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

Abstract

BackgroundOxygen 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 in vitro 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 in vitro 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 in vitro investigation of oxygen deficiency.MethodsHuman 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.ResultsThe 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.ConclusionThe results of our study indicate that the enzymatic model can find application for in vitro investigation of tumor hypoxia, despite limitations that need to be considered in the experimental design.

Enzyme-immobilized hydrogels to create hypoxia for in vitro cancer cell culture

“…However, no expression of LOX was detected (control group included); there is also a lack of literature for LOX expression with Molm14 cells or the sister Molm13 cell line. Nonetheless, similar data for both CA9 and LOX expression was reported by Askoxylakis et al where GOX and CAT were dissolved in culture media to generate hypoxia for culturing head and neck squamous carcinoma cells (Askoxylakis et al, 2011).…”

“…This was most likely attributed to the accumulation of cytotoxic byproduct, namely gluconic acid and H 2 O 2 . This is not a unique problem to our system as other GOX/CAT-induced hypoxia culture systems (e.g., soluble or membrane-bound enzyme) also exhibited decreased cytocompatibility after 24 hours of culture (Askoxylakis et al, 2011;Huang et al, 2013;Zitta et al, 2012). One potential solution for this drawback is to replace the GOX-immobilized hydrogel and the CAT supplemented media frequently.…”

“…Some recent work has started to explore the ability of GOX/CAT reactions to induce hypoxia for in vitro cell culture (Askoxylakis et al, 2011;Baumann et al, 2008;Huang et al, 2013;Li et al, 2016;Millonig et al, 2009;Mueller et al, 2009;Rajan et al, 2013;Sobotta et al, 2013). The GOX/CAT system has also been adapted to 3D printed inserts (Li et al, 2016) where GOX and CAT were coated on printed disks and the degrees of solution hypoxia were controlled by the distance between the enzyme-immobilized disks and the solution in the culture plate.…”

“…This system has been used to induce hypoxia in solutions and in microfluidic devices (Askoxylakis et al, 2011;Baumann et al, 2008;Huang et al, 2013;Li et al, 2016;Millonig et al, 2009;Mueller et al, 2009;Rajan et al, 2013;Sobotta et al, 2013;Zitta et al, 2012). The use of GOX/CAT is beneficial in that the system provides a rapid onset of hypoxia (usually within a few minutes) (Askoxylakis et al, 2011;Baumann et al, 2008;Huang et al, 2013;Li et al, 2016;Millonig et al, 2009;Mueller et al, 2009;Rajan et al, 2013;Sobotta et al, 2013;Zitta et al, 2012). One drawback to any GOX system, however, is the production of hydrogen peroxide, a reactive oxygen species (ROS) (Fruehauf and Meyskens, 2007) whose accumulation would not only cause undesired cellular response but also inactivate both GOX and CAT (Hielscher and Gerecht, 2015;Pal et al, 2000;Trachootham et al, 2009;Tse and Gough, 1987).…”

A novel oxygen tension programmable microfluidic system (oPROMs) for in vitro cell biology studies

“…However, this approach presents some critical limitations, mainly in regard to oxygen diffusion and equilibration. Studies have demonstrated that it requires more than three hours for the medium inside the culture plate to equilibrate to the gas outside of it [3]. Therefore, the longer time required for culture medium to reach equilibrium and the lack of ability to generate oxygen gradients greatly limits the scope of application for such chambers.`…”