ABSTRACT
Gastroenteropancreatic (GEP) neuroendocrine neoplasm (NEN) that consists of neuroendocrine tumor and neuroendocrine carcinoma (NEC) is a lethal but under-investigated disease owing to its rarity. To fill the scarcity of clinically relevant models of GEP-NEN, we here established 25 lines of NEN organoids and performed their comprehensive molecular characterization. GEP-NEN organoids recapitulated pathohistological and functional phenotypes of the original tumors. Whole-genome sequencing revealed frequent genetic alterations in TP53 and RB1 in GEP-NECs, and characteristic chromosome-wide loss of heterozygosity in GEP-NENs. Transcriptome analysis identified molecular subtypes that are distinguished by the expression of distinct transcription factors. GEP-NEN organoids gained independence from the stem cell niche irrespective of genetic mutations. Compound knockout of TP53 and RB1, together with overexpression of key transcription factors, conferred on the normal colonic epithelium phenotypes that are compatible with GEP-NEN biology. Altogether, our study not only provides genetic understanding of GEP-NEN, but also connects its genetics and biological phenotypes.
Subject(s)
Biological Specimen Banks , Neuroendocrine Tumors/pathology , Organoids/pathology , Animals , Chromosomes, Human/genetics , Genotype , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Intestinal Neoplasms/genetics , Intestinal Neoplasms/pathology , Male , Mice , Models, Genetic , Mutation/genetics , Neuroendocrine Tumors/genetics , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Phenotype , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , Transcriptome/genetics , Whole Genome SequencingABSTRACT
Recent sequencing analyses have shed light on heterogeneous patterns of genomic aberrations in human gastric cancers (GCs). To explore how individual genetic events translate into cancer phenotypes, we established a biological library consisting of genetically engineered gastric organoids carrying various GC mutations and 37 patient-derived organoid lines, including rare genomically stable GCs. Phenotype analyses of GC organoids revealed divergent genetic and epigenetic routes to gain Wnt and R-spondin niche independency. An unbiased phenotype-based genetic screening identified a significant association between CDH1/TP53 compound mutations and the R-spondin independency that was functionally validated by CRISPR-based knockout. Xenografting of GC organoids further established the feasibility of Wnt-targeting therapy for Wnt-dependent GCs. Our results collectively demonstrate that multifaceted genetic abnormalities render human GCs independent of the stem cell niche and highlight the validity of the genotype-phenotype screening strategy in gaining deeper understanding of human cancers.
Subject(s)
Adenocarcinoma/pathology , Organoids/pathology , Stomach Neoplasms/pathology , Stomach/pathology , Thrombospondins/metabolism , Wnt Proteins/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Animals , Antigens, CD/genetics , Apoptosis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cadherins/genetics , Carcinogenesis , Cell Proliferation , Clustered Regularly Interspaced Short Palindromic Repeats , Humans , Male , Mice , Mice, Inbred NOD , Mice, SCID , Mutation , Organoids/metabolism , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolism , Thrombospondins/genetics , Tumor Cells, Cultured , Tumor Suppressor Protein p53/genetics , Wnt Proteins/genetics , Xenograft Model Antitumor AssaysABSTRACT
The small intestine is the main organ for nutrient absorption, and its extensive resection leads to malabsorption and wasting conditions referred to as short bowel syndrome (SBS). Organoid technology enables an efficient expansion of intestinal epithelium tissue in vitro1, but reconstruction of the whole small intestine, including the complex lymphovascular system, has remained challenging2. Here we generate a functional small intestinalized colon (SIC) by replacing the native colonic epithelium with ileum-derived organoids. We first find that xenotransplanted human ileum organoids maintain their regional identity and form nascent villus structures in the mouse colon. In vitro culture of an organoid monolayer further reveals an essential role for luminal mechanistic flow in the formation of villi. We then develop a rat SIC model by repositioning the SIC at the ileocaecal junction, where the epithelium is exposed to a constant luminal stream of intestinal juice. This anatomical relocation provides the SIC with organ structures of the small intestine, including intact vasculature and innervation, villous structures, and the lacteal (a fat-absorbing lymphatic structure specific to the small intestine). The SIC has absorptive functions and markedly ameliorates intestinal failure in a rat model of SBS, whereas transplantation of colon organoids instead of ileum organoids invariably leads to mortality. These data provide a proof of principle for the use of intestinal organoids for regenerative purposes, and offer a feasible strategy for SBS treatment.
Subject(s)
Colon/cytology , Ileum/transplantation , Intestinal Mucosa/cytology , Organoids/transplantation , Regeneration , Regenerative Medicine/methods , Short Bowel Syndrome/therapy , Animals , Colon/blood supply , Colon/innervation , Colon/surgery , Disease Models, Animal , Heterografts , Humans , Ileum/cytology , Intestinal Mucosa/blood supply , Intestinal Mucosa/innervation , Intestinal Mucosa/surgery , Male , Organ Culture Techniques , Organoids/cytology , Rats , Rats, Inbred Lew , Short Bowel Syndrome/pathology , Short Bowel Syndrome/surgeryABSTRACT
Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases.
Subject(s)
Dyrk Kinases , Hedgehog Proteins , Protein Serine-Threonine Kinases , Protein-Tyrosine Kinases , Signal Transduction , Zinc Finger Protein Gli2 , Zinc Finger Protein Gli3 , Animals , Zinc Finger Protein Gli3/metabolism , Zinc Finger Protein Gli3/genetics , Zinc Finger Protein Gli2/metabolism , Zinc Finger Protein Gli2/genetics , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Mice , Protein-Tyrosine Kinases/metabolism , Protein-Tyrosine Kinases/genetics , Humans , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Kruppel-Like Transcription Factors/metabolism , Kruppel-Like Transcription Factors/genetics , Cell Proliferation , Cilia/metabolism , Smoothened Receptor/metabolism , Smoothened Receptor/genetics , Nuclear Proteins , Repressor ProteinsABSTRACT
With ageing, normal human tissues experience an expansion of somatic clones that carry cancer mutations1-7. However, whether such clonal expansion exists in the non-neoplastic intestine remains unknown. Here, using whole-exome sequencing data from 76 clonal human colon organoids, we identify a unique pattern of somatic mutagenesis in the inflamed epithelium of patients with ulcerative colitis. The affected epithelium accumulates somatic mutations in multiple genes that are related to IL-17 signalling-including NFKBIZ, ZC3H12A and PIGR, which are genes that are rarely affected in colon cancer. Targeted sequencing validates the pervasive spread of mutations that are related to IL-17 signalling. Unbiased CRISPR-based knockout screening in colon organoids reveals that the mutations confer resistance to the pro-apoptotic response that is induced by IL-17A. Some of these genetic mutations are known to exacerbate experimental colitis in mice8-11, and somatic mutagenesis in human colon epithelium may be causally linked to the inflammatory process. Our findings highlight a genetic landscape that adapts to a hostile microenvironment, and demonstrate its potential contribution to the pathogenesis of ulcerative colitis.
Subject(s)
Colitis, Ulcerative/genetics , Epithelium/metabolism , Interleukin-17/genetics , Mutation , Colitis, Ulcerative/metabolism , Humans , Interleukin-17/metabolism , Phenotype , Signal TransductionABSTRACT
BACKGROUND: Sodium picosulfate (SP)/magnesium citrate (MC) and polyethylene glycol (PEG) plus ascorbic acid are recommended by Western guidelines as laxative solutions for bowel preparation. Clinically, SP/MC has a slower post-dose defaecation response than PEG and is perceived as less cleansing; therefore, it is not currently used for major bowel cancer screening preparation. The standard formulation for bowel preparation is PEG; however, a large dose is required, and it has a distinctive flavour that is considered unpleasant. SP/MC requires a small dose and ensures fluid intake because it is administered in another beverage. Therefore, clinical trials have shown that SP/MC is superior to PEG in terms of acceptability. We aim to compare the novel bowel cleansing method (test group) comprising SP/MC with elobixibat hydrate and the standard bowel cleansing method comprising PEG plus ascorbic acid (standard group) for patients preparing for outpatient colonoscopy. METHODS: This phase III, multicentre, single-blind, noninferiority, randomised, controlled, trial has not yet been completed. Patients aged 40-69 years will be included as participants. Patients with a history of abdominal or pelvic surgery, constipation, inflammatory bowel disease, or severe organ dysfunction will be excluded. The target number of research participants is 540 (standard group, 270 cases; test group, 270 cases). The primary endpoint is the degree of bowel cleansing (Boston Bowel Preparation Scale [BBPS] score ≥ 6). The secondary endpoints are patient acceptability, adverse events, polyp/adenoma detection rate, number of polyps/adenomas detected, degree of bowel cleansing according to the BBPS (BBPS score ≥ 8), degree of bowel cleansing according to the Aronchik scale, and bowel cleansing time. DISCUSSION: This trial aims to develop a "patient-first" colon cleansing regimen without the risk of inadequate bowel preparation by using both elobixibat hydrate and SP/MC. TRIAL REGISTRATION: Japan Registry of Clinical Trials (jRCT; no. s041210067; 9 September 2021; https://jrct.niph.go.jp/ ), protocol version 1.5 (May 1, 2023).
Subject(s)
Citrates , Citric Acid , Dipeptides , Organometallic Compounds , Picolines , Polyethylene Glycols , Polyps , Thiazepines , Humans , Cathartics , Outpatients , Ascorbic Acid/adverse effects , Single-Blind Method , Colonoscopy/methods , Randomized Controlled Trials as Topic , Multicenter Studies as Topic , Clinical Trials, Phase III as TopicABSTRACT
Radiation enteritis (RE) is a prevalent complication of radiotherapy for pelvic malignant tumors, characterized by severe intestinal epithelial destruction and progressive submucosal fibrosis. However, little is known about the pathogenesis of this disease, and so far, there is no specific targeted therapy. Here, we report that CXCL16 is upregulated in the injured intestinal tissues of RE patients and in a mouse model. Genetic deletion of Cxcl16 mitigates fibrosis and promotes intestinal stem cell-mediated epithelial regeneration after radiation injury in mice. Mechanistically, CXCL16 functions on myofibroblasts through its receptor CXCR6 and activates JAK3/STAT3 signaling to promote fibrosis and, at the same time, to transcriptionally modulate the levels of BMP4 and hepatocyte growth factor (HGF) in myofibroblasts. Moreover, we find that CXCL16 and CXCR6 auto- and cross-regulate themselves in positive feedback loops. Treatment with CXCL16 neutralizing monoclonal antibody attenuates fibrosis and improves the epithelial repair in RE mouse model. Our findings emphasize the important role of CXCL16 in the progression of RE and suggest that CXCL16 signaling could be a potential therapeutic target for RE. © 2022 The Pathological Society of Great Britain and Ireland.
Subject(s)
Chemokine CXCL16 , Enteritis , Radiation Injuries , Animals , Mice , Chemokine CXCL16/metabolism , Enteritis/etiology , Enteritis/metabolism , Fibrosis , Radiation Injuries/genetics , Receptors, CXCR6 , RegenerationABSTRACT
OBJECTIVES: Despite recent advances in endoscopic equipment and diagnostic techniques, early detection of ulcerative colitis-associated neoplasia (UCAN) remains difficult because of the complex background of the inflamed mucosa of ulcerative colitis and the morphologic diversity of the lesions. We aimed to describe the main diagnostic patterns for UCAN in our cohort, including lateral extension surrounding flat lesions. METHODS: Sixty-three lesions in 61 patients with flat-type dysplasia that were imaged with dye chromoendoscopy (DCE) were included in this analysis. These DCE images were analyzed to clarify the dye-chromoendoscopic imaging characteristics of flat dysplasia, and the lesions were broadly classified into dysplastic and nondysplastic mucosal patterns. RESULTS: Dysplastic mucosal patterns were classified into two types: small round patterns with round to roundish structures, and mesh patterns with intricate mesh-like structures. Lesions with a nondysplastic mucosal pattern were divided into two major types: a ripple-like type and a gyrus-like type. Of note, 35 lesions (55.6%) had a small round pattern, and 51 lesions (80.9%) had some type of mesh pattern. About 70% of lesions with small round patterns and 49% of lesions with mesh patterns were diagnosed as high-grade dysplasia or carcinoma, while about 30% of lesions with small round patterns and 51% of lesions with mesh patterns were diagnosed as low-grade dysplasia. CONCLUSION: When a characteristic mucosal pattern, such as a small round or mesh pattern, is found by DCE, the possibility of UCAN should be considered.
Subject(s)
Colitis, Ulcerative , Humans , Colitis, Ulcerative/diagnostic imaging , Colitis, Ulcerative/pathology , Indigo Carmine , Colonoscopy/methods , Carmine , HyperplasiaABSTRACT
BACKGROUND & AIMS: In the mouse intestinal epithelium, Lgr5+ stem cells are vulnerable to injury, owing to their predominantly cycling nature, and their progenies de-differentiate to replenish the stem cell pool. However, how human colonic stem cells behave in homeostasis and during regeneration remains unknown. METHODS: Transcriptional heterogeneity among colonic epithelial cells was analyzed by means of single-cell RNA sequencing analysis of human and mouse colonic epithelial cells. To trace the fate of human colonic stem or differentiated cells, we generated LGR5-tdTomato, LGR5-iCasase9-tdTomato, LGR5-split-Cre, and KRT20-ERCreER knock-in human colon organoids via genome engineering. p27+ dormant cells were further visualized with the p27-mVenus reporter. To analyze the dynamics of human colonic stem cells in vivo, we orthotopically xenotransplanted fluorescence-labeled human colon organoids into immune-deficient mice. The cell cycle dynamics in xenograft cells were evaluated using 5-ethynyl-2'-deoxyuridine pulse-chase analysis. The clonogenic capacity of slow-cycling human stem cells or differentiated cells was analyzed in the context of homeostasis, LGR5 ablation, and 5-fluorouracil-induced mucosal injury. RESULTS: Single-cell RNA sequencing analysis illuminated the presence of nondividing LGR5+ stem cells in the human colon. Visualization and lineage tracing of slow-cycling LGR5+p27+ cells and orthotopic xenotransplantation validated their homeostatic lineage-forming capability in vivo, which was augmented by 5-FU-induced mucosal damage. Transforming growth factor-ß signaling regulated the quiescent state of LGR5+ cells. Despite the plasticity of differentiated KRT20+ cells, they did not display clonal growth after 5-FU-induced injury, suggesting that occupation of the niche environment by LGR5+p27+ cells prevented neighboring differentiated cells from de-differentiating. CONCLUSIONS: Our results highlight the quiescent nature of human LGR5+ colonic stem cells and their contribution to post-injury regeneration.
Subject(s)
Receptors, G-Protein-Coupled , Stem Cells , Humans , Mice , Animals , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Stem Cells/metabolism , Colon/metabolism , Intestinal Mucosa/metabolism , Fluorouracil , Transforming Growth Factors/metabolismABSTRACT
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the disease COVID-19 can lead to serious symptoms, such as severe pneumonia, in the elderly and those with underlying medical conditions. While vaccines are now available, they do not work for everyone and therapeutic drugs are still needed, particularly for treating life-threatening conditions. Here, we showed nasal delivery of a new, unmodified camelid single-domain antibody (VHH), termed K-874A, effectively inhibited SARS-CoV-2 titers in infected lungs of Syrian hamsters without causing weight loss and cytokine induction. In vitro studies demonstrated that K-874A neutralized SARS-CoV-2 in both VeroE6/TMPRSS2 and human lung-derived alveolar organoid cells. Unlike other drug candidates, K-874A blocks viral membrane fusion rather than viral attachment. Cryo-electron microscopy revealed K-874A bound between the receptor binding domain and N-terminal domain of the virus S protein. Further, infected cells treated with K-874A produced fewer virus progeny that were less infective. We propose that direct administration of K-874A to the lung could be a new treatment for preventing the reinfection of amplified virus in COVID-19 patients.
Subject(s)
Antibodies, Viral/administration & dosage , Antiviral Agents/administration & dosage , COVID-19 , Single-Domain Antibodies/administration & dosage , Virus Attachment/drug effects , Administration, Intranasal , Animals , Chlorocebus aethiops , Cricetinae , Disease Models, Animal , Humans , Mesocricetus , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/immunology , Vero CellsABSTRACT
The bacterium Leptothrix cholodnii generates cell chains encased in sheaths that are composed of woven nanofibrils. The nanofibrils are mainly composed of glycoconjugate repeats, and several glycosyltransferases (GTs) are required for its biosynthesis. However, only one GT (LthA) has been identified to date. In this study, we screened spontaneous variants of L. cholodnii SP6 to find those that form smooth colonies, which is one of the characteristics of sheathless variants. Genomic DNA sequencing of an isolated variant revealed an insertion in the locus Lcho_0972, which encodes a putative GT family 8 protein. We thus designated this protein LthB and characterized it using deletion mutants and antibodies. LthB localized adjacent to the cell envelope. ΔlthB cell chains were nanofibril free and thus sheathless, indicating that LthB is involved in nanofibril biosynthesis. Unlike the ΔlthA mutant and the wild-type strain, which often generate planktonic cells, most ΔlthB organisms presented as long cell chains under static conditions, resulting in deficient pellicle formation, which requires motile planktonic cells. These results imply that sheaths are not required for elongation of cell chains. Finally, calcium depletion, which induces cell chain breakage due to sheath loss, abrogated the expression of LthA, but not LthB, suggesting that these GTs cooperatively participate in glycoconjugate biosynthesis under different signaling controls. IMPORTANCE In recent years, the regulation of cell chain elongation of filamentous bacteria via extracellular signals has attracted attention as a potential strategy to prevent clogging of water distribution systems and filamentous bulking of activated sludge in industrial settings. However, a fundamental understanding of the ecology of filamentous bacteria remains elusive. Since sheath formation is associated with cell chain elongation in most of these bacteria, the molecular mechanisms underlying nanofibril sheath formation, including the intracellular signaling cascade in response to extracellular stimuli, must be elucidated. Here, we isolated a sheathless variant of L. cholodnii SP6 and thus identified a novel glycosyltransferase, LthB. Although mutants with deletions of lthA, encoding another GT, and lthB were both defective for nanofibril formation, they exhibited different phenotypes of cell chain elongation and pellicle formation. Moreover, LthA expression, but not LthB expression, was influenced by extracellular calcium, which is known to affect nanofibril formation, indicating the functional diversities of LthA and LthB. Such molecular insights are critical for a better understanding of ecology of filamentous bacteria, which, in turn, can be used to improve strategies to control filamentous bacteria in industrial facilities.
Subject(s)
Glycosyltransferases , Leptothrix , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Leptothrix/physiology , Calcium/metabolism , Sequence Analysis, DNA , Glycoconjugates/metabolismABSTRACT
Inflammatory bowel disease (IBD) is a chronic relapsing-remitting inflammatory disease of the gastrointestinal tract with an unknown etiology, and its incidence is increasing worldwide. Recent advances in immunomodulatory therapeutic agents such as biologics and small-molecule inhibitors have improved the prognosis of patients with IBD. However, some patients are refractory and resistant to these immunomodulatory therapies, and new therapies are needed. Given the importance of the intestinal epithelium in IBD pathogenesis, the difficulty of culturing intestinal epithelial cells (IECs) for long periods remains an obstacle in IBD research. Over the past 15 years, intestinal stem cells have been identified, and the in vivo microenvironment, called the niche, required for their maintenance has been elucidated, making the permanent culture of IECs possible. Recapitulating the niche in vitro, the intestinal epithelium forms 3-dimensional structures called organoids that simulate the intestinal epithelium in vivo. The intestinal epithelium plays an important role in the intestinal barrier and immunomodulatory functions and serves as a physical structure that separates the intestinal lumen from the body. Recent studies have revealed that functional disruption of the intestinal epithelium is closely related to the pathogenesis of IBD, and IBD research using organoids has attracted attention. In this review, we discuss the application of adult tissue-derived organoids culture technology to elucidate the pathogenesis of IBD and to develop novel therapies, including regenerative treatments.
Subject(s)
Inflammatory Bowel Diseases , Organoids , Adult , Epithelial Cells/pathology , Humans , Inflammatory Bowel Diseases/pathology , Inflammatory Bowel Diseases/therapy , Intestinal Mucosa/pathology , Intestines , Organoids/pathologyABSTRACT
BACKGROUND AND AIMS: Endoscopic remission is known to be defined as a Mayo endoscopic subscore (MES) of ≤1 in patients with ulcerative colitis (UC). However, some individuals experience relapse even after showing endoscopic remission under white-light imaging (WLI), and no tool exists that can detect these individuals. The aim of this study was to clarify the usefulness of texture and color enhancement imaging (TXI) in the assessment of inflammation in patients with UC. METHODS: This was a prospective, single-arm, observational study conducted at a university hospital. From January 2021 to December 2021, 146 UC patients with endoscopic remission were enrolled. Images were evaluated by WLI, TXI, and pathologic evaluation, followed by prognostic studies. The primary endpoint of the study was the cumulative relapse of UC in each TXI score. The secondary endpoints were the association between TXI and pathologic scores, predictors of relapse, and interobserver agreement between the MES and TXI scores. RESULTS: Patients with TXI score 2 had significantly lower UC relapse-free rates than did those with TXI scores 0-1 (log-rank test, P < .01). When pathologic remission was defined as Matts grade ≤2, the rate of pathologic remission decreased significantly with higher TXI scores (P = .01). In multivariate analysis, TXI score 2 was the only risk factor for UC relapse (P < .01; hazard ratio, 4.16; 95% confidence interval, 1.72-10.04). Interobserver agreement on the TXI score was good (κ = 0.597-0.823). CONCLUSION: TXI can be used to identify populations with poor prognosis in MES 1, for whom treatment intensification has been controversial.
Subject(s)
Colitis, Ulcerative , Humans , Colitis, Ulcerative/pathology , Colonoscopy/methods , Prospective Studies , Intestinal Mucosa/pathology , Prognosis , Severity of Illness IndexABSTRACT
The cancer stem cell (CSC) theory highlights a self-renewing subpopulation of cancer cells that fuels tumour growth. The existence of human CSCs is mainly supported by xenotransplantation of prospectively isolated cells, but their clonal dynamics and plasticity remain unclear. Here, we show that human LGR5+ colorectal cancer cells serve as CSCs in growing cancer tissues. Lineage-tracing experiments with a tamoxifen-inducible Cre knock-in allele of LGR5 reveal the self-renewal and differentiation capacity of LGR5+ tumour cells. Selective ablation of LGR5+ CSCs in LGR5-iCaspase9 knock-in organoids leads to tumour regression, followed by tumour regrowth driven by re-emerging LGR5+ CSCs. KRT20 knock-in reporter marks differentiated cancer cells that constantly diminish in tumour tissues, while reverting to LGR5+ CSCs and contributing to tumour regrowth after LGR5+ CSC ablation. We also show that combined chemotherapy potentiates targeting of LGR5+ CSCs. These data provide insights into the plasticity of CSCs and their potential as a therapeutic target in human colorectal cancer.
Subject(s)
Cell Tracking , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Molecular Targeted Therapy , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/pathology , Receptors, G-Protein-Coupled/metabolism , Animals , Cell Differentiation , Cell Lineage , Cell Proliferation , Cell Self Renewal , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Gene Knock-In Techniques , Humans , Keratin-20/genetics , Keratin-20/metabolism , Male , Mice , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/transplantation , Organoids/metabolism , Organoids/pathology , Organoids/transplantation , Receptors, G-Protein-Coupled/genetics , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Local resection, including endoscopic resection, is recommended for rectal neuroendocrine tumors (NETs) < 15 mm in patients without risk factors for metastasis, though the short- and long-term outcomes are unclear. AIMS: This study investigates the efficacy of endoscopic resection for rectal NETs < 15 mm. METHODS: The short- and long-term outcomes of patients with rectal NETs < 15 mm who underwent endoscopic resection and the outcomes of each endoscopic technique were analyzed. The tumors were stratified as < 10 mm (small-size group, SSG) and 10-14 mm (intermediate-size group, IMG). RESULTS: Overall, 139 lesions (SSG, n = 118; IMG, n = 21) were analyzed. All tumors were classified as G1 (n = 135) or G2 (n = 4) according to the 2019 World Health Organization grading criteria. The complete resection rate was not different between the groups (P = 0.151). Endoscopic submucosal dissection (ESD) and endoscopic submucosal resection with a ligation device (ESMR-L) achieved complete resection rates > 90% in the SSG. The ESMR-L procedure time (P < 0.001) and hospitalized period (P < 0.001) were significantly shorter than those of ESD. ESD achieved a complete resection rate of 80.0% in the IMG. The tumor size did not affect the overall survival or rate of lymph node/distant metastases. CONCLUSIONS: Endoscopic resection is a feasible and effective treatment for patients with rectal NETs < 15 mm without the risk factors of metastasis. ESMR-L and ESD are optimal techniques for resecting tumors smaller than 10 mm and 10-14 mm, respectively.
Subject(s)
Endoscopic Mucosal Resection , Neuroendocrine Tumors , Rectal Neoplasms , Humans , Neuroendocrine Tumors/surgery , Neuroendocrine Tumors/pathology , Retrospective Studies , Rectal Neoplasms/pathology , Endoscopic Mucosal Resection/methods , Treatment Outcome , Lymphatic Metastasis/pathology , Intestinal Mucosa/pathologyABSTRACT
OBJECTIVES: Large mucosal defects following gastric endoscopic submucosal dissection (ESD) cause postoperative bleeding. To address this limitation and ensure closure of large mucosal defects, we developed the reopenable clip-over-the-line method (ROLM) using a reopenable clip and nylon line. The purpose of this study was to evaluate the feasibility of the ROLM for closure of large mucosal defects following gastric ESD in a prospective, consecutive series of cases. METHODS: We performed the ROLM on 50 consecutive patients with gastric mucosal defects at the Ise Red Cross Hospital and Mie Prefectural Shima Hospital. The time to complete the ROLM, numbers of clips and lines required, size of defect, and closure success rate were measured, and postoperative adverse events were recorded. RESULTS: In all, 50 lesions were included in this study period between July 2021 and March 2022. The success rates of defect closure and defect closure without submucosal dead space of the ROLM were both 100% (50/50), with a median ROLM time of 30 (range, 14-35) min and a median resected specimen major axis of 45 (range, 31-73) mm. The median number of reopenable clips used was 31 (range, 10-93). Following gastric ESD, two cases of post-ESD bleeding were observed during the follow-up periods. CONCLUSION: Our results suggest that ROLM is a feasible strategy for complete closure of mucosal defects post-ESD without submucosal dead space. Future comparative studies with post-ESD bleeding rate as the main outcome are desirable to evaluate the efficacy of ROLM.
Subject(s)
Endoscopic Mucosal Resection , Humans , Endoscopic Mucosal Resection/adverse effects , Endoscopic Mucosal Resection/methods , Feasibility Studies , Prospective Studies , Gastric Mucosa/surgery , Surgical Instruments , Treatment Outcome , Retrospective StudiesABSTRACT
Endoscopic submucosal dissection is an established method for complete resection of large and early gastrointestinal tumors. However, methods to reduce bleeding, perforation, and other adverse events after endoscopic resection (ER) have not yet been defined. Mucosal defect closure is often performed endoscopically with a clip. Recently, reopenable clips and large-teeth clips have also been developed. The over-the-scope clip enables complete defect closure by withdrawing the endoscope once and attaching the clip. Other methods involve attaching the clip-line or a ring with an anchor to appose the edges of the mucosal defect, followed by the use of an additional clip for defect closure. Since clips are limited by their grasping force and size, other methods, such as endoloop closure, endoscopic ligation with O-ring closure, and the reopenable clip over-the-line method, have been developed. In recent years, techniques often utilized for full-thickness ER of submucosal tumors have been widely used in full-thickness defect closure. Specialized devices and techniques for defect closure have also been developed, including the curved needle and line, stitches, and an endoscopic tack and suture device. These clips and suture devices are applied for defect closure in emergency endoscopy, accidental perforations, and acute and chronic fistulas. Although endoscopic defect closure with clips has a high success rate, endoscopists need to simplify and promote endoscopic closure techniques to prevent adverse events after ER.
Subject(s)
Endoscopic Mucosal Resection , Gastrointestinal Neoplasms , Humans , Suture Techniques , Endoscopy, Gastrointestinal/adverse effects , Wound Closure Techniques , Gastrointestinal Neoplasms/etiology , Surgical InstrumentsABSTRACT
BACKGROUND AND AIMS: Diffuse-type gastric cancer (GC) is currently subdivided into signet-ring cell carcinoma (SRCC) and non-SRCC, referred to as poorly cohesive carcinoma not otherwise specified (PCC-NOS). Although these subtypes are considered to be independent, they often coexist in the same tumors, raising a question of whether they clonally differ or not. To tackle this question, we established an experimental platform for human diffuse GC that enables accurate modeling of histologic subtypes. METHODS: Seven patient-derived diffuse GC organoid lines were established, characterized by histopathologic analysis, in situ hybridization, and gene expression analysis. For genetic modeling of diffuse GC, we knocked out CDH1 and/or TP53 in human normal gastric organoids. Green fluorescent protein-labeled GC organoids were xenotransplanted into immune-deficient mice for in vivo assessment. RESULTS: PCC-NOS organoids transformed into SRCC-like structures on removal of Wnt and R-spondin from the culture medium. This morphologic change paralleled downregulation of Wnt-target and gastric stem cell genes, including LGR5, and elevation of differentiation markers, such as KRT20 and MUCs. The association between Wnt target gene expression and histologic subtypes was confirmed in 3 patient-derived GC tissues. In vivo, single clone-derived organoids formed tumors that comprised 2 distinct histologic compartments, each corresponding to SRCC and PCC-NOS. The transition from PCC-NOS to SRCC histology reflected the abundance of surrounding R-spondin-expressing fibroblasts. CONCLUSIONS: SRCC and PCC-NOS were clonally identical, and their morphology was regulated by extracellular Wnt and R-spondin expression. Our results decoded how genetic mutations and the tumor environment shape pathohistologic and biologic phenotypes in human diffuse GCs.
Subject(s)
Carcinoma, Signet Ring Cell/parasitology , Gastric Mucosa/pathology , Stomach Neoplasms/pathology , Wnt Signaling Pathway , Aged , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Cadherins/genetics , Cadherins/metabolism , Carcinoma, Signet Ring Cell/genetics , Female , Gastric Mucosa/cytology , Gene Knockout Techniques , Humans , Male , Mice , Middle Aged , Organoids/pathology , Primary Cell Culture , RNA-Seq , Stomach Neoplasms/genetics , Thrombospondins , Tumor Cells, Cultured , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Exome Sequencing , Xenograft Model Antitumor AssaysABSTRACT
The bacterium Leptothrix cholodnii generates filaments encased in a sheath comprised of woven nanofibrils. In static liquid culture, L. cholodnii moves toward the air-liquid interface, where it forms porous pellicles. Observations of aggregation at the interface reveal that clusters consisting of only a few bacteria primarily grow by netting free cells. These growing clusters hierarchically enlarge through the random docking of other small clusters. We find that the bacteria swim using their polar flagellum toward the interface, where their sheath assists them in intertwining with others and thereby promotes the formation of small clusters. In contrast, sheathless hydrophobic mutant cells get stuck to the interface. We find that the nanofibril sheath is vital for robust pellicle formation as it lowers cell surface hydrophobicity by 60%, thereby reducing their adsorption and enabling cells to move toward and stick together at the air-liquid interface. IMPORTANCE Efficient and sustainable management of water resources is becoming a fundamental issue for supporting growing populations and for developing economic activity. Fundamental to this management is the treatment of wastewater. Microorganisms are the active component of activated sludge that is employed in the biodegradation process of many wastewater treatment facilities. However, uncontrolled growth of filamentous bacteria such as Sphaerotilus often results in filamentous bulking, lowering the efficiency of water treatment systems. To prevent this undesirable condition, strategies based on a fundamental understanding of the ecology of filamentous bacteria are required. Although the filamentous bacterium Leptothrix cholodnii, which is closely related to Sphaerotilus, is a minor inhabitant of activated sludge, its complete genome sequence is known, making gene manipulation relatively easy. Moreover, L. cholodnii generates porous pellicles under static conditions, which may be a characteristic of filamentous bulking. We show that both swimming motility and nanofibril-mediated air-liquid interface attachment are required for porous pellicle formation. These insights are critical for a better understanding of the characteristics of filamentous bulking and might improve strategies to control activated sludge.
Subject(s)
Leptothrix , Sewage/microbiology , Porosity , Wastewater , Bacteria/metabolismABSTRACT
BACKGROUND & AIMS: Traditional serrated adenomas (TSAs) are rare colorectal polyps with unique histologic features. Fusions in R-spondin genes have been found in TSAs, but it is not clear whether these are sufficient for TSA development, due to the lack of a chromosome engineering platform for human tissues. We studied the effects of fusions in R-spondin genes and other genetic alterations found in TSA using CRISPR-Cas9-mediated chromosome and genetic modification of human colonic organoids. METHODS: We introduced chromosome rearrangements that involve R-spondin genes into human colonic organoids, with or without disruption of TP53, using CRISPR-Cas9 (chromosome-engineered organoids). We then knocked a mutation into BRAF encoding the V600E substitution and overexpressed the GREM1 transgene; the organoids were transplanted into colons of NOG mice and growth of xenograft tumors was measured. Colon tissues were collected and analyzed by immunohistochemistry or in situ hybridization. We also established 2 patient-derived TSA organoid lines and characterized their genetic features and phenotypes. We inserted a bicistronic cassette expressing a dimerizer-inducible suicide gene and fluorescent marker downstream of the LGR5 gene in the chromosome-engineered organoids; addition of the dimerizer eradicates LGR5+ cells. Some tumor-bearing mice were given intraperitoneal injections of the dimerizer to remove LGR5-expressing cells. RESULTS: Chromosome engineering of organoids required disruption of TP53 or culture in medium containing IGF1 and FGF2. In colons of mice, organoids that expressed BRAFV600E and fusions in R-spondin genes formed flat serrated lesions. Patient-derived TSA organoids grew independent of exogenous R-spondin, and 1 line grew independent of Noggin. Organoids that overexpressed GREM1, in addition to BRAFV600E and fusions in R-spondin genes, formed polypoid tumors in mice that had histologic features similar to TSAs. Xenograft tumors persisted after loss of LGR5-expressing cells. CONCLUSIONS: We demonstrated efficient chromosomal engineering of human normal colon organoids. We introduced genetic and chromosome alterations into human colon organoids found in human TSAs; tumors grown from these organoids in mice had histopathology features of TSAs. This model might be used to study progression of human colorectal tumors with RSPO fusion gene and GREM1 overexpression.