Investigation of tumor hypoxia using a two-enzyme system for in vitro generation of oxygen deficiency

<p>Abstract</p> <p>Background</p> <p>Oxygen deficiency in tumor tissue is associated with a malign phenotype, characterized by high invasiveness, increased metastatic potential and poor prognosis. Hypoxia chambers are the established standard model for <it>in vitro </it>studies on tumor hypoxia. An enzymatic hypoxia system (GOX/CAT) based on the use of glucose oxidase (GOX) and catalase (CAT) that allows induction of stable hypoxia for <it>in vitro </it>approaches more rapidly and with less operating expense has been introduced recently. Aim of this work is to compare the enzymatic system with the established technique of hypoxia chamber in respect of gene expression, glucose metabolism and radioresistance, prior to its application for <it>in vitro </it>investigation of oxygen deficiency.</p> <p>Methods</p> <p>Human head and neck squamous cell carcinoma HNO97 cells were incubated under normoxic and hypoxic conditions using both hypoxia chamber and the enzymatic model. Gene expression was investigated using Agilent microarray chips and real time PCR analysis. ¹⁴C-fluoro-deoxy-glucose uptake experiments were performed in order to evaluate cellular metabolism. Cell proliferation after photon irradiation was investigated for evaluation of radioresistance under normoxia and hypoxia using both a hypoxia chamber and the enzymatic system.</p> <p>Results</p> <p>The microarray analysis revealed a similar trend in the expression of known HIF-1 target genes between the two hypoxia systems for HNO97 cells. Quantitative RT-PCR demonstrated different kinetic patterns in the expression of carbonic anhydrase IX and lysyl oxidase, which might be due to the faster induction of hypoxia by the enzymatic system. ¹⁴C-fluoro-deoxy-glucose uptake assays showed a higher glucose metabolism under hypoxic conditions, especially for the enzymatic system. Proliferation experiments after photon irradiation revealed increased survival rates for the enzymatic model compared to hypoxia chamber and normoxia, indicating enhanced resistance to irradiation. While the GOX/CAT system allows independent investigation of hypoxia and oxidative stress, care must be taken to prevent acidification during longer incubation.</p> <p>Conclusion</p> <p>The results of our study indicate that the enzymatic model can find application for <it>in vitro </it>investigation of tumor hypoxia, despite limitations that need to be considered in the experimental design.</p

Hypoxia Potentiates Glioma-Mediated Immunosuppression

Glioblastoma multiforme (GBM) is a lethal cancer that exerts potent immune suppression. Hypoxia is a predominant feature of GBM, but it is unclear to the degree in which tumor hypoxia contributes to this tumor-mediated immunosuppression. Utilizing GBM associated cancer stem cells (gCSCs) as a treatment resistant population that has been shown to inhibit both innate and adaptive immune responses, we compared immunosuppressive properties under both normoxic and hypoxic conditions. Functional immunosuppression was characterized based on production of immunosuppressive cytokines and chemokines, the inhibition of T cell proliferation and effector responses, induction of FoxP3+ regulatory T cells, effect on macrophage phagocytosis, and skewing to the immunosuppressive M2 phenotype. We found that hypoxia potentiated the gCSC-mediated inhibition of T cell proliferation and activation and especially the induction of FoxP3+T cells, and further inhibited macrophage phagocytosis compared to normoxia condition. These immunosuppressive hypoxic effects were mediated by signal transducer and activator of transcription 3 (STAT3) and its transcriptionally regulated products such as hypoxia inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF). Inhibitors of STAT3 and HIF-1α down modulated the gCSCs' hypoxia-induced immunosuppressive effects. Thus, hypoxia further enhances GBM-mediated immunosuppression, which can be reversed with therapeutic inhibition of STAT3 and HIF-1α and also helps to reconcile the disparate findings that immune therapeutic approaches can be used successfully in model systems but have yet to achieve generalized successful responses in the vast majority of GBM patients by demonstrating the importance of the tumor hypoxic environment