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1.
Artículo en Inglés | MEDLINE | ID: mdl-39320093

RESUMEN

COVID-19 commonly presents as pneumonia, with those most severely affected progressing to respiratory failure. Patient responses to SARS-CoV-2 infection are varied, with comorbidities acting as contributors to varied outcomes. Focusing on one such major comorbidity, we assessed whether pharmacological induction of Type I Diabetes Mellitus (T1DM) would increase the severity of lung injury in a murine model of COVID-19 pneumonia utilizing wild type mice infected with mouse-adapted SARS-CoV-2. Hyperglycemic mice exhibited increased weight loss and reduced blood oxygen saturation in comparison to their euglycemic counterparts, suggesting that these animals indeed experienced more severe lung injury. Transcriptomic analysis revealed a significant impairment of the adaptive immune response in the lungs of diabetic mice compared to those of control. In order to expand the options available for tissue analysis due to biosafety restrictions, we employed a new technique to digest highly fixed tissue into a single cell suspension, originally designed for scRNA-Seq, which we then adapted for flow cytometric analysis. Flow immunophenotyping and scRNA-Seq confirmed impaired recruitment of T cells into the lungs of T1DM animals. Additionally, scRNA-Seq revealed a distinct, highly inflammatory macrophage profile in the diabetic cohort that correlates with the more severe infection these mice experienced clinically, allowing insight into a possible mechanism for this phenomenon. Recognizing the near certainty that respiratory viruses will continue to present significant public health concerns for the foreseeable future, our study provides key insights into how T1DM results in a much more severe infection and identifies possible targets to ameliorate comorbidity-associated severe disease.

2.
EMBO Rep ; 23(11): e55209, 2022 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-36120829

RESUMEN

The intestinal epithelium exhibits a rapid and efficient regenerative response to injury. Emerging evidence supports a model where plasticity of differentiated cells, particularly those in the secretory lineages, contributes to epithelial regeneration upon ablation of injury-sensitive stem cells. However, such facultative stem cell activity is rare within secretory populations. Here, we ask whether specific functional properties predict facultative stem cell activity. We utilize in vivo labeling combined with ex vivo organoid formation assays to evaluate how cell age and autophagic state contribute to facultative stem cell activity within secretory lineages. Strikingly, we find that cell age (time elapsed since cell cycle exit) does not correlate with secretory cell plasticity. Instead, high autophagic vesicle content predicts plasticity and resistance to DNA damaging injury independently of cell lineage. Our findings indicate that autophagic status prior to injury serves as a lineage-agnostic marker for the prospective identification of facultative stem cells.


Asunto(s)
Mucosa Intestinal , Células Madre , Estudios Prospectivos , Células Madre/metabolismo , Linaje de la Célula , Diferenciación Celular/genética
3.
Carcinogenesis ; 44(2): 182-195, 2023 05 26.
Artículo en Inglés | MEDLINE | ID: mdl-37014121

RESUMEN

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of human malignancy, often displaying limited therapeutic response. Here, we examine the non-steroidal anti-inflammatory drug diclofenac (DCF) as a novel therapeutic agent in ESCC using complementary in vitro and in vivo models. DCF selectively reduced viability of human ESCC cell lines TE11, KYSE150, and KYSE410 as compared with normal primary or immortalized esophageal keratinocytes. Apoptosis and altered cell cycle profiles were documented in DCF-treated TE11 and KYSE 150. In DCF-treated TE11, RNA-Sequencing identified differentially expressed genes and Ingenuity Pathway Analysis predicted alterations in pathways associated with cellular metabolism and p53 signaling. Downregulation of proteins associated with glycolysis was documented in DCF-treated TE11 and KYSE150. In response to DCF, TE11 cells further displayed reduced levels of ATP, pyruvate, and lactate. Evidence of mitochondrial depolarization and superoxide production was induced by DCF in TE11 and KYSE150. In DCF-treated TE11, the superoxide scavenger MitoTempo improved viability, supporting a role for mitochondrial reactive oxygen species in DCF-mediated toxicity. DCF treatment resulted in increased expression of p53 in TE11 and KYSE150. p53 was further identified as a mediator of DCF-mediated toxicity in TE11 as genetic depletion of p53 partially limited apoptosis in response to DCF. Consistent with the anticancer activity of DCF in vitro, the drug significantly decreased tumor burdene in syngeneic ESCC xenograft tumors and 4-nitroquinoline 1-oxide-mediated ESCC lesions in vivo. These preclinical findings identify DCF as an experimental therapeutic that should be explored further in ESCC.


Asunto(s)
Antineoplásicos , Diclofenaco , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Humanos , Antineoplásicos/farmacología , Apoptosis , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Diclofenaco/farmacología , Diclofenaco/uso terapéutico , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/metabolismo , Superóxidos/metabolismo , Superóxidos/farmacología , Superóxidos/uso terapéutico , Carga Tumoral , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
4.
Cell Mol Gastroenterol Hepatol ; 18(1): 15-40, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38452871

RESUMEN

BACKGROUND & AIMS: Autophagy plays roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelial homeostasis. METHODS: We generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histologic and biochemical analyses. We fluorescence-activated cell sorted esophageal basal cells based on fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID and then subjected these cells to transmission electron microscopy, image flow cytometry, three-dimensional organoid assays, RNA sequencing, and cell cycle analysis. Three-dimensional organoids were subjected to passaging, single-cell RNA sequencing, cell cycle analysis, and immunostaining. RESULTS: Genetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells under homeostatic conditions and also was associated with significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Esophageal basal cells with high AV level (Cyto-IDHigh) displayed limited organoid formation capability on initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-IDLow). RNA sequencing suggested increased autophagy in Cyto-IDHigh esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. Single-cell RNA sequencing of three-dimensional organoids generated by Cyto-IDLow and Cyto-IDHigh cells identified expansion of 3 cell populations and enrichment of G2/M-associated genes in the Cyto-IDHigh group. Ki67 expression was also increased in organoids generated by Cyto-IDHigh cells, including in basal cells localized beyond the outermost cell layer. CONCLUSIONS: Autophagy contributes to maintenance of the esophageal proliferation-differentiation gradient. Esophageal basal cells with high AV level exhibit limited proliferation and generate three-dimensional organoids with enhanced self-renewal capacity.


Asunto(s)
Autofagia , Proliferación Celular , Homeostasis , Ratones Noqueados , Organoides , Animales , Ratones , Organoides/metabolismo , Esófago/patología , Esófago/citología , Esófago/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Proteína 7 Relacionada con la Autofagia/metabolismo , Proteína 7 Relacionada con la Autofagia/genética , 4-Nitroquinolina-1-Óxido , Autorrenovación de las Células , Mucosa Esofágica/patología , Mucosa Esofágica/metabolismo , Mucosa Esofágica/citología , Análisis de la Célula Individual
5.
Front Allergy ; 4: 1086032, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37064719

RESUMEN

Introduction: Under homeostatic conditions, esophageal epithelium displays a proliferation/differentiation gradient that is generated as proliferative basal cells give rise to suprabasal cells then terminally differentiated superficial cells. This proliferation/differentiation gradient is often perturbed in esophageal pathologies. Basal cell hyperplasia may occur in patients with gastroesophageal reflux disease (GERD), a condition in which acid from the stomach enters the esophagus, or eosinophilic esophagitis (EoE), an emerging form of food allergy. While GERD is a primary risk factor for esophageal cancer, epidemiological data suggests that EoE patients do not develop esophageal cancer. Methods: In order to investigate the impact of EoE and esophageal cancer specifically on the cellular landscape of esophageal epithelium, we perform single cell RNA-sequencing in murine models of EoE and esophageal cancer, specifically esophageal squamous cell carcinoma (ESCC). We further evaluate modules of co-expressed genes in EoE- and ESCC-enriched epithelial cell clusters. Finally, we pair EoE and ESCC murine models to examine the functional relationship between these pathologies. Results: In mice with either EoE or ESCC, we find expansion of cell populations as compared to normal esophageal epithelium. In mice with EoE, we detect distinct expansion of 4 suprabasal populations coupled with depletion of 2 basal populations. By contrast, mice with ESCC display unique expansion of 2 basal populations and 1 suprabasal population, as well as depletion of 2 suprabasal populations. Senescence, glucocorticoid receptor signaling, and granulocyte-macrophage colony-stimulating factor pathways are associated with EoE-enriched clusters while pathways associated with cell proliferation and metabolism are identified in ESCC-enriched clusters. Finally, our in vivo data demonstrate that exposure to EoE inflammation limits tumor burden of esophageal carcinogenesis. Discussion: Our findings provide the first functional investigation of the relationship between EoE and esophageal cancer and suggest that esophageal epithelial remodeling events occurring in response to EoE inflammation may limit esophageal carcinogenesis. This investigation may have future implications for leveraging allergic inflammation-associated alterations in epithelial biology to prevent and/or treat esophageal cancer.

6.
bioRxiv ; 2023 Sep 22.
Artículo en Inglés | MEDLINE | ID: mdl-37781581

RESUMEN

Background & Aims: Autophagy has been demonstrated to play roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelium under homeostatic conditions. Methods: We generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histological and biochemical analyses. We FACS sorted esophageal basal cells based upon fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID, then subjected these cells to transmission electron microscopy, image flow cytometry, 3D organoid assays, RNA-Sequencing (RNA-Seq), and cell cycle analysis. 3D organoids were subjected to passaging, single cell (sc) RNA-Seq, cell cycle analysis, and immunostaining. Results: Genetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells. Esophageal basal cells with high AV level (Cyto-ID High ) displayed limited organoid formation capability upon initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-ID Low ). RNA-Seq suggested increased autophagy in Cyto- ID High esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. scRNA-Seq of 3D organoids generated by Cyto-ID Low and Cyto- ID High cells identified expansion of 3 cell populations, enrichment of G2/M-associated genes, and aberrant localization of cell cycle-associated genes beyond basal cell populations in the Cyto- ID High group. Ki67 expression was also increased in organoids generated by Cyto-ID High cells, including in cells beyond the basal cell layer. Squamous epithelial-specific autophagy inhibition induced significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Conclusions: High AV level identifies esophageal epithelium with limited proliferation and enhanced self-renewal capacity that contributes to maintenance of the esophageal proliferation- differentiation gradient in vivo .

7.
bioRxiv ; 2023 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-37745612

RESUMEN

In pancreatic ductal adenocarcinoma (PDAC), the fibroblastic stroma constitutes most of the tumor mass and is remarkably devoid of functional blood vessels. This raises an unresolved question of how PDAC cells obtain essential metabolites and water-insoluble lipids. We have found a critical role for cancer-associated fibroblasts (CAFs) in obtaining and transferring lipids from blood-borne particles to PDAC cells via trogocytosis of CAF plasma membranes. We have also determined that CAF-expressed phospholipid scramblase anoctamin 6 (ANO6) is an essential CAF trogocytosis regulator required to promote PDAC cell survival. During trogocytosis, cancer cells and CAFs form synapse-like plasma membranes contacts that induce cytosolic calcium influx in CAFs via Orai channels. This influx activates ANO6 and results in phosphatidylserine exposure on CAF plasma membrane initiating trogocytosis and transfer of membrane lipids, including cholesterol, to PDAC cells. Importantly, ANO6-dependent trogocytosis also supports the immunosuppressive function of pancreatic CAFs towards cytotoxic T cells by promoting transfer of excessive amounts of cholesterol. Further, blockade of ANO6 antagonizes tumor growth via disruption of delivery of exogenous cholesterol to cancer cells and reverses immune suppression suggesting a potential new strategy for PDAC therapy.

8.
Front Allergy ; 3: 983412, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36591561

RESUMEN

Subepithelial fibrosis occurs in a subset of eosinophilic esophagitis (EoE) patients and is associated with esophageal stricture. While mechanisms driving EoE fibrosis remain incompletely understood, findings from experimental systems support roles for epithelial-fibroblast crosstalk in this type of tissue remodeling. The current paradigm presents EoE as a progressive fibrostenotic disease in which aged patients develop fibrosis as a function of disease chronicity. In the current study we provide evidence that altered epithelial biology in the aging esophagus may also contribute to EoE-associated fibrosis. We find that induction of EoE inflammation in young and aged mice using the MC903/Ovalbumin protocol for the same time period results in increased lamina propria thickness uniquely in aged animals. Additionally, epithelial cells from aged mice less efficiently limit fibroblast contractility in collagen plug contraction assays compared to those from their young counterparts. Finally, to identify potential mechanisms through which aged esophageal epithelial cells may stimulate fibrotic remodeling, we perform cytokine array experiments in young and aged mice. These studies are significant as identification of age-associated factors that contribute to fibrotic remodeling may aid in the design of strategies toward early detection, prevention, and therapy of fibrostenotic EoE.

9.
Nat Commun ; 13(1): 2167, 2022 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-35443762

RESUMEN

Although morphologic progression coupled with expression of specific molecular markers has been characterized along the esophageal squamous differentiation gradient, the molecular heterogeneity within cell types along this trajectory has yet to be classified at the single cell level. To address this knowledge gap, we perform single cell RNA-sequencing of 44,679 murine esophageal epithelial, to identify 11 distinct cell populations as well as pathways alterations along the basal-superficial axis and in each individual population. We evaluate the impact of aging upon esophageal epithelial cell populations and demonstrate age-associated mitochondrial dysfunction. We compare single cell transcriptomic profiles in 3D murine organoids and human esophageal biopsies with that of murine esophageal epithelium. Finally, we employ pseudotemporal trajectory analysis to develop a working model of cell fate determination in murine esophageal epithelium. These studies provide comprehensive molecular perspective on the cellular heterogeneity of murine esophageal epithelium in the context of homeostasis and aging.


Asunto(s)
Neoplasias Esofágicas , Transcriptoma , Animales , Células Epiteliales , Epitelio/metabolismo , Neoplasias Esofágicas/patología , Esófago/patología , Humanos , Ratones , Análisis de la Célula Individual , Transcriptoma/genética
10.
Cancers (Basel) ; 11(11)2019 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-31683722

RESUMEN

Esophageal cancer is among the most aggressive forms of human malignancy with five-year survival rates of <20%. Autophagy is an evolutionarily conserved catabolic process that degrades and recycles damaged organelles and misfolded proteins to maintain cellular homeostasis. While alterations in autophagy have been associated with carcinogenesis across tissues, cell type- and context-dependent roles for autophagy have been reported. Herein, we review the current knowledge related to autophagy in esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC), the two most common subtypes of esophageal malignancy. We explore roles for autophagy in the development and progression of ESCC and EAC. We then continue to discuss molecular markers of autophagy as they relate to esophageal patient outcomes. Finally, we summarize current literature examining roles for autophagy in ESCC and EAC response to therapy and discuss considerations for the potential use of autophagy inhibitors as experimental therapeutics that may improve patient outcomes in esophageal cancer.

11.
Adv Cancer Res ; 144: 95-135, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31349905

RESUMEN

Esophageal squamous cell carcinoma (ESCC) is among the most deadly forms of human malignancy characterized by late stage diagnosis, metastasis, therapy resistance and frequent recurrence. Clinical management of ESCC remains challenging and the disease presently lacks approved targeted therapeutics. However, emerging data from recent clinical and translational investigations hold great promise for future progress toward improving patient outcomes in this deadly disease. Here, we review current clinical perspectives in ESCC epidemiology, pathophysiology, and clinical care, highlighting recent advances with potential to impact ESCC prevention, diagnosis and management. We further provide an overview of recent translational investigations contributing to our understanding of the molecular mechanisms underlying ESCC development, progression and therapy response, including insights gained from genetic studies and various murine model systems. Finally, we discuss future perspectives in the clinical and translational realms, along with remaining hurdles that must be overcome to eradicate ESCC.


Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/terapia , Carcinoma de Células Escamosas de Esófago/diagnóstico , Carcinoma de Células Escamosas de Esófago/terapia , Animales , Manejo de la Enfermedad , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas de Esófago/genética , Humanos , Factores de Riesgo
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