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1.
Cell ; 187(12): 3056-3071.e17, 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38848678

RESUMO

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.


Assuntos
Homeostase , Mucosa Intestinal , Receptores Acoplados a Proteínas G , Regeneração , Células-Tronco , Animais , Células-Tronco/metabolismo , Células-Tronco/citologia , Camundongos , Mucosa Intestinal/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Intestinos/citologia , Diferenciação Celular , Camundongos Endogâmicos C57BL , Células Epiteliais/metabolismo , Análise de Célula Única , Masculino
2.
Gut ; 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-39353725

RESUMO

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.

3.
bioRxiv ; 2024 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-38496544

RESUMO

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.

4.
Cell Mol Gastroenterol Hepatol ; 17(3): 321-346, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-37898454

RESUMO

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.


Assuntos
Células Endoteliais , Intestinos , Células Mieloides , Proteína Vermelha Fluorescente , Regeneração , Animais , Feminino , Masculino , Camundongos , Ciclo-Oxigenase 2 , Prostaglandinas
5.
bioRxiv ; 2024 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-39257805

RESUMO

Dclk1 expression defines a rare population of cells in the normal pancreas whose frequency is increased at early stages of pancreatic tumorigenesis. The identity and the precise roles of Dclk1 expressing cells in pancreas have been matter of debate, although evidence suggests their involvement in a number of key functions, including regeneration and neoplasia. We employed a recently developed Dclk1 reporter mouse model and single cell RNAseq analysis to define Dclk1 expressing cells in normal pancreas and pancreatic neoplasia. In normal pancreas, Dclk1 epithelial expression identifies subsets of ductal, islet and acinar cells. In pancreatic neoplasia, Dclk1 expression identifies five epithelial cell populations, among which acinar-to-ductal metaplasia (ADM)-like cells and tuft-like cells are predominant. These two cell populations play opposing roles in pancreatic neoplasia, with Dclk1+ ADM-like cells sustaining tumor growth while Dclk1+ tuft-like cells restraining tumor progression. The differentiation of Kras mutant acinar cells into Dclk1+ tuft-like cells requires the activation of the transcription factor SPIB and is further supported by a cellular paracrine loop involving cancer group 2 innate lymphoid cells (ILC2) and cancer activated fibroblasts (CAFs) that provide IL13 and IL33, respectively. In turn, Dclk1+ tuft-like cells release angiotensinogen that plays protective roles against pancreatic neoplasia. Overall, our study provides novel insights on the biology of Dclk1+ cells in normal pancreas and unveils a protective axis against pancreatic neoplasia, involving CAFs, ILC2 and Dclk1+ tuft-like cells, which ultimately results in angiotensinogen release.

6.
STAR Protoc ; 5(1): 102836, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38219150

RESUMO

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.


Assuntos
Pâncreas , Hormônios Pancreáticos , Animais , Camundongos , Dissecação , Células Epiteliais , Perfilação da Expressão Gênica
7.
Cancer Discov ; 2024 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-39137067

RESUMO

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.

8.
bioRxiv ; 2024 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-39416177

RESUMO

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.

9.
J Oncol ; 2023: 6413796, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36778918

RESUMO

Background: Gastrointestinal stromal tumor (GIST) originates from a pacemaker cell, the Cajal cell. However, little is known about the cancer neuroscience in GIST. In this study, we aimed to elucidate the clinical and biological roles of adrenoceptor beta 2 (ADRB2) in GIST. Methods: Immunohistochemistry was used to evaluate the expression of ADRB2 in GIST tissues. The biological effects of ADRB2 on GIST cell proliferation, migration, invasion, and apoptosis were explored using Cell Counting Kit -8, plate colony formation assay, transwell invasion assay, and flow cytometry. We also explored the growth and metastasis of xenograft tumors in nude mice. Western blotting was used to quantify protein expression and phosphorylation. Results: ADRB2 is generally highly expressed in GIST. High ADRB2 expression was significantly associated with risk level, tumor size, mitotic count, and metastasis. Overexpression of ADRB2 promoted GIST cell proliferation, migration, invasion, and apoptosis, while silencing ADRB2 expression showed the opposite effects. Furthermore, we found that silencing endogenous ADRB2 inhibited GIST progression and metastasis in nude mice. ADRB2-induced ETV1 upregulation enhanced the activation of c-KIT. Conclusion: ADRB2 plays an important role in the proliferation and metastasis of GIST and is expected to be a potential target for the treatment of GIST.

10.
Sci Total Environ ; 904: 166799, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-37673270

RESUMO

Airborne antibiotic-resistant bacteria (ARB) can critically impact human health. We performed resistome profiling of 283 personal airborne exposure samples from 15 participants spanning 890 days and 66 locations. We found a greater diversity and abundance of airborne bacteria community and antibiotic resistomes in spring than in winter, and temperature contributed largely to the difference. A total of 1123 bacterial genera were detected, with 16 genera dominating. Of which, 7/16 were annotated as major antibiotic resistance gene (ARG) hosts. The participants were exposed to a highly dynamic collection of ARGs, including 322 subtypes conferring resistance to 18 antibiotic classes dominated by multidrug, macrolide-lincosamide-streptogramin, ß-lactam, and fosfomycin. Unlike the overall community-level bacteria exposure, an extremely high abundance of specific ARG subtypes, including lunA and qacG, were found in some samples. Staphylococcus was the predominant genus in the bacterial community, serving as a primary bacterial host for the ARGs. The annotation of ARG-carrying contigs indicated that humans and companion animals were major reservoirs for ARG-carrying Staphylococcus. This study contextualized airborne antibiotic resistomes in the precision medicine framework through longitudinal personal monitoring, which can have broad implications for human health.


Assuntos
Antibacterianos , Genes Bacterianos , Humanos , Antagonistas de Receptores de Angiotensina , Inibidores da Enzima Conversora de Angiotensina , Bactérias
11.
Cell Stem Cell ; 30(8): 1091-1109.e7, 2023 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-37541213

RESUMO

While adult pancreatic stem cells are thought not to exist, it is now appreciated that the acinar compartment harbors progenitors, including tissue-repairing facultative progenitors (FPs). Here, we study a pancreatic acinar population marked by trefoil factor 2 (Tff2) expression. Long-term lineage tracing and single-cell RNA sequencing (scRNA-seq) analysis of Tff2-DTR-CreERT2-targeted cells defines a transit-amplifying progenitor (TAP) population that contributes to normal homeostasis. Following acute and chronic injury, Tff2+ cells, distinct from FPs, undergo depopulation but are eventually replenished. At baseline, oncogenic KrasG12D-targeted Tff2+ cells are resistant to PDAC initiation. However, KrasG12D activation in Tff2+ cells leads to survival and clonal expansion following pancreatitis and a cancer stem/progenitor cell-like state. Selective ablation of Tff2+ cells prior to KrasG12D activation in Mist1+ acinar or Dclk1+ FP cells results in enhanced tumorigenesis, which can be partially rescued by adenoviral Tff2 treatment. Together, Tff2 defines a pancreatic TAP population that protects against Kras-driven carcinogenesis.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/genética , Fator Trefoil-2/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Pâncreas/metabolismo , Células Acinares/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo
12.
Cell Mol Gastroenterol Hepatol ; 11(4): 1119-1138, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33249238

RESUMO

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.


Assuntos
Medula Óssea/patologia , Colite/patologia , Células-Tronco Hematopoéticas/patologia , Histamina/metabolismo , Histidina Descarboxilase/fisiologia , Inflamação/patologia , Intestinos/patologia , Células Mieloides/patologia , Animais , Medula Óssea/imunologia , Medula Óssea/metabolismo , Colite/etiologia , Colite/metabolismo , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Intestinos/imunologia , Intestinos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/imunologia , Células Mieloides/metabolismo , Transdução de Sinais
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