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1.
Nature ; 608(7924): 784-794, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35798028

RESUMO

Cancer relapse after chemotherapy remains a main cause of cancer-related death. Although the relapse is thought to result from the propagation of resident cancer stem cells1, a lack of experimental platforms that enable the prospective analysis of cancer stem cell dynamics with sufficient spatiotemporal resolution has hindered the testing of this hypothesis. Here we develop a live genetic lineage-tracing system that allows the longitudinal tracking of individual cells in xenotransplanted human colorectal cancer organoids, and identify LGR5+ cancer stem cells that exhibit a dormant behaviour in a chemo-naive state. Dormant LGR5+ cells are marked by the expression of p27, and intravital imaging provides direct evidence of the persistence of LGR5+p27+ cells during chemotherapy, followed by clonal expansion. Transcriptome analysis reveals that COL17A1-a cell-adhesion molecule that strengthens hemidesmosomes-is upregulated in dormant LGR5+p27+ cells. Organoids in which COL17A1 is knocked out lose the dormant LGR5+p27+ subpopulation and become sensitive to chemotherapy, which suggests that the cell-matrix interface has a role in the maintenance of dormancy. Chemotherapy disrupts COL17A1 and breaks the dormancy in LGR5+p27+ cells through FAK-YAP activation. Abrogation of YAP signalling prevents chemoresistant cells from exiting dormancy and delays the regrowth of tumours, highlighting the therapeutic potential of YAP inhibition in preventing cancer relapse. These results offer a viable therapeutic approach to overcome the refractoriness of human colorectal cancer to conventional chemotherapy.


Assuntos
Neoplasias do Colo , Células-Tronco Neoplásicas , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem da Célula , Proliferação de Células , Rastreamento de Células , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Quinase 1 de Adesão Focal/metabolismo , Perfilação da Expressão Gênica , Xenoenxertos , Humanos , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/patologia , Colágenos não Fibrilares/metabolismo , Organoides/metabolismo , Organoides/patologia , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Transcrição/metabolismo , Colágeno Tipo XVII
2.
Gastroenterology ; 163(5): 1391-1406.e24, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35963362

RESUMO

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.


Assuntos
Receptores Acoplados a Proteínas G , Células-Tronco , Humanos , Camundongos , Animais , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/metabolismo , Colo/metabolismo , Mucosa Intestinal/metabolismo , Fluoruracila , Fatores de Crescimento Transformadores/metabolismo
3.
J Cell Sci ; 131(16)2018 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-30072444

RESUMO

The role of the actin cytoskeleton in the sequence of physiological epithelial repair in the intact epithelium has yet to be elucidated. Here, we explore the role of actin in gastric repair in vivo and in vitro gastric organoids (gastroids). In response to two-photon-induced cellular damage of either an in vivo gastric or in vitro gastroid epithelium, actin redistribution specifically occurred in the lateral membranes of cells neighboring the damaged cell. This was followed by their migration inward to close the gap at the basal pole of the dead cell, in parallel with exfoliation of the dead cell into the lumen. The repair and focal increase of actin was significantly blocked by treatment with EDTA or the inhibition of actin polymerization. Treatment with inhibitors of myosin light chain kinase, myosin II, trefoil factor 2 signaling or phospholipase C slowed both the initial actin redistribution and the repair. While Rac1 inhibition facilitated repair, inhibition of RhoA/Rho-associated protein kinase inhibited it. Inhibitors of focal adhesion kinase and Cdc42 had negligible effects. Hence, initial actin polymerization occurs in the lateral membrane, and is primarily important to initiate dead cell exfoliation and cell migration to close the gap.


Assuntos
Actinas/metabolismo , Mucosa Gástrica/lesões , Organoides/lesões , Multimerização Proteica/fisiologia , Reepitelização/fisiologia , Estômago/citologia , Animais , Movimento Celular , Células Cultivadas , Células Epiteliais/fisiologia , Feminino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Organoides/citologia , Organoides/fisiologia , Polimerização , Regeneração/fisiologia , Estômago/lesões
4.
J Physiol ; 597(10): 2673-2690, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30912855

RESUMO

KEY POINTS: Determining the signalling cascade of epithelial repair, using murine gastric organoids, allows definition of regulatory processes intrinsic to epithelial cells, at the same time as validating and dissecting the signalling cascade with more precision than is possible in vivo Following single cell damage, intracellular calcium selectively increases within cells adjacent to the damage site and is essential for promoting repair. Trefoil factor 2 (TFF2) acts via chemokine C-X-C receptor 4 and epidermal growth factor receptor signalling, including extracellular signal-regulated kinase activation, to drive calcium mobilization and promote gastric repair. Sodium hydrogen exchanger 2, although essential for repair, acts downstream of TFF2 and calcium mobilization. ABSTRACT: The gastric mucosa of the stomach is continually exposed to environmental and physiological stress factors that can cause local epithelial damage. Although much is known about the complex nature of gastric wound repair, the stepwise process that characterizes epithelial restitution remains poorly defined. The present study aimed to determine the effectors that drive gastric epithelial repair using a reductionist culture model. To determine the role of trefoil factor 2 (TFF2) and intracellular calcium (Ca2+ ) mobilization in gastric restitution, gastric organoids were derived from TFF2 knockout (KO) mice and yellow Cameleon-Nano15 (fluorescent calcium reporter) transgenic mice, respectively. Inhibitors and recombinant protein were used to determine the upstream and downstream effectors of gastric restitution following photodamage (PD) to single cells within the gastric organoids. Single cell PD resulted in parallel events of dead cell exfoliation and migration of intact neighbouring cells to restore a continuous epithelium in the damage site. Under normal conditions following PD, Ca2+ levels increased within neighbour migrating cells, peaking at ∼1 min, suggesting localized Ca2+ mobilization at the site of cell protrusion/migration. TFF2 KO organoids exhibit delayed repair; however, this delay can be rescued by the addition of exogenous TFF2. Inhibition of epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK)1/2 or a TFF2 receptor, chemokine C-X-C receptor 4 (CXCR4), resulted in significant delay and dampened Ca2+ mobilization. Inhibition of sodium hydrogen exchanger 2 (NHE2) caused significant delay but did not affect Ca2+ mobilization. A similar delay was observed in NHE2 KO organoids. In TFF2 KO gastric organoids, the addition of exogenous TFF2 in the presence of EGFR or CXCR4 inhibition was unable to rescue repair. The present study demonstrates that intracellular Ca2+ mobilization occurs within gastric epithelial cells adjacent to the damage site to promote repair by mechanisms that involve TFF2 signalling via CXCR4, as well as activation of EGFR and ERK1/2. Furthermore NHE2 is shown to be important for efficient repair and to operate via a mechanism either downstream or independent of calcium mobilization.


Assuntos
Cálcio/metabolismo , Organoides/metabolismo , Receptores CXCR4/metabolismo , Fator Trefoil-2/metabolismo , Animais , Cálcio/farmacologia , Epitélio , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores CXCR4/genética , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Estômago , Fator Trefoil-2/administração & dosagem , Fator Trefoil-2/genética , Fator Trefoil-2/farmacologia
5.
Infect Immun ; 87(9)2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31262979

RESUMO

Helicobacter pylori is a pathogen that chronically colonizes the stomachs of approximately half of the world's population and contributes to the development of gastric inflammation. We demonstrated previously in vivo that H. pylori uses motility to preferentially colonize injury sites in the mouse stomach. However, the chemoreceptor responsible for sensing gastric injury has not yet been identified. In this study, we utilized murine gastric organoids (gastroids) and mutant H. pylori strains to investigate the components necessary for H. pylori chemotaxis. High-intensity 730-nm light (two-photon photodamage) was used to cause single-cell damage in gastroids, and repair of the damage was monitored over time; complete repair occurred within ∼10 min in uninfected gastroids. Wild-type H. pylori accumulated at the damage site after gastric damage induction. In contrast, mutants lacking motility (ΔmotB) or chemotaxis (ΔcheY) did not accumulate at the injury site. Using mutants lacking individual chemoreceptors, we found that only TlpB was required for H. pylori accumulation, while TlpA, TlpC, and TlpD were dispensable. All strains that were able to accumulate at the damage site limited repair. When urea (an identified chemoattractant sensed by TlpB) was microinjected into the gastroid lumen, it prevented the accumulation of H. pylori at damage sites. Overall, our findings demonstrate that H. pylori colonizes and limits repair at damage sites via chemotactic motility that requires the TlpB chemoreceptor to sense signals generated by gastric epithelial cells.


Assuntos
Proteínas de Bactérias/fisiologia , Fatores Quimiotáticos/farmacologia , Quimiotaxia/fisiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/efeitos dos fármacos , Gastropatias/microbiologia , Animais , Modelos Animais de Doenças , Mucosa Gástrica/microbiologia , Camundongos
6.
Biochem Biophys Res Commun ; 488(1): 171-176, 2017 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-28483523

RESUMO

We investigated the effects of essential amino acids on intestinal stem cell proliferation and differentiation using murine small intestinal organoids (enteroids) from the jejunum. By selectively removing individual essential amino acids from culture medium, we found that 24 h of methionine (Met) deprivation markedly suppressed cell proliferation in enteroids. This effect was rescued when enteroids cultured in Met deprivation media for 12 h were transferred to complete medium, suggesting that Met plays an important role in enteroid cell proliferation. In addition, mRNA levels of the stem cell marker leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) decreased in enteroids grown in Met deprivation conditions. Consistent with this observation, Met deprivation also attenuated Lgr5-EGFP fluorescence intensity in enteroids. In contrast, Met deprivation enhanced mRNA levels of the enteroendocrine cell marker chromogranin A (ChgA) and markers of K cells, enterochromaffin cells, goblet cells, and Paneth cells. Immunofluorescence experiments demonstrated that Met deprivation led to an increase in the number of ChgA-positive cells. These results suggest that Met deprivation suppresses stem cell proliferation, thereby promoting differentiation. In conclusion, Met is an important nutrient in the maintenance of intestinal stem cells and Met deprivation potentially affects cell differentiation.


Assuntos
Aminoácidos Essenciais/farmacologia , Diferenciação Celular/efeitos dos fármacos , Metionina/farmacologia , Organoides/química , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Jejuno/química , Camundongos , Camundongos Endogâmicos C57BL
7.
Methods Mol Biol ; 2650: 141-153, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37310630

RESUMO

The intestinal epithelium maintains self-renewal and differentiation capacities via coordination of key signaling pathways, including the Wnt, bone morphogenetic protein (BMP), epidermal growth factor (EGF), and Notch signaling pathways. Based on this understanding, a combination of stem cell niche factors, EGF, Noggin, and the Wnt agonist R-spondin was shown to enable the growth of mouse intestinal stem cells and the formation of organoids with indefinite self-renewal and full differentiation capacity. Two small-molecule inhibitors, including a p38 inhibitor and a TGF-beta inhibitor, were added to propagate cultured human intestinal epithelium but at the cost of differentiation capacity. There have been improvements in culture conditions to overcome these issues. Substitution of the EGF and a p38 inhibitor with insulin-like growth factor-1 (IGF-1) and fibroblast growth factor-2 (FGF-2) enabled multilineage differentiation. Monolayer culture with mechanical flow to the apical epithelium promoted the formation of villus-like structures with mature enterocyte gene expression. Here, we summarize our recent technological improvements in human intestinal organoid culture that will deepen the understanding of intestinal homeostasis and diseases.


Assuntos
Fator de Crescimento Epidérmico , Organoides , Humanos , Animais , Camundongos , Diferenciação Celular , Intestinos , Mucosa Intestinal , Fator 2 de Crescimento de Fibroblastos
8.
Cell Rep ; 42(3): 112212, 2023 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-36870059

RESUMO

Human lung cancer is a constellation of tumors with various histological and molecular properties. To build a preclinical platform that covers this broad disease spectrum, we obtained lung cancer specimens from multiple sources, including sputum and circulating tumor cells, and generated a living biobank consisting of 43 lines of patient-derived lung cancer organoids. The organoids recapitulated the histological and molecular hallmarks of the original tumors. Phenotypic screening of niche factor dependency revealed that EGFR mutations in lung adenocarcinoma are associated with the independence from Wnt ligands. Gene engineering of alveolar organoids reveals that constitutive activation of EGFR-RAS signaling provides Wnt independence. Loss of the alveolar identity gene NKX2-1 confers Wnt dependency, regardless of EGFR signal mutation. Sensitivity to Wnt-targeting therapy can be stratified by the expression status of NKX2-1. Our results highlight the potential of phenotype-driven organoid screening and engineering for the fabrication of therapeutic strategies to combat cancer.


Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Humanos , Adenocarcinoma de Pulmão/metabolismo , Bancos de Espécimes Biológicos , Receptores ErbB/metabolismo , Genótipo , Neoplasias Pulmonares/patologia , Organoides/metabolismo , Fenótipo
9.
Cytotechnology ; 73(4): 669-682, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34349355

RESUMO

Although tumor necrosis factor-α (TNF-α) is a known major inflammatory mediator in inflammatory bowel disease (IBD) and has various effects on intestinal epithelial cell (IEC) homeostasis, the changes in IECs in the early inflammatory state induced during short-time treatment (24 h) with TNF-α remain unclear. In this study, we investigated TNF-α-induced alterations in IECs in the early inflammatory state using mouse jejunal organoids (enteroids). Of the inflammatory cytokines, i.e., TNF-α, IL-1ß, IL-6, and IL-17, only TNF-α markedly increased the mRNA level of macrophage inflammatory protein 2 (MIP-2; the mouse homologue of interleukin-8), which is induced in the early stages of inflammation. TNF-α stimulation (3 h and 6 h) decreased the mRNA level of the stem cell markers leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) and polycomb group ring finger 4 and the progenitor cell marker prominin-1, which is also known as CD133. In addition, TNF-α treatment (24 h) decreased the number of Lgr5-positive cells and enteroid proliferation. TNF-α stimulation at 3 h and 6 h also decreased the mRNA level of chromogranin A and mucin 2, which are respective markers of enteroendocrine and goblet cells. Moreover, enteroids treated with TNF-α (24 h) not only decreased the integrity of tight junctions and cytoskeletal components but also increased intercellular permeability in an influx test with fluorescent dextran, indicating disrupted intestinal barrier function. Taken together, our findings indicate that short-time treatment with TNF-α promotes the inflammatory response and decreases intestinal stem cell activity and barrier function. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00487-y.

10.
Toxins (Basel) ; 12(10)2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32987679

RESUMO

The different effects of deoxynivalenol (DON) on intestinal barrier and stem cells by its route of exposure remain less known. We explored the toxic effects of DON on intestinal barrier functions and stem cells after DON microinjection (luminal exposure) or addition to a culture medium (basolateral exposure) using three-dimensional mouse intestinal organoids (enteroids). The influx test using fluorescein-labeled dextran showed that basolateral DON exposure (1 micromolar (µM) disrupted intestinal barrier functions in enteroids compared with luminal DON exposure at the same concentration. Moreover, an immunofluorescence experiment of intestinal epithelial proteins, such as E-cadherin, claudin, zonula occludens-1 (ZO-1), and occludin, exhibited that only basolateral DON exposure broke down intestinal epithelial integrity. A time-lapse analysis using enteroids from leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5)-enhanced green fluorescence protein (EGFP) transgenic mice and 5-ethynyl-2-deoxyuridine (EdU) assay indicated that only the basolateral DON exposure, but not luminal DON exposure, suppressed Lgr5+ stem cell count and proliferative cell ratio, respectively. These results revealed that basolateral DON exposure has larger impacts on intestinal barrier function and stem cells than luminal DON exposure. This is the first report that DON had different impacts on intestinal stem cells depending on the administration route. In addition, RNA sequencing analysis showed different expression of genes among enteroids after basolateral and luminal DON exposure.


Assuntos
Células Epiteliais/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Tricotecenos/toxicidade , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Regulação da Expressão Gênica , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Jejuno/metabolismo , Jejuno/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Organoides , Permeabilidade , Células-Tronco/metabolismo , Células-Tronco/patologia , Fatores de Tempo
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