exported HtrA proteases represent highly attractive targets for antibacterial treatment by inhibiting their proteolytic activity or application in vaccine development. Hide abstract