Human gastric multi-regional assembloids for functional parietal maturation and patient-specific modelling of antral foveolar hyperplasia.

Gastric Organoids: Progress and Remaining Challenges.

Background and aimReliable in vitro derivation of mature subsets of human gastric epithelial cells has been challenging to achieve in 3D culture. Small intestinal organoids have been shown to self-organise...

Recently, reports published in Nature Medicine and Cell have suggested the existence of a gut-bone axis based on studies of gene knockout mice. First, impaired gastric acid secretion was claimed to negatively affect calcium homeostasis and bone mass; which was based on the bone phenotype of cholecystokinin-B (or 2) receptor knockout mice. However, also histidine decarboxylase knockout mice suffered from impaired gastric acid secretion, while exhibiting a very different bone phenotype. This argues against the view that lack of gastric acid causes bone loss. Second, circulating serotonin was claimed to inhibit bone formation. This claim was based on the observation that mice deficient in low-density lipoprotein receptor-related protein 5 (Lrp5) exhibited bone loss coupled with accelerated serotonin synthesis. Lrp5 was claimed to control bone formation by a link involving duodenal serotonin synthesis. However, the accelerated serotonin synthesis in Lrp5 knockouts occurred after the onset of bone loss, a sequence of events that does not suggest a causal relationship between elevated serotonin in blood and bone loss. Moreover, pharmacological inhibition of serotonin synthesis prevented bone loss following ovariectomy in wild-type mice and rats, an observation that does not support the existence of Lrp5 pathway in the serotonin-bone axis.

Gastric organoid is the organotypic cultures of gastric stem cells or pluripotent stem cells. Gastric organoid is comprised of all major types of gastric epithelial cells and represent the architecture and function remarkably similar to those of the gastric epithelium, faithfully recapitulating the functional gastric epithelium ex vivo. As ideal basic experimental model, gastric organoid has advantages over animal models and conventional cell model in many aspects. Gastric organoid derived from human gastric tissue, in particular, allows the investigation of the function of human stomach in the ex vivo setting. It has now been applied in the field of formation and physiology of the stomach, Helicobacter pylori infection-associated diseases, research of the pathogenic gene, screening and development of drugs, and regenerative medicine. What is more, as an innovative pre-clinical cancer model, gastric cancer organoid has provided important insights in the development of gastric cancer and screening of antitumor drugs, such as simulating the occurrence and development of gastric cancer, screening and development of antitumor drugs, personalized medication and targeted therapy for gastric cancer, and combined application with patient-derived xenograft. In this review, we summarize the establishment and application of gastric and gastric cancer organoids, especially in modeling gastric cancer, basic research and drug development.

Organoids are three-dimensional in vitro models of human disease developed from benign and malignant gastrointestinal tissues with tremendous potential for personalized medicine applications. We sought to determine whether gastric cancer patient-derived organoids (PDOs) could be safely established from endoscopic biopsies for rapid drug screening. Patients underwent esophagogastroduodenoscopy (EGD) for surveillance or staging and had additional forceps biopsies taken for PDO creation. Cancer tissues from operative specimens were also used to create PDOs. To address potential tumor heterogeneity, we performed low-coverage whole-genome sequencing of endoscopic-derived PDOs with paired surgical PDOs and whole-tumor lysates. The stability of genomic alterations in endoscopic organoids was assessed by next-generation sequencing and nested polymerase chain reaction (PCR) assay. The feasibility and potential accuracy of drug sensitivity screening with endoscopic-derived PDOs were also evaluated. Gastric cancer PDOs (n = 15) were successfully established from EGD forceps biopsies (n = 8) and surgical tissues (n = 7) from five patients with gastric adenocarcinoma. Low-coverage whole-genomic profiling of paired EGD and surgical PDOs along with whole-tumor lysates demonstrated absence of tumor heterogeneity. Nested PCR assay identified similar KRAS alterations in primary tumor and paired organoids. Drug sensitivity testing of endoscopic-derived PDOs displayed standard dose-response curves to current gastric cancer cytotoxic therapies. Our study results demonstrate the feasibility of developing gastric cancer PDOs from EGD biopsies. These results also indicate that endoscopic-derived PDOs are accurate surrogates of the primary tumor and have the potential for drug sensitivity screening and personalized medicine applications.

We are all committed to extending the knowledge of textiles. These papers offer outstanding examples of how museum curators, an academic, and a conservator have expanded audiences and brought new materials to telling the textile story. Major themes have emerged from their papers, and are worthy of further consideration. First, it is quite apparent that professionals in museums need to work together. Harold Mailand has shown what the conservator can bring to the curator in the interpretation of textiles. I contend that textile department and costume departments should work together, and collaborate with other departments in their institutions. I am sure that many do this now.. Second, it is clear from Gayle Strege that costumes should be thought of as part of textile collections, and that flat textiles, those that are really draped clothing, should be viewed as such, literally. Again, this is an opportunity for collaboration. In university textiles classes students should have the opportunity to become engaged with the finished product, which could be an upholstered chair or a swimsuit. Karen Herbaugh, also made this clear for the museum exhibition. Why not see the whole picture from fiber through manufactured product and end use, and maybe even reuse. The story has a beginning and an end.

Introduction: Patient-derived organoids (PDOs) have become attractive tools for genetic studies, biomarker identification, drug screening, and preclinical evaluation of personalized medicine strategies. Previously, we created gastric cancer PDOs from esophagogastroduodenoscopy (EGD) biopsy specimens. As a continuation of our previous work, we sought to optimize methods for creating EGD-derived PDOs for drug sensitivity testing within clinically actionable time constraints. Methods: We previously established a standard operating protocol for creating PDOs from EGD specimens of patients with gastric cancer. To optimize the development of PDOs, we enrolled patients with gastric adenocarcinoma undergoing EGD at Markey Cancer Center. During diagnostic EGDs, additional research biopsies were collected for creation of gastric cancer PDOs and placed in ice-cold organoid medium. We employed different dissociation methods based on the texture of the biopsy tissues. Briefly, soft tissues were washed and isolated in a chelating solution to release the glands, while the hard tissues were digested with collagenase and dispase to release epithelium. Glands or epithelia cells were collected, resuspended in BME (basement membrane extract), and plated in 2 wells of 24-well chambers. On day 4-6, PDOs were dissociated and passaged using manual pipetting. For passage 2 on day 9-11, PDOs were isolated into smaller and uniform-sized PDOs or single cells with both mechanical and enzymatic (TripLE Express) dissociation and plated in a 96-well plate at 10uL (500-1000 cells)/well. To test drug sensitivities, PDOs were treated on day 12 with current standard-of-care cytotoxic combination chemotherapies (e.g., FLOT, ECF, FOLFIRI, and FOLFOX) or solvent control. We tested concentrations determined by the Cmax and AUC of each drug associated with a single highest recommended dose in drug product label. We performed LIVE/DEAD assay on CellInsight CX7 High-Content Screening (HCS) Platform (ThermoFisher) and CellTiter Glo luminescent cell viability assay consecutively to measure response of PDOs to chemotherapy regimens. Result: Consistent with our prior experience, we successfully developed EGD-derived gastric cancer PDOs from patients undergoing diagnostic EGD. Using our modified technique for PDO creation and expansion, we obtained sufficient and uniform-sized PDOs at the second passage on day 10 and then treated these PDOs with current standard-of-care chemotherapy regimens to predict in vivo drug response. Creation of EGD-derived PDOs and drug sensitivity testing was feasible within two weeks of tissue collection. Conclusions: We have optimized methods to create, grow, and test EGD-derived PDOs within a clinically actionable time period. Our modified technique yielded higher concentration and more uniform-sized PDOs, providing sufficient biologic material for future implementation of rapid and comprehensive drug testing for neoadjuvant chemotherapy trials. Citation Format: Mei Gao, Miranda Lin, Wesam M Frandah, Moamen Gabr, Houssam E. Mardini, Michael Cavnar, Joseph Kim. Utilizing endoscopic-derived gastric cancer organoids for personalized neoadjuvant chemotherapy [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A21.

Gastric stem cells are adult stem cells found in the gastric tissues, which possess high self-renewal capability, proliferation rate and multiple differentiation capability. They can regenerate all the gastric mucosa epithelial cells. Gastric stem cells play an important role in the self-renewal and injury repair, making epithelium of gastric mucosa in the dynamic balance and maintaining the integrity of gastric mucosa. With the constant deepening of stem cell research, the application of gastric stem cells provides a new means for the study of gastric physiology and diseases. Since the first report by Barker in 2010, gastric organoids have soon become a model of interest and are highly desirable as tools for studying gastric diseases. As an optimal experimental model, gastric organoids are superior to animal model and conventional cell culture. Gastric organoids are comprised of all major types of gastric epithelial cells, represent the architecture and function remarkably similar to those of the gastric epithelium, faithfully recapitulating the functional gastric epithelium ex vivo. Especially gastric organoids derived from the human body, which allow the investigation of the function of human stomach in the ex vivo setting. In this review, research progresses of gastric stem cells and their application in establishment of gastric organoids are summarized. Key words: Stem cell; Gastric stem cell; Gastric organoid; Model

Background EndoFaster® analyzes gastric juice in real time during gastroscopy allowing the detection of hypo-achlorhydric conditions, like corpus atrophic gastritis. Narrow-band imaging (NBI) endoscopy allows to accurately detect and perform target biopsies in areas of intestinal metaplasia, a histological change often associated to corpus atrophic gastritis. Aims To compare the diagnostic accuracy of EndoFaster® with histological evaluation for corpus atrophic gastritis through high-resolution (HR) NBI targeted biopsies. Methods Prospective study on consecutive adult patients undergoing gastroscopy between April and November 2018. Patients in therapy with proton pump inhibitors, previous gastric surgery, and/or known gastric neoplasia were excluded. At the beginning of gastroscopy, gastric juice was aspirated and analyzed by EndoFaster® in 15 seconds. Endoscopists were blinded to the report of EndoFaster®. Evaluation of gastric mucosa in HR-white light was firstly performed, then with HR-NBI allowing to perform targeted biopsies on areas suspected for intestinal metaplasia; otherwise, biopsies were performed according to the updated Sydney System protocol and sent for histopathological evaluation. Results Overall, 124 patients were included [64% F; 56 (18-85) years]. Corpus atrophic gastritis was present in 41.9% of patients. EndoFaster® showed an accuracy for corpus atrophic gastritis diagnosis, compared to histopathological evaluation as gold standard, of 87.1% and a sensitivity, specificity, PPV, and NPV of 78.8%, 93.1%, 89.1%, and 85.9%, respectively. pH showed a positive correlation with the severity score of atrophy (r = 0.67, 95% CI: 0.73-0.81, and p < 0.0001). EndoFaster® allowed to diagnose corpus atrophic gastritis in 3.7% of patients negative to NBI (corpus atrophic gastritis without intestinal metaplasia). Conclusion EndoFaster® seems a promising tool to diagnose corpus atrophic gastritis. The evaluation of hypo-achlorhydria during gastroscopy can address bioptic sampling in corpus atrophic gastritis patients without intestinal metaplasia.

An acidic environment in the stomach appears to enhance absorption of supplemental thyroxine. In this study, Italian researchers examined thyroxine

The effect of age and Billroth gastrectomy on absorption of desmethyldiazepam (DMDZ) from a single 20‐mg oral dose of its precursor, clorazepate (CZP) dipotassium, was assessed in 24 males. Six were healthy young controls (mean age, 24), 8 were elderly controls (mean age, 66.5 yr) with no gastrointestinal disease, and 10 were patients (mean age, 56) who had undergone Billroth gastrectomy at least 2 yr before. Absent or impaired gastric acid secretion was documented in 6 of the 10 postgastrectomy patients. Weight‐normalized area under the 48‐hr serum DMDZ concentration curve (WtN‐AUC‐48) among elderly controls (mean, 324 units) and gastrectomy patients (401 units) were similar and significantly less than those in young controls (603 units). Peak serum DMDZ concentrations in elderly controls and gastrectomy patients (185 and 216 ng/ml, respectively) were also lower and reached later after the dose (2.2 and 2.3 hr) than in young controls (371 ng/ml; 1.1 hr). Age per se rather than gastrectomy or acid secretion status explained by far most of the overall variability in WtN‐AUC‐48 and peak DMDZ concentration. Thus normal gastric acidity is not essential for absorption of CZP‐derived DMDZ. The appearance of DMDZ in the systemic circulation is reduced in elderly males, irrespective of surgery or gastric acid secretion. This could be explained by age‐related reduction in conversion of CZP to DMDZ or by more extensive distribution of DMDZ in the elderly.

Basal gastric acid output was reduced in 9 out of 14 infants and young children with malnutrition compared with 21 age-matched controls. In all the patients the response of the gastric mucosa to stimulation by pentagastrin was impaired, and gastritis of variable severity was present in 8 out of the 9 patients in who biopsies were performed. Impaired gastric acid secretion probably contributes towards bacterial overgrowth and diarrhoeal diseases in malnourished children.

An inadequate supply of fresh tissue is a major limitation of three-dimensional patient-derived gastric organoid research. We propose that tissue procurement for organoid culture could be increased by developing a cold storage shipment protocol for fresh surgical tissues. Sixty stomach specimens from C57BL/6J mice were resected, of which forty-five were stored in Hank's Balanced Salt (HBSS), University of Wisconsin (UW), or Histidine-Tryptophan-Ketoglutarate (HTK) solutions for subsequent organoid culture. Stomachs were dissociated and processed into gastric organoids as fresh tissue or after transport at 4 °C for 24 or 48 h. All gastric organoid cultures were established and maintained for 10 passages. Cold storage for 24 or 48 h did not significantly affect organoid viability. Although cold storage was associated with decreased organoid growth rate, there were no differences in viability, cytotoxic dose response, or LGR5 and TROY stem cell gene expression compared to organoids prepared from fresh tissue. As a proof of concept, six human gastric cancer organoids were established after 24 or 48 h of storage. Patient-derived gastric organoids from mouse and human gastric tissue can be established after 48 h of cold ischemia. Our method, which only requires ice packs, standard shipping containers, and HBSS is feasible and reliable. This method does not affect the reliability of downstream dose-response assays and maintains organoid viability for at least 10 passages. The shipment of fresh tissue for organoid procurement could serve to enhance multicenter collaboration and achieve more elaborate or controlled organoid experimentation.

Programmed cell death promotes homeostatic cell turnover in the epithelium but is dysregulated in cancer. The glycosyltransferase ST6Gal-I is known to block homeostatic apoptosis through α2,6-linked sialylation of the death receptor TNFR1 in many cell types. However, its role has not been investigated in gastric epithelial cells or gastric tumorigenesis. We determined that human gastric antral epithelium rarely expressed ST6Gal-I, but the number of ST6Gal-I-expressing epithelial cells increased significantly with advancing premalignancy leading to cancer. The mRNA expression levels of ST6GAL-I and SOX9 in human gastric epithelial cells correlated positively with one another through the premalignancy cascade, indicating that increased epithelial cell expression of ST6Gal-I is associated with premalignant progression. To determine the functional impact of increased ST6Gal-I, we generated human gastric antral organoids from epithelial stem cells and differentiated epithelial monolayers from gastric organoids. Gastric epithelial stem cells strongly expressed ST6Gal-I, suggesting a novel biomarker of stemness. In contrast, organoid-derived epithelial monolayers expressed markedly reduced ST6Gal-I and underwent TNF-induced, caspase-mediated apoptosis, consistent with homeostasis. Conversely, epithelial monolayers generated from gastric cancer stem cells retained high levels of ST6Gal-I and resisted TNF-induced apoptosis, supporting prolonged survival. Protection from TNF-induced apoptosis depended on ST6Gal-I overexpression, because forced ST6Gal-I overexpression in normal gastric stem cell-differentiated monolayers inhibited TNF-induced apoptosis, and cleavage of α2,6-linked sialic acids from gastric cancer organoid-derived monolayers restored susceptibility to TNF-induced apoptosis. These findings implicate up-regulated ST6Gal-I expression in blocking homeostatic epithelial cell apoptosis in gastric cancer pathogenesis, suggesting a mechanism for prolonged epithelioid tumor cell survival.

BackgroundGastric cancer ranks as the fifth most common malignancy worldwide. Of note, this entity shows a rising incidence rate over the last decades. Surgical resection and adjuvant chemotherapy are the...