Physioxic human cell culture improves viability, metabolism, and mitochondrial morphology while reducing DNA damage

Timpano, Sara; Guild, Brianna D.; Specker, Erin J.; Melanson, Gaelan; Medeiros, Philip J.; Sproul, Shannon L.J.; Uniacke, James

doi:10.1096/fj.201802279r

Cited by 21 publications

(17 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Studies in 2D cell line cultures indicated an optimal physioxic range low enough to reduce oxidative damage but high enough for efficient oxidative metabolism for different cell types. ( Ferguson et al, 2018 ; Timpano et al, 2019 ). Finding an optimal oxygen concentration that mirrors the in vivo physiological range in colon (<1%–3%), might require some optimization depending on the type of experiments, organoid density and differentiation protocol used.…”

Section: Discussionmentioning

confidence: 99%

Patient Derived Colonoids as Drug Testing Platforms–Critical Importance of Oxygen Concentration

et al. 2021

View full text Add to dashboard Cite

Treatment of inflammatory bowel disease (IBD) is challenging, with a series of available drugs each helping only a fraction of patients. Patients may face time-consuming drug trials while the disease is active, thus there is an unmet need for biomarkers and assays to predict drug effect. It is well known that the intestinal epithelium is an important factor in disease pathogenesis, exhibiting physical, biochemical and immunologic driven barrier dysfunctions. One promising test system to study effects of existing or emerging IBD treatments targeting intestinal epithelial cells (IECs) is intestinal organoids (“mini-guts”). However, the fact that healthy intestinal epithelium is in a physiologically hypoxic state has largely been neglected, and studies with intestinal organoids are mainly performed at oxygen concentration of 20%. We hypothesized that lowering the incubator oxygen level from 20% to 2% would recapitulate better the in vivo physiological environment of colonic epithelial cells and enhance the translational value of intestinal organoids as a drug testing platform. In the present study we examine the effects of the key IBD cytokines and drug targets TNF/IL17 on human colonic organoids (colonoids) under atmospheric (20%) or reduced (2%) O2. We show that colonoids derived from both healthy controls and IBD-patients are viable and responsive to IBD-relevant cytokines at 2% oxygen. Because chemokine release is one of the important immunoregulatory traits of the epithelium that may be fine-tuned by IBD-drugs, we also examined chemokine expression and release at different oxygen concentrations. We show that chemokine responses to TNF/IL17 in organoids display similarities to inflamed epithelium in IBD-patients. However, inflammation-associated genes induced by TNF/IL17 were attenuated at low oxygen concentration. We detected substantial oxygen-dependent differences in gene expression in untreated as well as TNF/IL17 treated colonoids in all donors. Further, for some of the IBD-relevant cytokines differences between colonoids from healthy controls and IBD patients were more pronounced in 2% O2 than 20% O2. Our results strongly indicate that an oxygen concentration similar to the in vivo epithelial cell environment is of essence in experimental pharmacology.

show abstract

Section: Discussionmentioning

confidence: 99%

Patient Derived Colonoids as Drug Testing Platforms–Critical Importance of Oxygen Concentration

et al. 2021

View full text Add to dashboard Cite

show abstract

“…Here we provide detailed evidence of intertumoral heterogeneity at the transcriptional level by performing in vitro hypoxia studies using several human-derived cell lines, which allowed us to identify a robust common hypoxia signature ( Figure S1d ), despite the heterogeneity. Importantly, we used 5% O 2 as a physiological oxygen control, rather than 21% O 2 , more accurately representing the in vivo oxygenation levels, which can influence viable cell number, metabolism, and mitochondrial function [ 13 ]. By analyzing differential gene expression at three different oxygen concentrations ( Figure 1 b), it is clear that the vast majority of transcriptional changes had already occurred under physioxia, with a very modest number of genes differentially regulated between 5% O 2 and 1% O 2 .…”

Section: Discussionmentioning

confidence: 99%

“…Tumor hypoxia drives malignancy by promoting chemo-and radiotherapy resistance, an immunosuppressive microenvironment, cancer cell stemness, angiogenesis, and metabolic modulation [9][10][11]. The study of tumor hypoxia in vitro frequently uses cell cultures exposed to atmospheric conditions (21% O 2 ) as a control, although this does not represent any physiological oxygen fraction found in vivo [12] and does not always recapitulate cellular functions under physioxia [13]. Physiological oxygen availability is tissue-dependent, with 2%-9% O 2 (10-40 mmHg) being reported for the healthy brain [14].…”

Section: Introductionmentioning

confidence: 99%

An Experimentally Defined Hypoxia Gene Signature in Glioblastoma and Its Modulation by Metformin

Tardón

Marinari

Migliorini³

et al. 2020

Biology

View full text Add to dashboard Cite

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor, characterized by a high degree of intertumoral heterogeneity. However, a common feature of the GBM microenvironment is hypoxia, which can promote radio- and chemotherapy resistance, immunosuppression, angiogenesis, and stemness. We experimentally defined common GBM adaptations to physiologically relevant oxygen gradients, and we assessed their modulation by the metabolic drug metformin. We directly exposed human GBM cell lines to hypoxia (1% O2) and to physioxia (5% O2). We then performed transcriptional profiling and compared our in vitro findings to predicted hypoxic areas in vivo using in silico analyses. We observed a heterogenous hypoxia response, but also a common gene signature that was induced by a physiologically relevant change in oxygenation from 5% O2 to 1% O2. In silico analyses showed that this hypoxia signature was highly correlated with a perinecrotic localization in GBM tumors, expression of certain glycolytic and immune-related genes, and poor prognosis of GBM patients. Metformin treatment of GBM cell lines under hypoxia and physioxia reduced viable cell number, oxygen consumption rate, and partially reversed the hypoxia gene signature, supporting further exploration of targeting tumor metabolism as a treatment component for hypoxic GBM.

show abstract

“…Immunofluorescence and microscopy. Immunofluorescence and microscopy were performed as previously described (55). For mitochondrial shape and fusion, images were analyzed using Volocity (PerkinElmer).…”

Section: Methodsmentioning

confidence: 99%

DEAD Box Protein Family Member DDX28 Is a Negative Regulator of Hypoxia-Inducible Factor 2α- and Eukaryotic Initiation Factor 4E2-Directed Hypoxic Translation

Evagelou

Bebenek

Specker

et al. 2020

Molecular and Cellular Biology

Self Cite

View full text Add to dashboard Cite

Hypoxia is a deficiency in oxygen delivery to tissues and is connected to physiological and pathophysiological processes such as embryonic development and cancer. The master regulators of oxygen homeostasis in mammalian cells are the heterodimeric hypoxia-inducible transcription factors 1 and 2 (HIF-1 and HIF-2, respectively). The oxygen-labile HIF-2α subunit has been implicated not only in transcription but also as a regulator of eukaryotic initiation factor 4E2 (eIF4E2)-directed hypoxic translation. Here, we have identified the DEAD box protein family member DDX28 as an interactor and negative regulator of HIF-2α that suppresses HIF-2α’s ability to activate eIF4E2-directed translation. Stable silencing of DDX28 via short hairpin RNA (shRNA) in hypoxic human U87MG glioblastoma cells caused an increase of eIF4E2 binding to the m7GTP cap structure and the translation of eIF4E2 target mRNAs (including the HIF-2α mRNA itself). DDX28 depletion elevated nuclear and cytoplasmic HIF-2α protein, but HIF-2α transcriptional activity did not increase, possibly due to its already high nuclear abundance in hypoxic control cells. Depletion of DDX28 conferred a proliferative advantage to hypoxic, but not normoxic, cells. DDX28 protein levels are reduced in several cancers, including gliomas, relative to levels in normal tissue. Therefore, we uncover a regulatory mechanism for this potential tumor suppressor in the repression of HIF-2α- and eIF4E2-mediated translation activation of oncogenic mRNAs.

show abstract

Physioxic human cell culture improves viability, metabolism, and mitochondrial morphology while reducing DNA damage

Cited by 21 publications

References 42 publications

Patient Derived Colonoids as Drug Testing Platforms–Critical Importance of Oxygen Concentration

Patient Derived Colonoids as Drug Testing Platforms–Critical Importance of Oxygen Concentration

An Experimentally Defined Hypoxia Gene Signature in Glioblastoma and Its Modulation by Metformin

DEAD Box Protein Family Member DDX28 Is a Negative Regulator of Hypoxia-Inducible Factor 2α- and Eukaryotic Initiation Factor 4E2-Directed Hypoxic Translation

Contact Info

Product

Resources

About