Amphiregulin Switches Progenitor Cell Fate for Lineage Commitment During Gastric Mucosal Regeneration.

BACKGROUND & AIMS: Isthmic progenitors, tissue-specific stem cells in the stomach corpus, maintain mucosal homeostasis by balancing between proliferation and differentiation to gastric epithelial lineages. The progenitor cells rapidly adopt an active state in response to mucosal injury. However, it remains unclear how the isthmic progenitor cell niche is controlled during the regeneration of damaged epithelium. METHODS: We recapitulated tissue recovery process after acute mucosal injury in the mouse stomach. Bromodeoxyuridine incorporation was used to trace newly generated cells during the injury and recovery phases. To define the epithelial lineage commitment process during recovery, we performed single-cell RNA-sequencing on epithelial cells from the mouse stomachs. We validated the effects of amphiregulin (AREG) on mucosal recovery, using recombinant AREG treatment or AREG-deficient mice. RESULTS: We determined that an epidermal growth factor receptor ligand, AREG, can control progenitor cell lineage commitment. Based on the identification of lineage-committed subpopulations in the corpus epithelium through single-cell RNA-sequencing and bromodeoxyuridine incorporation, we showed that isthmic progenitors mainly transition into short-lived surface cell lineages but are less frequently committed to long-lived parietal cell lineages in homeostasis. However, mucosal regeneration after damage directs the lineage commitment of isthmic progenitors towards parietal cell lineages. During recovery, AREG treatment promoted repopulation with parietal cells, while suppressing surface cell commitment of progenitors. In contrast, transforming growth factor-α did not alter parietal cell regeneration, but did induce expansion of surface cell populations. AREG deficiency impairs parietal cell regeneration but increases surface cell commitment. CONCLUSIONS: These data demonstrate that different epidermal growth factor receptor ligands can distinctly regulate isthmic progenitor-driven mucosal regeneration and lineage commitment.

Factors Impacting Academic Productivity and Satisfaction of Surgeon-scientists: A Nationwide Survey.

OBJECTIVE: To identify factors related to research success for academic surgeons. SUMMARY BACKGROUND DATA: Many recognize mounting barriers to scientific success for academic surgeons, but little is known about factors that predict success for individual surgeons. METHODS: A phase 1 survey was emailed to department chairpersons at highly funded US departments of surgery. Participating chairpersons distributed a phase 2 survey to their faculty surgeons. Training- and faculty-stage exposures and demographic data were collected and compared with participant-reported measures of research productivity. Five primary measures of productivity were assessed including number of grants applied for, grants funded, papers published, first/senior author papers published, and satisfaction in research. RESULTS: Twenty chairpersons and 464 faculty surgeons completed the survey, and 444 faculty responses were included in the final analysis. Having a research-focused degree was significantly associated with more grants applied for (PhD, incidence rate ratio (IRR)=6.93; masters, IRR=4.34) and funded (PhD, IRR=4.74; masters, IRR=4.01) compared to surgeons with only clinical degrees (all P<0.01). Having a formal research mentor was significantly associated with more grants applied for (IRR=1.57, P=0.03) and higher satisfaction in research (IRR=2.22, P<0.01). Contractually protected research time was significantly associated with more grants applied for (IRR=3.73), grants funded (IRR=2.14), papers published (IRR=2.12), first/senior authors published (IRR=1.72), and research satisfaction (Odds ratio=2.15) (all P<0.01). The primary surgeon-identified barrier to research productivity was lack of protection from clinical burden. CONCLUSIONS: Surgeons pursuing research-focused careers should consider the benefits of attaining a research-focused degree, negotiating for contractually protected research time, and obtaining formal research mentorship.

CEACAM5 and TROP2 define metaplastic and dysplastic transitions in human antral gastric precancerous lesions and tumors.

BACKGROUND: Mucosal gastric atrophy and intestinal metaplasia (IM) increase the risk for the development of gastric cancer (GC) as they represent a field for development of dysplasia and intestinal-type gastric adenocarcinoma. METHODS: We have investigated the expression of two dysplasia markers, CEACAM5 and TROP2, in human antral IM and gastric tumors to assess their potential as molecular markers. RESULTS: In the normal antral mucosa, weak CEACAM5 and TROP2 expression was only observed in the foveolar epithelium, while inflamed antrum exhibited increased expression of both markers. Complete IM exhibited weak CEACAM5 expression at the apical surface, but no basolateral TROP2 expression. On the other hand, incomplete IM demonstrated high levels of both CEACAM5 and TROP2 expression. Notably, incomplete IM with dysplastic morphology (dysplastic incomplete IM) exhibited higher levels of CEACAM5 and TROP2 expression compared to incomplete IM without dysplastic features (simple incomplete IM). In addition, dysplastic incomplete IM showed diminished SOX2 and elevated CDX2 expression compared to simple incomplete IM. CEACAM5 and TROP2 positivity in incomplete IM was similar to that of gastric adenomas and GC. Significant association was found between CEACAM5 and TROP2 positivity and histology of GC. CONCLUSIONS: These findings support the concept that incomplete IM is more likely associated with GC development. Overall, our study provides evidence of the heterogeneity of gastric IM and the distinct expression profiles of CEACAM5 and TROP2 in dysplastic incomplete IM. Our findings support the potential use of CEACAM5 and TROP2 as molecular markers for identifying individuals with a higher risk of GC development in the context of incomplete IM.

The Fibroblast Landscape in Stomach Carcinogenesis.

Numerous recent studies using single cell RNA sequencing and spatial transcriptomics have shown the vast cell heterogeneity, including epithelial, immune, and stromal cells, present in the normal human stomach and at different stages of gastric carcinogenesis. Fibroblasts within the metaplastic and dysplastic mucosal stroma represent key contributors to the carcinogenic microenvironment in the stomach. The heterogeneity of fibroblast populations is present in the normal stomach, but plasticity within these populations underlies their alterations in association with both metaplasia and dysplasia. In this review, we summarize and discuss efforts over the past several years to study the fibroblast components in human stomach from normal to metaplasia, dysplasia, and cancer. In the stomach, myofibroblast populations increase during late phase carcinogenesis and are a source of matrix proteins. PDGFRA-expressing telocyte-like cells are present in normal stomach and expand during metaplasia and dysplasia in close proximity with epithelial lineages, likely providing support for both normal and metaplastic progenitor niches. The alterations in fibroblast transcriptional signatures across the stomach carcinogenesis process indicate that fibroblast populations are likely as plastic as epithelial populations during the evolution of carcinogenesis.

Modeling the cell biology of monogenetic intestinal epithelial disorders.

Monogenetic variants are responsible for a range of congenital human diseases. Variants in genes that are important for intestinal epithelial function cause a group of disorders characterized by severe diarrhea and loss of nutrient absorption called congenital diarrheas and enteropathies (CODEs). CODE-causing genes include nutrient transporters, enzymes, structural proteins, and vesicular trafficking proteins in intestinal epithelial cells. Several severe CODE disorders result from the loss-of-function in key regulators of polarized endocytic trafficking such as the motor protein, Myosin VB (MYO5B), as well as STX3, STXBP2, and UNC45A. Investigations of the cell biology and pathophysiology following loss-of-function in these genes have led to an increased understanding of both homeostatic and pathological vesicular trafficking in intestinal epithelial cells. Modeling different CODEs through investigation of changes in patient tissues, coupled with the development of animal models and patient-derived enteroids, has provided critical insights into the enterocyte differentiation and function. Linking basic knowledge of cell biology with the phenotype of specific patient variants is a key step in developing effective treatments for rare monogenetic diseases. This knowledge can also be applied more broadly to our understanding of common epithelial disorders.

Regulation of metaplasia and dysplasia in the stomach by the stromal microenvironment.

Research on the microenvironment associated with gastric carcinogenesis has focused on cancers of the stomach and often underestimates premalignant stages such as metaplasia and dysplasia. Since epithelial interactions with T cells, macrophages, and type 2 innate lymphoid cells (ILC2s) are indispensable for the formation of precancerous lesions in the stomach, understanding the cellular interactions that promote gastric precancer warrants further investigation. Although various types of immune cells have been shown to play important roles in gastric carcinogenesis, it remains unclear how stromal cells such as fibroblasts influence epithelial transformation in the stomach, especially during precancerous stages. Fibroblasts exist as distinct populations across tissues and perform different functions depending on the expression patterns of cell surface markers and secreted factors. In this review, we provide an overview of known microenvironmental components in the stroma with an emphasis on fibroblast subpopulations and their roles during carcinogenesis in tissues including breast, pancreas, and stomach. Additionally, we offer insights into potential targets of tumor-promoting fibroblasts and identify open areas of research related to fibroblast plasticity and the modulation of gastric carcinogenesis.

Dynamic tuft cell expansion during gastric metaplasia and dysplasia.

Tuft cells are chemosensory cells associated with luminal homeostasis, immune response, and tumorigenesis in the gastrointestinal tract. We aimed to elucidate alterations in tuft cell populations during gastric atrophy and tumorigenesis in humans with correlative comparison to relevant mouse models. Tuft cell distribution was determined in human stomachs from organ donors and in gastric pathologies including Ménétrier's disease, Helicobacter pylori gastritis, intestinal metaplasia (IM), and gastric tumors. Tuft cell populations were examined in Lrig1-KrasG12D , Mist1-KrasG12D , and MT-TGFα mice. Tuft cells were evenly distributed throughout the entire normal human stomach, primarily concentrated in the isthmal region in the fundus. Ménétrier's disease stomach showed increased tuft cells. Similarly, Lrig1-Kras mice and mice overexpressing TGFα showed marked foveolar hyperplasia and expanded tuft cell populations. Human stomach with IM or dysplasia also showed increased tuft cell numbers. Similarly, Mist1-Kras mice had increased numbers of tuft cells during metaplasia and dysplasia development. In human gastric cancers, tuft cells were rarely observed, but showed positive associations with well-differentiated lesions. In mouse gastric cancer xenografts, tuft cells were restricted to dysplastic well-differentiated mucinous cysts and were lost in less differentiated cancers. Taken together, tuft cell populations increased in atrophic human gastric pathologies, metaplasia, and dysplasia, but were decreased in gastric cancers. Similar findings were observed in mouse models, suggesting that, while tuft cells are associated with precancerous pathologies, their loss is most associated with the progression to invasive cancer.

Telocyte Recruitment During the Emergence of a Metaplastic Niche in the Stomach.

BACKGROUND & AIM: Telocytes, a recently identified type of subepithelial interstitial cell, have garnered attention for their potential roles in tissue homeostasis and repair. However, their contribution to gastric metaplasia remains unexplored. This study elucidates the role of telocytes in the development of metaplasia within the gastric environment. METHODS: To investigate the presence and behavior of telocytes during metaplastic transitions, we used drug-induced acute injury models (using DMP-777 or L635) and a genetically engineered mouse model (Mist1-Kras). Lineage tracing via the Foxl1-CreERT2;R26R-tdTomato mouse model was used to track telocyte migratory dynamics. Immunofluorescence staining was used to identify telocyte markers and evaluate their correlation with metaplasia-related changes. RESULTS: We confirmed the existence of FOXL1+/PDGFRα+ double-positive telocytes in the stomach's isthmus region. As metaplasia developed, we observed a marked increase in the telocyte population. The distribution of telocytes expanded beyond the isthmus to encompass the entire gland and closely reflected the expansion of the proliferative cell zone. Rather than a general response to mucosal damage, the shift in telocyte distribution was associated with the establishment of a metaplastic cell niche at the gland base. Furthermore, lineage-tracing experiments highlighted the active recruitment of telocytes to the emerging metaplastic cell niche, and we observed expression of Wnt5a, Bmp4, and Bmp7 in PDGFRα+ telocytes. CONCLUSIONS: These results suggest that telocytes contribute to the evolution of a gastric metaplasia niche. The dynamic behavior of these stromal cells, their responsiveness to metaplastic changes, and potential association with Wnt5a, Bmp4, and Bmp7 signaling emphasize the significance of telocytes in tissue adaptation and repair.

Interleukin 13 Promotes Maturation and Proliferation in Metaplastic Gastroids.

BACKGROUND & AIMS: Type 2 innate lymphoid cells (ILC2s) and interleukin-13 (IL-13) promote the onset of spasmolytic polypeptide-expressing metaplasia (SPEM) cells. However, little is known about molecular effects of IL-13 in SPEM cells. We now sought to establish a reliable organoid model, Meta1 gastroids, to model SPEM cells in vitro. We evaluated cellular and molecular effects of ILC2s and IL-13 on maturation and proliferation of SPEM cells. METHODS: We performed single-cell RNA sequencing to characterize Meta1 gastroids, which were derived from stomachs of Mist1-Kras transgenic mice that displayed pyloric metaplasia. Cell sorting was used to isolate activated ILC2s from stomachs of IL-13-tdTomato reporter mice treated with L635. Three-dimensional co-culture was used to determine the effects of ILC2s on Meta1 gastroids. Mouse normal or metaplastic (Meta1) and human metaplastic gastroids were cultured with IL-13 to evaluate cell responses. Air-Liquid Interface culture was performed to test long-term culture effects of IL-13. In silico analysis determined possible STAT6-binding sites in gene promoter regions. STAT6 inhibition was performed to corroborate STAT6 role in SPEM cells maturation. RESULTS: Meta1 gastroids showed the characteristics of SPEM cell lineages in vitro even after several passages. We demonstrated that co-culture with ILC2s or IL-13 treatment can induce phosphorylation of STAT6 in Meta1 and normal gastroids and promote the maturation and proliferation of SPEM cell lineages. IL-13 up-regulated expression of mucin-related proteins in human metaplastic gastroids. Inhibition of STAT6 blocked SPEM-related gene expression in Meta1 gastroids and maturation of SPEM in both normal and Meta1 gastroids. CONCLUSIONS: IL-13 promotes the maturation and proliferation of SPEM cells consistent with gastric mucosal regeneration.

Coronavirus M Protein Trafficking in Epithelial Cells Utilizes a Myosin Vb Splice Variant and Rab10.

The membrane (M) glycoprotein of coronaviruses (CoVs) serves as the nidus for virion assembly. Using a yeast two-hybrid screen, we identified the interaction of the cytosolic tail of Murine Hepatitis Virus (MHV-CoV) M protein with Myosin Vb (MYO5B), specifically with the alternative splice variant of cellular MYO5B including exon D (MYO5B+D), which mediates interaction with Rab10. When co-expressed in human lung epithelial A549 and canine kidney epithelial MDCK cells, MYO5B+D co-localized with the MHV-CoV M protein, as well as with the M proteins from Porcine Epidemic Diarrhea Virus (PEDV-CoV), Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome 2 (SARS-CoV-2). Co-expressed M proteins and MYO5B+D co-localized with endogenous Rab10 and Rab11a. We identified point mutations in MHV-CoV M that blocked the interaction with MYO5B+D in yeast 2-hybrid assays. One of these point mutations (E121K) was previously shown to block MHV-CoV virion assembly and its interaction with MYO5B+D. The E to K mutation at homologous positions in PEDV-CoV, MERS-CoV and SARS-CoV-2 M proteins also blocked colocalization with MYO5B+D. The knockdown of Rab10 blocked the co-localization of M proteins with MYO5B+D and was rescued by re-expression of CFP-Rab10. Our results suggest that CoV M proteins traffic through Rab10-containing systems, in association with MYO5B+D.