Isolation of Epithelial, Endothelial, and Immune Cells from Lungs of Transgenic Mice with Oncogene-Induced Lung Adenocarcinomas

Bantikassegn, Amlak; Song, Xiaoling; Politi, Katerina

doi:10.1165/rcmb.2014-0312ma

Cited by 46 publications

(36 citation statements)

References 41 publications

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“…In this report, we describe novel methods to enrich and isolate four cell populations simultaneously from pediatric human lung of suitable quality for further analyses. Obtaining dissociated lung single-cell suspensions and isolating one or two particular cell types have been previously described (2,5,8,11,16,17,26,29). Here we present a technique to 1) efficiently isolate single-cell suspensions from lung lobes with high yield and viability and then 2) enrich four major distal lung cell subpopulations based on distinct patterns of membrane protein expression.…”

Section: Discussionmentioning

confidence: 99%

Dissociation, cellular isolation, and initial molecular characterization of neonatal and pediatric human lung tissues

Bandyopadhyay

Huyck

Misra

et al. 2018

American Journal of Physiology-Lung Cellular and Molecular Phys

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Human lung morphogenesis begins by embryonic life and continues after birth into early childhood to form a complex organ with numerous morphologically and functionally distinct cell types. Pulmonary organogenesis involves dynamic changes in cell proliferation, differentiation, and migration of specialized cells derived from diverse embryonic lineages. Studying the molecular and cellular processes underlying formation of the fully functional lung requires isolating distinct pulmonary cell populations during development. We now report novel methods to isolate four major pulmonary cell populations from pediatric human lung simultaneously. Cells were dissociated by protease digestion of neonatal and pediatric lung and isolated on the basis of unique cell membrane protein expression patterns. Epithelial, endothelial, nonendothelial mesenchymal, and immune cells were enriched by fluorescence-activated cell sorting. Dead cells and erythrocytes were excluded by 7-aminoactinomycin D uptake and glycophorin-A (CD235a) expression, respectively. Leukocytes were identified by membrane CD45 (protein tyrosine phosphatase, receptor type C), endothelial cells by platelet endothelial cell adhesion molecule-1 (CD31) and vascular endothelial cadherin (CD144), and both were isolated. Thereafter, epithelial cell adhesion molecule (CD326)-expressing cells were isolated from the endothelial- and immune cell-depleted population to enrich epithelial cells. Cells lacking these membrane markers were collected as "nonendothelial mesenchymal" cells. Quantitative RT-PCR and RNA sequencing analyses of population specific transcriptomes demonstrate the purity of the subpopulations of isolated cells. The method efficiently isolates major human lung cell populations that we announce are now available through the National Heart, Lung, and Blood Institute Lung Molecular Atlas Program (LungMAP) for their further study.

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Section: Discussionmentioning

confidence: 99%

Dissociation, cellular isolation, and initial molecular characterization of neonatal and pediatric human lung tissues

Bandyopadhyay

Huyck

Misra

et al. 2018

American Journal of Physiology-Lung Cellular and Molecular Phys

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“…The cytoplasms of cells were granulated and the vacuolization of the inclusion bodies was also observed. Separation of endothelium and epithelium from the basement membrane of the alveoli was also observed, which is indicative of injury (10). This result may well be correlated with the increase in the levels of tumor marker enzymes, especially lactate dehydrogenase (LDH), which is also a marker of tissue injury (11).…”

Section: Discussionmentioning

confidence: 99%

Ultrastructural changes during lung carcinogenesis-modulation by curcumin and quercetin

Wang

Zhang

et al. 2016

Oncology Letters

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The aim of the present study was to examine the effectiveness of curcumin and quercetin in modulating ultrastructural changes during lung carcinogenesis. A total of 24 male laka mice were divided into the normal control, benzo[a]pyrene (BP)-treated, BP+curcumin-treated, BP+quercetin- treated, and BP+curcumin+quercetin-treated groups (n=6 per group). Lung carcinogenesis was induced by a single intraperitoneal injection of BP [100 mg/kg of body weight (b.wt.)]. Curcumin was supplemented to mice at a dose level of 60 mg/kg of b.wt. in drinking water and quercetin was given at a dose level of 40 mg/kg of b.wt. in drinking water. The ultrastructure of BP-treated mice revealed disruptions in cellular integrity together with nuclear deformation and premature mitochondrial aging. Notably, supplementation with phytochemicals individually resulted in improvement of the ultra-histoarchitecture of BP-treated mice although the improvement was much greater with combined supplementation of phytochemicals. Furthermore, BP treatment revealed alterations in lung histoarchitecture, which, however, were improved appreciably following combined supplementation with curcumin and quercetin. The results of the present study show that, combined supplementation with curcumin and quercetin effectively preserved the histoarchitecture as well as ultra-histoarchitecture during BP-induced lung carcinogenesis in mice.

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“…Isolation of type II pneumocyte from mouse lung. Type 2 pneumocytes were isolated from mouse lung following an adaptation of a previously described protocol (51). Briefly, a euthanized mouse was dissected, renal arteries sectioned, and lungs perfused with PBS to remove blood.…”

Section: Author Contributionsmentioning

confidence: 99%

O-GlcNAcylation is required for mutant KRAS-induced lung tumorigenesis

Taparra¹,

Wang²,

Malek³

et al. 2018

Journal of Clinical Investigation

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Mutant KRAS drives glycolytic flux in lung cancer, potentially impacting aberrant protein glycosylation. Recent evidence suggests aberrant KRAS drives flux of glucose into the hexosamine biosynthetic pathway (HBP). HBP is required for various glycosylation processes, such as protein N- or O-glycosylation and glycolipid synthesis. However, its function during tumorigenesis is poorly understood. One contributor and proposed target of KRAS-driven cancers is a developmentally conserved epithelial plasticity program called epithelial-mesenchymal transition (EMT). Here we showed in novel autochthonous mouse models that EMT accelerated KrasG12D lung tumorigenesis by upregulating expression of key enzymes of the HBP pathway. We demonstrated that HBP was required for suppressing KrasG12D-induced senescence, and targeting HBP significantly delayed KrasG12D lung tumorigenesis. To explore the mechanism, we investigated protein glycosylation downstream of HBP and found elevated levels of O-linked β-N-acetylglucosamine (O-GlcNAcylation) posttranslational modification on intracellular proteins. O-GlcNAcylation suppressed KrasG12D oncogene-induced senescence (OIS) and accelerated lung tumorigenesis. Conversely, loss of O-GlcNAcylation delayed lung tumorigenesis. O-GlcNAcylation of proteins SNAI1 and c-MYC correlated with the EMT-HBP axis and accelerated lung tumorigenesis. Our results demonstrated that O-GlcNAcylation was sufficient and required to accelerate KrasG12D lung tumorigenesis in vivo, which was reinforced by epithelial plasticity programs.

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Isolation of Epithelial, Endothelial, and Immune Cells from Lungs of Transgenic Mice with Oncogene-Induced Lung Adenocarcinomas

Cited by 46 publications

References 41 publications

Dissociation, cellular isolation, and initial molecular characterization of neonatal and pediatric human lung tissues

Dissociation, cellular isolation, and initial molecular characterization of neonatal and pediatric human lung tissues

Ultrastructural changes during lung carcinogenesis-modulation by curcumin and quercetin

O-GlcNAcylation is required for mutant KRAS-induced lung tumorigenesis

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