Establishment of gastrointestinal assembloids to study the interplay between epithelial crypts and their mesenchymal niche.

The cellular organization of gastrointestinal crypts is orchestrated by different cells of the stromal niche but available in vitro models fail to fully recapitulate the interplay between epithelium and stroma. Here, we establish a colon assembloid system comprising the epithelium and diverse stromal cell subtypes. These assembloids recapitulate the development of mature crypts resembling in vivo cellular diversity and organization, including maintenance of a stem/progenitor cell compartment in the base and their maturation into secretory/absorptive cell types. This process is supported by self-organizing stromal cells around the crypts that resemble in vivo organization, with cell types that support stem cell turnover adjacent to the stem cell compartment. Assembloids that lack BMP receptors either in epithelial or stromal cells fail to undergo proper crypt formation. Our data highlight the crucial role of bidirectional signaling between epithelium and stroma, with BMP as a central determinant of compartmentalization along the crypt axis.

R-spondin/YAP axis promotes gastric oxyntic gland regeneration and Helicobacter pylori-associated metaplasia in mice.

The stomach corpus epithelium is organized into anatomical units that consist of glands and pits. Mechanisms that control the cellular organization of corpus glands and enable their recovery upon injury are not well understood. R-spondin 3 (RSPO3) is a WNT-signaling enhancer that regulates stem cell behavior in different organs. Here, we investigated the function of RSPO3 in the corpus during homeostasis, upon chief and/or parietal cell loss, and during chronic Helicobacter pylori infection. Using organoid culture and conditional mouse models, we demonstrate that RSPO3 is a critical driver of secretory cell differentiation in the corpus gland toward parietal and chief cells, while its absence promoted pit cell differentiation. Acute loss of chief and parietal cells induced by high dose tamoxifen - or merely the depletion of LGR5+ chief cells - caused an upregulation of RSPO3 expression, which was required for the initiation of a coordinated regenerative response via the activation of yes-associated protein (YAP) signaling. This response enabled a rapid recovery of the injured secretory gland cells. However, in the context of chronic H. pylori infection, the R-spondin-driven regeneration was maintained long term, promoting severe glandular hyperproliferation and the development of premalignant metaplasia.

Gastric stem cells promote inflammation and gland remodeling in response to Helicobacter pylori via Rspo3-Lgr4 axis.

Helicobacter pylori is a pathogen that colonizes the stomach and causes chronic gastritis. Helicobacter pylori can colonize deep inside gastric glands, triggering increased R-spondin 3 (Rspo3) signaling. This causes an expansion of the "gland base module," which consists of self-renewing stem cells and antimicrobial secretory cells and results in gland hyperplasia. The contribution of Rspo3 receptors Lgr4 and Lgr5 is not well explored. Here, we identified that Lgr4 regulates Lgr5 expression and is required for H. pylori-induced hyperplasia and inflammation, while Lgr5 alone is not. Using conditional knockout mice, we reveal that R-spondin signaling via Lgr4 drives proliferation of stem cells and also induces NF-κB activity in the proliferative stem cells. Upon exposure to H. pylori, the Lgr4-driven NF-κB activation is responsible for the expansion of the gland base module and simultaneously enables chemokine expression in stem cells, resulting in gland hyperplasia and neutrophil recruitment. This demonstrates a connection between R-spondin-Lgr and NF-κB signaling that links epithelial stem cell behavior and inflammatory responses to gland-invading H. pylori.

BMP feed-forward loop promotes terminal differentiation in gastric glands and is interrupted by H. pylori-driven inflammation.

Helicobacter pylori causes gastric inflammation, gland hyperplasia and is linked to gastric cancer. Here, we studied the interplay between gastric epithelial stem cells and their stromal niche under homeostasis and upon H. pylori infection. We find that gastric epithelial stem cell differentiation is orchestrated by subsets of stromal cells that either produce BMP inhibitors in the gland base, or BMP ligands at the surface. Exposure to BMP ligands promotes a feed-forward loop by inducing Bmp2 expression in the epithelial cells themselves, enforcing rapid lineage commitment to terminally differentiated mucous pit cells. H. pylori leads to a loss of stromal and epithelial Bmp2 expression and increases expression of BMP inhibitors, promoting self-renewal of stem cells and accumulation of gland base cells, which we mechanistically link to IFN-γ signaling. Mice that lack IFN-γ signaling show no alterations of BMP gradient upon infection, while exposure to IFN-γ resembles H. pylori-driven mucosal responses.

Epithelial response to IFN-γ promotes SARS-CoV-2 infection.

SARS-CoV-2, the agent that causes COVID-19, invades epithelial cells, including those of the respiratory and gastrointestinal mucosa, using angiotensin-converting enzyme-2 (ACE2) as a receptor. Subsequent inflammation can promote rapid virus clearance, but severe cases of COVID-19 are characterized by an inefficient immune response that fails to clear the infection. Using primary epithelial organoids from human colon, we explored how the central antiviral mediator IFN-γ, which is elevated in COVID-19, affects epithelial cell differentiation, ACE2 expression, and susceptibility to infection with SARS-CoV-2. In mouse and human colon, ACE2 is mainly expressed by surface enterocytes. Inducing enterocyte differentiation in organoid culture resulted in increased ACE2 production. IFN-γ treatment promoted differentiation into mature KRT20+ enterocytes expressing high levels of ACE2, increased susceptibility to SARS-CoV-2 infection, and resulted in enhanced virus production in infected cells. Similarly, infection-induced epithelial interferon signaling promoted enterocyte maturation and enhanced ACE2 expression. We here reveal a mechanism by which IFN-γ-driven inflammatory responses induce a vulnerable epithelial state with robust replication of SARS-CoV-2, which may have an impact on disease outcome and virus transmission.

BMI-adjusted prognosis of signet ring cell carcinoma in patients undergoing radical gastrectomy for gastric adenocarcinoma.

BACKGROUND: Compared with other histologic types, signet ring cell gastric carcinoma (SRC) has unique oncological characteristics, and its implication on the prognosis of gastric cancer patients remains unclear. The purpose of this study was to evaluate the prognostic impact of body mass index (BMI) on SRC patients. METHODS: A retrospective analysis was performed using the clinical records of 3342 patients with SRC or tubular adenocarcinoma who underwent radical gastrectomy between 2000 and 2014. Patients were divided into three groups according to histologic subtype: SRC, well-to-moderately differentiated adenocarcinoma (WMD), and poorly differentiated adenocarcinoma (PD). We compared the survival of SRC patients with that of tubular adenocarcinoma patients according to BMI. RESULTS: The 5-year survival of SRC was significantly worse than that of WMD (P < 0.001) but superior to that of PD (P < 0.001). BMI-stratified analysis showed that in the high-BMI group, the prognosis of SRC was similar to that of WMD (P > 0.05) and better than that of PD (P < 0.001). In normal-BMI patients, SRC had a worse prognosis than WMD (P < 0.001) but a more favorable prognosis than PD (P < 0.001). SRC among low-BMI patients displayed much poorer survival than did both WMD (P < 0.001) and PD (P = 0.005). Multivariate analysis indicated that the risk of death was the lowest in SRC patients with a high BMI and highest for SRC patients with a low BMI (low-BMI hazard ratio: SRC 1 vs. WMD 0.51 and PD 0.53). CONCLUSION: SRC has worse prognostic impact as BMI decreases. BMI leads to differing prognosis of SRC compared with tubular adenocarcinoma.

A modified subclassification to evaluate the survival of patients with N3 gastric cancer: an international database study.

BACKGROUND: The eighth TNM classification for gastric cancer categorizes N3 as N3a and N3b in the final pathologic stage. The cutoff for N3a/N3b is defined as 15 metastatic lymph nodes, but the rationale for this cutoff remains unclear. This study aimed to determine the optimal N3a/N3b cutoff and evaluate its prognostic significance. METHODS: An international database was constructed by combining data from patients with N3 gastric cancer and complete five-year follow-up data from the Surveillance, Epidemiology, and End Results program database (n = 1833) and the Fujian Medical University Union Hospital database (n = 920) (total n = 2753). A log-rank test was performed to determine the optimal N3a/N3b cutoff, and its prognostic significance was confirmed in a two-step multivariate analysis and compared to that of the eighth TNM. RESULTS: A cut-point analysis performed at each metastatic lymph node number identified the greatest survival difference between N3a and N3b at 13 metastatic lymph nodes (χ2 = 157.671, P = 3.65 × 10- 36). In patients with 14-15 metastatic lymph nodes, prognoses were significantly worse than those in patients with 7-13 metastatic lymph nodes (P < 0.001) but similar to those in patients with > 15 metastatic lymph nodes (P = 0.078). Therefore, patients with 14-15 metastatic lymph nodes were incorporated into a modified N3b classification. In the two-step multivariate analysis, the eighth N3 classification fell out of the model, while the modified N3 classification remained intact (HR 1.51, P < 0.001). Further analyses demonstrated that the modified TNM classification had superior homogeneity, discriminatory ability, and gradient monotonicity compared to the eighth TNM classification. CONCLUSIONS: For improved prognostic stratification, we recommend adjusting the cutoff for subclassification of N3 gastric cancer to 13 metastatic lymph nodes.

CDK5RAP3 suppresses Wnt/ß-catenin signaling by inhibiting AKT phosphorylation in gastric cancer.

BACKGROUND: CDK5RAP3 was initially isolated as a binding protein of the CDK5 activator p35. Although CDK5RAP3 has been shown to negatively regulate the Wnt/ß-catenin signaling pathway in gastric cancer by repressing GSK-3ß phosphorylation, its in-depth mechanism has not been determined. METHODS: Following CDK5RAP3 overexpression or knock down, CDK5RAP3 signaling pathways were investigated in gastric cancer cells by Western Blotting. Cell growth, invasion and migration were also evaluated in gastric cancer cell lines. We analyzed CDK5RAP3, AKT, p-AKT (Ser473), GSK-3ß and p-GSK-3ß (Ser9) expression in gastric tumor samples and adjacent non-tumor tissues from 295 patients using immunohistochemistry and Western Blotting. The prognostic significance of CDK5RAP3 and p-AKT (Ser473) was confirmed by a Log-rank test. RESULTS: Our study demonstrated that the expression of p-AKT (Ser473) and p-GSK-3ß (Ser9) was negatively correlated with CDK5RAP3 in stable gastric cancer cell lines. CDK5RAP3 repressed AKT phosphorylation, which promoted GSK-3ß phosphorylation, thereby suppressing ß-catenin protein expression and, consequently, gastric cancer. The protein level of CDK5RAP3 was markedly decreased in most gastric tumor tissues compared with adjacent non-tumor tissues, and the levels of p-AKT (Ser473) and p-GSK-3ß (Ser9) were also negatively correlated with those of CDK5RAP3. The prognostic value of CDK5RAP3 for overall survival was found to be dependent on AKT phosphorylation. CONCLUSION: Our results demonstrated that CDK5RAP3 negatively regulates the Wnt/ß-catenin signaling pathway by repressing AKT phosphorylation, which leads to better survival of patients with gastric cancer.

The prognostic relevance of parapyloric lymph node metastasis in Siewert type II/III adenocarcinoma of the esophagogastric junction.

BACKGROUND: The purpose of this study was to evaluate the prognosis of patients with Siewert type II/III adenocarcinoma of the esophagogastric junction (AEG) with parapyloric lymph node (No. 5 and 6 lymph nodes, PLN) metastasis and to determine the need for PLN dissection for patients with type II/III AEG. METHODS: A total of 1008 patients with type II/III AEG who underwent a transabdominal total gastrectomy were enrolled. The long-term surgical outcome of PLN-positive patients and the therapeutic value of PLN dissection were analyzed. RESULTS: There was no significant difference in the incidence of PLN metastasis between type II and III cancers (5.7% vs. 8.5%, P > 0.05). PLN metastasis was a significant prognostic factor for type II/III cancers (HR 1.63; P = 0.001). Among type II/III cancers, the 5-year survival of patients with PLN-positive cancers was much lower than that of patients with PLN-negative cancers (21.3% vs. 60.8%, P < 0.001). Even after radical resection, the 5-year survival of patients with stage I-III PLN-positive cancers was similar to that of patients with stage IV cancers without PLN metastasis (23.5% vs. 23.1%, P > 0.05). In the analysis of the therapeutic value of lymph node dissection in each station for type II and III cancers after radical resection, lymph nodes with the lowest therapeutic value index after No. 12a were No. 5 and 6 lymph nodes. CONCLUSIONS: Patients with type II/III AEG with PLN metastasis have a poor prognosis, similar to patients with stage IV disease. PLN dissection offers marginal therapeutic value for patients with type II/III AEG.

Baicalein inhibits migration and invasion of gastric cancer cells through suppression of the TGF-ß signaling pathway.

The transforming growth factor-ß (TGF-ß) signaling pathway exhibits an important role in cancer invasion and metastasis. Excessive expression of TGF-ß activates Smad4, leading to the upregulation of downstream metastasis-associated genes. Thus, the inhibition of the TGF-ß/Smad4 signaling pathway may be a novel strategy for treatment of cancer metastasis. Baicalein, a flavonoid derived from the root of Scutellaria baicalensis, has been reported to exert strong anti-tumor activity towards various types of cancer. In the present study the effect of baicalein on migration and invasion of cancer cells was evaluated using wound-healing and Transwell assays. In order to investigate the possible molecular mechanisms of the anti-metastatic effects of baicalein, quantitative polymerase chain reaction (qPCR) and western blot analyses were performed to examine the effect on the expression of TGF­ß, Smad4, N-cadherin, vimentin, ZEB1 and ZEB2. It was determined that baicalein inhibited the migration and invasion of AGS cells by suppressing the TGF-ß/Smad4 signaling pathway. In addition, baicalein treatment reduced the expression of the metastasis-associated N-cadherin, vimentin, ZEB1 and ZEB2, downstream target genes of the TGF­ß/Smad4 signaling pathway. Collectively, these results suggest that inhibition of the metastasis of cancer cells via inactivation of TGF-ß/Smad4 signaling is one of the mechanisms by which baicalein may treat cancer.