Isthmus progenitor cells contribute to homeostatic cellular turnover and support regeneration following intestinal injury.

The currently accepted intestinal epithelial cell organization model proposes that Lgr5+ crypt-base columnar (CBC) cells represent the sole intestinal stem cell (ISC) compartment. However, previous studies have indicated that Lgr5+ cells are dispensable for intestinal regeneration, leading to two major hypotheses: one favoring the presence of a quiescent reserve ISC and the other calling for differentiated cell plasticity. To investigate these possibilities, we studied crypt epithelial cells in an unbiased fashion via high-resolution single-cell profiling. These studies, combined with in vivo lineage tracing, show that Lgr5 is not a specific ISC marker and that stemness potential exists beyond the crypt base and resides in the isthmus region, where undifferentiated cells participate in intestinal homeostasis and regeneration following irradiation (IR) injury. Our results provide an alternative model of intestinal epithelial cell organization, suggesting that stemness potential is not restricted to CBC cells, and neither de-differentiation nor reserve ISC are drivers of intestinal regeneration.

p53 mutation biases squamocolumnar junction progenitor cells towards dysplasia rather than metaplasia in Barrett's oesophagus.

BACKGROUND: While p53 mutations occur early in Barrett's oesophagus (BE) progression to oesophageal adenocarcinoma (EAC), their role in gastric cardia stem cells remains unclear. OBJECTIVE: This study investigates the impact of p53 mutation on the fate and function of cardia progenitor cells in BE to EAC progression, particularly under the duress of chronic injury. DESIGN: We used a BE mouse model (L2-IL1ß) harbouring a Trp53 mutation (R172H) to study the effects of p53 on Cck2r+ cardia progenitor cells. We employed lineage tracing, pathological analysis, organoid cultures, single-cell RNA sequencing (scRNA-seq) and computational analyses to investigate changes in progenitor cell behaviour, differentiation patterns and tumour progression. Additionally, we performed orthotopic transplantation of sorted metaplastic and mutant progenitor cells to assess their tumourigenic potential in vivo. RESULTS: The p53 mutation acts as a switch to expand progenitor cells and inhibit their differentiation towards metaplasia, but only amidst chronic injury. In L2-IL1ß mice, p53 mutation increased progenitors expansion and lineage-tracing with a shift from metaplasia to dysplasia. scRNA-seq revealed dysplastic cells arise directly from mutant progenitors rather than progressing through metaplasia. In vitro, p53 mutation enhanced BE progenitors' organoid-forming efficiency, growth, DNA damage resistance and progression to aneuploidy. Sorted metaplastic cells grew poorly with no progression to dysplasia, while mutant progenitors gave rise to dysplasia in orthotopic transplantation. Computational analyses indicated that p53 mutation inhibited stem cell differentiation through Notch activation. CONCLUSIONS: p53 mutation contributes to BE progression by increasing expansion and fitness of undifferentiated cardia progenitors and preventing their differentiation towards metaplasia.

Adult enteric Dclk1-positive glial and neuronal cells reveal distinct responses to acute intestinal injury.

Intestinal ganglionic cells in the adult enteric nervous system (ENS) are continually exposed to stimuli from the surrounding microenvironment and need at times to respond to disturbed homeostasis following acute intestinal injury. The kinase DCLK1 and intestinal Dclk1-positive cells have been reported to contribute to intestinal regeneration. Although Dclk1-positive cells are present in adult enteric ganglia, their cellular identity and response to acute injury have not been investigated in detail. Here, we reveal the presence of distinct Dclk1-tdTom+/CD49b+ glial-like and Dclk1-tdTom+/CD49b- neuronal cell types in adult myenteric ganglia. These ganglionic cells demonstrate distinct patterns of tracing over time yet show a similar expansion in response to elevated serotonergic signaling. Interestingly, Dclk1-tdTom+ glial-like and neuronal cell types appear resistant to acute irradiation injury-mediated cell death. Moreover, Dclk1-tdTom+/CD49b+ glial-like cells show prominent changes in gene expression profiles induced by injury, in contrast to Dclk1-tdTom+/CD49b- neuronal cell types. Finally, subsets of Dclk1-tdTom+/CD49b+ glial-like cells demonstrate prominent overlap with Nestin and p75NTR and strong responses to elevated serotonergic signaling or acute injury. These findings, together with their role in early development and their neural crest-like gene expression signature, suggest the presence of reserve progenitor cells in the adult Dclk1 glial cell lineage.NEW & NOTEWORTHY The kinase DCLK1 identifies glial-like and neuronal cell types in adult murine enteric ganglia, which resist acute injury-mediated cell death yet differ in their cellular response to injury. Interestingly, Dclk1-labeled glial-like cells show prominent transcriptional changes in response to injury and harbor features reminiscent of previously described enteric neural precursor cells. Our data thus add to recently emerging evidence of reserve cellular plasticity in the adult enteric nervous system.

Trefoil factor family 2 inhibits cholangiocarcinogenesis by regulating the PTEN pathway in mice.

Trefoil factor family 2 (TFF2) is one of three trefoil factor family proteins and is expressed abundantly in the gastrointestinal epithelium. Recent studies have shown that TFF2 acts as a tumor suppressor in gastric and pancreatic carcinogenesis; however, little is known about its function in cholangiocarcinogenesis. To investigate the function of TFF2 in cholangiocellular carcinoma (CCC), immunohistochemistry of surgically resected human CCC samples was performed. TFF2 expression was upregulated in the early stage and lost in the late stage of cholangiocarcinogenesis, suggesting the association of TFF2 and CCC. A TFF2 expression vector was then transfected into a CCC cell line (HuCCT1) in vitro, revealing that TFF2 functions as a tumor suppressor not only by inhibiting proliferation and invasion but also by promoting the apoptosis of cancer cells. In addition, PTEN signaling activity was downregulated by TFF2, suggesting an association between TFF2 and PTEN. Next, hepatic carcinogenesis model mice (KC; albumin-Cre/Lox-Stop-Lox KRASG12D) were bred with TFF2-knockout mice to generate a TFF2-deficient mouse model (KC/TFF2-/-). Although the incidence of hepatocellular carcinoma was not different between KC/TFF2-/- mice and control mice, biliary intraepithelial neoplasm (BilIN), the precursor of CCC, was frequently found in the biliary epithelium of KC/TFF2-/- mice. Immunohistochemistry revealed that BilIN samples from these mice did not express PTEN. In addition, two KC/TFF2-/- mice developed CCC adjacent to BilIN, suggesting that TFF2 functions to inhibit the development of CCC in vivo. These results indicate that TFF2 acts as a tumor suppressor to inhibit the development of CCC by regulating PTEN activity.

Trefoil Factor Family 1 Inhibits the Development of Hepatocellular Carcinoma by Regulating ß-Catenin Activation.

BACKGROUND AND AIMS: Recent studies have suggested that trefoil factor family 1 (TFF1) functions as a tumor suppressor in gastric and pancreatic carcinogenesis. APPROACH AND RESULTS: To investigate the role of TFF1 in hepatocarcinogenesis, we performed immunohistochemical staining of surgically resected human liver samples, transfected a TFF1 expression vector into hepatocellular carcinoma (HCC) cell lines, and employed a mouse model of spontaneous HCC development (albumin-cyclization recombination/Lox-Stop-Lox sequence-Kirsten rat sarcoma viral oncogene homologG12D [KC]); the model mouse strain was bred with a TFF1-knockout mouse strain to generate a TFF1-deficient HCC mouse model (KC/TFF1-/- ). TFF1 expression was found in some human samples with HCC. Interestingly, TFF1-positive cancer cells showed a staining pattern contradictory to that of proliferating cell nuclear antigen, and aberrant DNA hypermethylation in TFF1 promoter lesions was detected in HCC samples, indicating the tumor-suppressive role of TFF1. In vitro, induction of TFF1 expression resulted in impaired proliferative activity and enhanced apoptosis in HCC cell lines (HuH7, HepG2, and HLE). These anticancer effects of TFF1 were accompanied by the loss of nuclear ß-catenin expression, indicating inactivation of the ß-catenin signaling pathway by TFF1. In vivo, TFF1 deficiency in KC mice accelerated the early development and growth of HCC, resulting in poor survival rates. In addition, immunohistochemistry revealed that the amount of nuclear-localized ß-catenin was significantly higher in KC/TFF1-/- mice than in KC mice and that human HCC tissue showed contradictory expression patterns for ß-catenin and TFF1, confirming the in vitro observations. CONCLUSIONS: TFF1 might function as a tumor suppressor that inhibits the development of HCC by regulating ß-catenin activity.

Single-Incision Laparoscopic Splenectomy for an Unruptured Aneurysm of the Splenic Artery.

OBJECTIVE: We describe our experience of single-incision laparoscopic splenectomy (SILS) for an unruptured aneurysm of the splenic artery. CLINICAL PRESENTATION AND INTERVENTION: A 73-year-old woman was diagnosed as having a splenic aneurysm which grew from 14 to 22 mm in diameter within 2 years. Due to a contrast agent allergy, transcatheter arterial embolization could not be performed; therefore, SILS was performed with a 4-cm Z-shaped incision. The operative time and intraoperative blood loss were 132 min and 27 mL, respectively. The patient was discharged 4 days after surgery. CONCLUSION: In selected cases, SILS is a suitable and safe procedure for an unruptured aneurysm of the splenic artery.

Nociceptive neurons interact directly with gastric cancer cells via a CGRP/Ramp1 axis to promote tumor progression.

Cancer cells have been shown to exploit neurons to modulate their survival and growth, including through establishment of neural circuits within the central nervous system (CNS) 1-3 . Here, we report a distinct pattern of cancer-nerve interactions between the peripheral nervous system (PNS) and gastric cancer (GC). In multiple GC mouse models, nociceptive nerves demonstrated the greatest degree of nerve expansion in an NGF-dependent manner. Neural tracing identified CGRP+ peptidergic neurons as the primary gastric sensory neurons. Three-dimensional co-culture models showed that sensory neurons directly connect with gastric cancer spheroids through synapse-like structures. Chemogenetic activation of sensory neurons induced the release of calcium into the cytoplasm of cancer cells, promoting tumor growth and metastasis. Pharmacological ablation of sensory neurons or treatment with CGRP inhibitors suppressed tumor growth and extended survival. Depolarization of gastric tumor membranes through in vivo optogenetic activation led to enhanced calcium flux in nodose ganglia and CGRP release, defining a cancer cell-peptidergic neuronal circuit. Together, these findings establish the functional connectivity between cancer and sensory neurons, identifying this pathway as a potential therapeutic target.

Fecal DNA analysis coupled with the sighting records re-expanded a known distribution of dugongs in Ryukyu Islands after half a century.

DNA analysis of large herbivore feces samples collected from seagrass beds at two distant sites (Irabu Island in Miyako Islands and Kushi in Okinawa Island) in the Ryukyu Islands proved that some of these feces were from dugongs, which had been treated in recent studies as extinct in this region since the last stranding of a deceased individual in 2019. In addition, local knowledge of sightings of animals thought to be dugongs and confirmed cases of dugong feeding trails since 2010 were compiled to estimate its recent distribution. This is the first scientific report on the presence of this mammal in the Ryukyu Islands within the last four years, and particularly in the Miyako Islands within the last half-century. As the Ryukyu Islands are known to be the northern limit of the dugong's fragmented distribution in East Asia, conservation efforts are therefore needed.

Microbial-Dependent Recruitment of Immature Myeloid Cells Promotes Intestinal Regeneration.

BACKGROUND & AIMS: The intestinal epithelium functions both in nutrient absorption and as a barrier, separating the luminal contents from a network of vascular, fibroblastic, and immune cells underneath. After injury to the intestine, multiple cell populations cooperate to drive regeneration of the mucosal barrier, including lymphatic endothelial cells (LECs). A population of granulocytic immature myeloid cells (IMCs), marked by Hdc, participate in regeneration of multiple organs such as the colon and central nervous system, and their contribution to intestinal regeneration was investigated. METHODS: By using male and female histidine decarboxylase (Hdc) green fluorescent reporter (GFP) mice, we investigated the role of Hdc+ IMCs in intestinal regeneration after exposure to 12 Gy whole-body irradiation. The movement of IMCs was analyzed using flow cytometry and immunostaining. Ablation of Hdc+ cells using the HdcCreERT2 tamoxifen-inducible recombinase Cre system, conditional knockout of Prostaglandin-endoperoxidase synthase 2 (Ptgs2) in Hdc+ cells using HdcCre; Ptgs2 floxed mice, and visualization of LECs using Prox1tdTomato mice also was performed. The role of microbial signals was investigated by knocking down mice gut microbiomes using antibiotic cocktail gavages. RESULTS: We found that Hdc+ IMCs infiltrate the injured intestine after irradiation injury and promote epithelial regeneration in part by modulating LEC activity. Hdc+ IMCs express Ptgs2 (encoding cyclooxygenase-2/COX-2), and enables them to produce prostaglandin E2. Prostaglandin E2 acts on the prostaglandin E2 receptor 4 receptor (EP4) on LECs to promote lymphangiogenesis and induce the expression of proregenerative factors including R-spondin 3. Depletion of gut microbes leads to reduced intestinal regeneration by impaired recruitment of IMCs. CONCLUSIONS: Altogether, our results unveil a critical role for IMCs in intestinal repair by modulating LEC activity and implicate gut microbes as mediators of intestinal regeneration.

An optimized protocol for isolation of murine pancreatic single cells with high yield and purity.

Here, we present a protocol for rapidly isolating single cells from the mouse pancreas, minimizing damage caused by digestive enzymes in exocrine cells. We guide you through steps to optimize the dissection sequence, enzyme composition, and operational procedures, resulting in high yields of viable pancreatic single cells. This protocol can be applied across a wide range of research areas, including single-cell sequencing, gene expression profiling, primary cell culture, and even the development of spheroids or organoids. For complete details on the use and execution of this protocol, please refer to Jiang et al. (2023).1.

Neuro-mesenchymal interaction mediated by a ß2 adrenergic-nerve growth factor feedforward loop promotes colorectal cancer progression.

Cancer-associated fibroblasts (CAFs) and nerves, components of the tumor microenvironment, have each been shown to directly promote gastrointestinal cancers. However, it remains unknown whether these cells interact with each other to regulate cancer progression. We found that in colorectal cancer (CRC) norepinephrine induces ADRB2-dependent nerve growth factor (NGF) secretion from CAFs, which in turn increases intra-tumor sympathetic innervation and norepinephrine accumulation. Adrenergic stimulation accelerates CRC growth through ADRA2A/Gi-mediated activation of Yes-Associated Protein (YAP). NGF from CAFs directly enhances CRC cell growth via the PI3K/AKT pathway. Treatment with a tropomyosin receptor kinase (Trk) inhibitor decreased YAP and AKT activation and CRC progression in mice. In human CRC, high NGF expression is associated with the mesenchymal-like tumor subtype and poor patient survival. These findings suggest a central role for reciprocal CAF-nerve crosstalk in promoting CRC progression. Blocking this feedforward loop with a Trk inhibitor may represent a potential therapeutic approach for CRC.

A CXCR4 partial agonist improves immunotherapy by targeting polymorphonuclear myeloid-derived suppressor cells and cancer-driven granulopoiesis.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are pathologically activated neutrophils that potently impair immunotherapy responses. The chemokine receptor CXCR4, a central regulator of hematopoiesis, represents an attractive PMN-MDSC target1. Here, we fused a secreted CXCR4 partial agonist TFF2 to mouse serum albumin (MSA) and demonstrated that TFF2-MSA peptide synergized with anti-PD-1 to induce tumor regression or eradication, inhibited distant metastases, and prolonged survival in multiple gastric cancer (GC) models. Using histidine decarboxylase (Hdc)-GFP transgenic mice to track PMN-MDSC in vivo , we found TFF2-MSA selectively reduced the immunosuppressive Hdc-GFP + CXCR4 hi tumor PMN-MDSCs while preserving proinflammatory neutrophils, thereby boosting CD8 + T cell-mediated anti-tumor response together with anti-PD-1. Furthermore, TFF2-MSA systemically reduced PMN-MDSCs and bone marrow granulopoiesis. In contrast, CXCR4 antagonism plus anti-PD-1 failed to provide a similar therapeutic benefit. In GC patients, expanded PMN-MDSCs containing a prominent CXCR4 + LOX-1 + subset are inversely correlated with the TFF2 level and CD8 + T cells in circulation. Collectively, our studies introduce a strategy of using CXCR4 partial agonism to restore anti-PD-1 sensitivity in GC by targeting PMN-MDSCs and granulopoiesis.

Premalignant pancreatic cells seed stealth metastasis in distant organs in mice.

Recent findings suggest that the dissemination of tumor cells occurs at the early stage of breast and pancreatic carcinogenesis, which is known as early dissemination. The evidence of early dissemination has been demonstrated predominantly in the bloodstream and bone marrow; however, limited evidence has revealed the existence and behavior of disseminated cells in distant organs. Here, we show that premalignant pancreatic cells seed distant stealth metastasis that eventually develops into manifest metastasis. By analyzing lineage-labeled pancreatic cancer mouse models (KPCT/TFF1KO; Pdx1-Cre/LSL-KRASG12D/LSL-p53R172H/LSL-tdTomato/TFF1KO), we found that premalignant pancreatic cells, rather than mature malignant cells, were prone to enter the bloodstream and reside in the bone marrow, liver, and lung. While these metastatic cells exhibited the characteristics of the cells of host organs and did not behave as malignant cells, they underwent malignant transformation and formed distinct tumors. Surprisingly, the manifestation of distant metastasis occurred even before tumor development in the primary site. Our data revealed that disseminated premalignant cells reside stealthily in distant organs and evolve in parallel with the progression of the primary tumor. These observations suggest that we must rebuild a therapeutic strategy for metastatic pancreatic cancer.

Acute Intestinal Inflammation Depletes/Recruits Histamine-Expressing Myeloid Cells From the Bone Marrow Leading to Exhaustion of MB-HSCs.

BACKGROUND & AIMS: Histidine decarboxylase (HDC), the histamine-synthesizing enzyme, is expressed in a subset of myeloid cells but also marks quiescent myeloid-biased hematopoietic stem cells (MB-HSCs) that are activated upon myeloid demand injury. However, the role of MB-HSCs in dextran sulfate sodium (DSS)-induced acute colitis has not been addressed. METHODS: We investigated HDC+ MB-HSCs and myeloid cells by flow cytometry in acute intestinal inflammation by treating HDC-green fluorescent protein (GFP) male mice with 5% DSS at various time points. HDC+ myeloid cells in the colon also were analyzed by flow cytometry and immunofluorescence staining. Knockout of the HDC gene by using HDC-/-; HDC-GFP and ablation of HDC+ myeloid cells by using HDC-GFP; HDC-tamoxifen-inducible recombinase Cre system; diphtheria toxin receptor (DTR) mice was performed. The role of H2-receptor signaling in acute colitis was addressed by treatment of DSS-treated mice with the H2 agonist dimaprit dihydrochloride. Kaplan-Meier survival analysis was performed to assess the effect on survival. RESULTS: In acute colitis, rapid activation and expansion of MB-HSC from bone marrow was evident early on, followed by a gradual depletion, resulting in profound HSC exhaustion, accompanied by infiltration of the colon by increased HDC+ myeloid cells. Knockout of the HDC gene and ablation of HDC+ myeloid cells enhance the early depletion of HDC+ MB-HSC, and treatment with H2-receptor agonist ameliorates the depletion of MB-HSCs and resulted in significantly increased survival of HDC-GFP mice with acute colitis. CONCLUSIONS: Exhaustion of bone marrow MB-HSCs contributes to the progression of DSS-induced acute colitis, and preservation of quiescence of MB-HSCs by the H2-receptor agonist significantly enhances survival, suggesting the potential for therapeutic utility.

Up-regulation of hepatic heme oxygenase-1 expression by locally induced interleukin-6 in rats administered carbon tetrachloride intraperitoneally.

We previously reported that interleukin-6 (IL-6) was locally produced in the early period after intraperitoneal (i.p.) or subcutaneous carbon tetrachloride (CCl4) administration, but not after oral (p.o.) administration. In the present study, we focused on the up-regulation of stress-inducible proteins induced by IL-6 after i.p. CCl4 administration. The expression of heme oxygenase-1 (HO-1) (EC 1.14.99.3) mRNA and protein were induced more in rats administered CCl4 via the i.p. route, compared with the p.o. route; however, expression of heat shock protein (HSP) 72 and HSP90 mRNA were increased to similar extents in both experimental groups. The induction of HO-1 mRNA and protein after i.p. CCl4 administration were significantly reduced after pretreatment with anti-rat IL-6 antibody. Activation of the signal transducer and activator of transcription factor 3 (STAT3), which promotes HO-1 expression, peaked together with plasma levels of IL-6 after i.p. CCl4 administration, suggesting that hepatic HO-1 expression was increased by IL-6 via the Janus kinase/STAT3 pathway. The present data indicate that hepatic HO-1 is up-regulated by endogenously produced IL-6, in addition to its up-regulation by heme derived from cytochrome P450 which has already been reported in rats administered i.p. CCl4. The up-regulation of hepatic HO-1 expression may reduce the tissue injury to livers caused by CCl4.

Laparoscopic surgery for the treatment of a ruptured aneurysm of the right gastric artery: a case report.

Laparoscopic surgery for the treatment of a ruptured visceral artery aneurysm is recognized as a challenging procedure. Here, we describe our experience with laparoscopic surgery to treat a ruptured aneurysm of the right gastric artery. A 72-year-old woman was diagnosed with intra-abdominal hemorrhage caused by a ruptured aneurysm of the right gastric artery. Transcatheter arterial embolization failed because the right gastric artery could not be cannulated. Therefore, we performed laparoscopic surgery. Using laparoscopy, we detected that the bleeding from the aneurysm had ceased; thus, the planned procedure was successful. The operative time and intraoperative blood loss were 100 min and 5 mL, respectively. The patient was discharged 7 days after surgery. Laparoscopic surgery after the failure of transcatheter arterial embolization is a suitable and safe procedure for ruptured visceral artery aneurysms, provided the circulatory dynamics are stable as a result of the temporary cessation of bleeding from the ruptured aneurysm.

Interleukin-6 production by peritoneal mesothelial cells and its regulation by inflammatory factors in rats administered carbon tetrachloride intraperitoneally.

We previously reported that a high level of interleukin-6 (IL-6), which is protective against CCl(4)-induced hepatotoxicity, is produced in the peritoneal cavity in the early period after ip carbon tetrachloride (CCl(4)) administration. The objective of this study was to identify the tissues and cells involved in IL-6 production and clarify the mechanisms underlying its regulation. IL-6 mRNA levels increased significantly in the serous membranes of the mesentery and peritoneum, but not in the parenchymal organs including liver, kidney and spleen, 3 h after ip CCl(4) administration. Peritoneal mesothelial cells (PMCs), a major cell population in serous membranes, were isolated from rat peritoneal walls by trypsin digestion and cultured with peritoneal exudate fluid (PEF) from CCl(4)-administered rats. PMCs produced a high level of IL-6 in the presence of PEF recovered 0.5 h after ip CCl(4) administration. Analyses of PEF revealed that the levels of prostaglandin E(2) (PGE(2)), histamine, IL-1alpha, IL-1beta and tumor necrosis factor-alpha (TNF-alpha) increased immediately after ip CCl(4) administration. These inflammatory factors, except for histamine, stimulated IL-6 production to varying degrees, in the following order: IL-1alpha>IL-1beta>TNF-alpha>>PGE(2). In summary, the present study indicates that the high level of IL-6 observed in the rat peritoneal cavity after ip CCl(4) administration is at least partially produced by PMCs stimulated cooperatively with IL-1alpha, IL-1beta, TNF-alpha and PGE(2). These inflammatory factors may be released from tissues or cells either stimulated or injured directly by CCl(4).