Oxygen Consumption Rate In Cells Research Articles

Scanning electrochemical microscopy for determining oxygen consumption rates of cells in hydrogel fibers fabricated using an extrusion 3D bioprinter

Three-dimensional (3D)-cultured cells have attracted the attention of researchers in tissue engineering- and drug screening-related fields. Among them, 3D cellular fibers have attracted significant attention because they can be stacked to prepare more complex tissues and organs. Cellular fibers are widely fabricated using extrusion 3D bioprinters. For these applications, it is necessary to evaluate cellular activities, such as the oxygen consumption rate (OCR), which is one of the major metabolic activities. We previously reported the use of scanning electrochemical microscopy (SECM) to evaluate the OCRs of cell spheroids. However, the SECM approach has not yet been applied to hydrogel fibers prepared using the bioprinters. To the best of our knowledge, this is the first study to evaluate the OCR of cellular fibers printed by extrusion 3D bioprinters. First, the diffusion theory was discussed to address this issue. Next, diffusion models were simulated to compare realistic models with this theory. Finally, the OCRs of MCF-7 cells in the printed hydrogel fibers were evaluated as a proof of concept. Our proposed approach could potentially be used to evaluate the OCRs of tissue-engineered fibers for organ transplantation and drug screening using in-vitro models.

Anthelmintic nitazoxanide protects against experimental pulmonary fibrosis.

Nitazoxanide is a therapeutic anthelmintic drug. Our previous studies found that nitazoxanide and its metabolite tizoxanide activated adenosine 5'-monophosphate-activated protein kinase (AMPK) and inhibited signal transducer and activator of transcription 3 (STAT3) signals. As AMPK activation and/or STAT3 inhibition are targets for treating pulmonary fibrosis, we hypothesized that nitazoxanide would be effective in experimental pulmonary fibrosis. The mitochondrial oxygen consumption rate of cells was measured by using the high-resolution respirometry system Oxygraph-2K. The mitochondrial membrane potential of cells was evaluated by tetramethyl rhodamine methyl ester (TMRM) staining. The target protein levels were measured by using western blotting. The mice pulmonary fibrosis model was established through intratracheal instillation of bleomycin. The examination of the lung tissues changes were carried out using haematoxylin and eosin (H&E), and Masson staining. Nitazoxanide and tizoxanide activated AMPK and inhibited STAT3 signalling in human lung fibroblast cells (MRC-5 cells). Nitazoxanide and tizoxanide inhibited transforming growth factor-β1 (TGF-β1)-induced proliferation and migration of MRC-5 cells, collagen-I and α-smooth muscle cell actin (α-SMA) expression, and collagen-I secretion from MRC-5 cells. Nitazoxanide and tizoxanide inhibited epithelial-mesenchymal transition (EMT) and inhibited TGF-β1-induced Smad2/3 activation in mouse lung epithelial cells (MLE-12 cells). Oral administration of nitazoxanide reduced the bleomycin-induced mice pulmonary fibrosis and, in the established bleomycin-induced mice, pulmonary fibrosis. Delayed nitazoxanide treatment attenuated the fibrosis progression. Nitazoxanide improves the bleomycin-induced pulmonary fibrosis in mice, suggesting a potential application of nitazoxanide for pulmonary fibrosis treatment in the clinic.

FOXA2 suppresses PKM2 transcription and affects the Wnt/β-catenin activity to block aerobic glycolysis in thyroid carcinoma.

Aerobic glycolysis is critical for the energy metabolism of cancer cells. This study focuses on the regulation of forkhead box A2 (FOXA2) on pyruvate kinase M2 (PKM2) and their effects on the glycolytic activity and malignant phenotype of thyroid carcinoma (THCA) cells. By analysing four Gene Expression Omnibus datasets and querying bioinformatics systems, we obtained FOXA2 as a poorly expressed transcription factor in THCA. Later, we validated decreased mRNA and protein levels of FOXA2 in THCA cells by quantitative polymerase chain reaction and western blot assays. FOXA2 upregulation in THCA cells suppressed the glucose uptake and lactate production, and it reduced the extracellular acidification rate, but increased the oxygen consumption rate of cells. Meanwhile, the FOXA2 overexpression blocked the proliferation and mobility, and the tumourigenic activity of cancer cells. The chromatin immunoprecipitation and luciferase assays showed that FOXA2 bound to PKM2 promoter and suppressed the transcription of PKM2, which was highly expressed in THCA cells. Further upregulation of PKM2 elevated the β-catenin, c-Myc and cyclin D1 levels and restored the glycolytic activity as well as the malignant properties of cancer cells. Collectively, this work reveals that FOXA2 suppresses aerobic glycolysis and progression of THCA by blocking PKM2 transcription and inactivating the Wnt/β-catenin pathway.

Measurement of oxygen consumption rates of human renal proximal tubule cells in an array of organ-on-chip devices to monitor drug-induced metabolic shifts

Measurement of cell metabolism in moderate-throughput to high-throughput organ-on-chip (OOC) systems would expand the range of data collected for studying drug effects or disease in physiologically relevant tissue models. However, current measurement approaches rely on fluorescent imaging or colorimetric assays that are focused on endpoints, require labels or added substrates, and lack real-time data. Here, we integrated optical-based oxygen sensors in a high-throughput OOC platform and developed an approach for monitoring cell metabolic activity in an array of membrane bilayer devices. Each membrane bilayer device supported a culture of human renal proximal tubule epithelial cells on a porous membrane suspended between two microchannels and exposed to controlled, unidirectional perfusion and physiologically relevant shear stress for several days. For the first time, we measured changes in oxygen in a membrane bilayer format and used a finite element analysis model to estimate cell oxygen consumption rates (OCRs), allowing comparison with OCRs from other cell culture systems. Finally, we demonstrated label-free detection of metabolic shifts in human renal proximal tubule cells following exposure to FCCP, a drug known for increasing cell oxygen consumption, as well as oligomycin and antimycin A, drugs known for decreasing cell oxygen consumption. The capability to measure cell OCRs and detect metabolic shifts in an array of membrane bilayer devices contained within an industry standard microtiter plate format will be valuable for analyzing flow-responsive and physiologically complex tissues during drug development and disease research.

Evaluation of Effects of Selenium Nanoparticles as an Occupational and Environmental Chemical Hazard on Cellular Bioenergetic Processes

Introduction: High-volume manufacturing of selenium and a widespread use of its compounds pose potential risks to human health. Certain copper production processes emit selenium-containing nanoparticles. Objective: To assess health effects of selenium oxide nanoparticles as an industrial and environmental toxicant. Materials and methods: Selenium oxide nanoparticles (SeO NPs) were obtained by laser ablation. Their toxicity was studied both in vitro on human lung-derived embryonic fibroblasts (FLEH-104 cell line) by assaying adenosine triphosphate (ATP) bioluminescence and the rate of oxygen consumption, and in vivo on outbred albino rats by analyzing ultrastructural changes in tissues using electron microscopy, measuring succinate dehydrogenase activity of blood lymphocytes, and conducting a blood-based metabolomic test. Results: The in vitro experiment showed a decrease in ATP bioluminescence by 75.9 % and in the oxygen consumption rate of cells by 79.8 % in the incubation medium with 100 μg/mL concentration of SeO NPs. In the in vivo experiment, succinate dehydrogenase activity of blood lymphocytes decreased inversely with the increasing dose by 10.12 %, 14.0 %, 15.9 % compared to the control animals in the SeO NPs 0.1, SeO NPs 0.5, and SeO NPs 1 exposure groups, respectively. The study of ultrastructural changes in liver tissue showed a smaller number of normal mitochondria (7.78 % less in the SeO NP 1 group) compared to the controls while the metabolomic test revealed decreased acylcarnitines and increased lysophosphatidylinositols following the exposure to SeO NPs (p > 0.05). Conclusion: The results of our in vitro and in vivo studies showed adverse effects of SeO NPs on bioenergetics processes in cells involving at least two mechanisms: disruption of mitochondrial β-oxidation of fatty acid and inactivation of succinate dehydrogenase. The fundamental role of the latter in the mitochondrial electron transport chain makes its vitally important for most multicellular organisms. Our findings can serve as a rationale for assessing selenium-containing nanoparticles as a chemical hazard and searching for approaches to managing their health risks.

Abstract 2154: PARP7 inhibitor RBN-2397 increases tumoral IFN signaling leading to various tumor cell intrinsic effects and tumor regressions in mouse models

Abstract Targeting cytosolic nucleic acid sensing pathways to activate the Type I interferon (IFN) response is an emerging therapeutic strategy being explored in oncology. The PARP family consists of seventeen enzymes that regulate fundamental biological processes including response to cellular stress. PARP7 (TIPARP) is a stress-induced mono-ART that catalyzes the transfer of a single unit of ADP-ribose onto substrates (MARylation) to regulate their function and plays a role in suppressing the Type I IFN response in tumor cells (Gozgit 2021 Cancer Cell). RBN-2397 is the first potent and selective small molecule inhibitor of PARP7 catalytic function. To investigate the cell autonomous effects of PARP7 inhibition, we performed a cell line screen to identify PARP7 dependent cancer cell lines. We found that treatment of a subset of lines across several cancers led to a robust decrease in cell viability. Additionally, dosing of tumor bearing mice led to complete regressions in NCI-H1373 lung cancer xenografts. To investigate the mechanism of action (MOA) leading to decreased cell viability, we treated NCI-H1373 cells with RBN-2397 and found accumulation of cells in the G0/G1 phase of the cell cycle indicative of a cell cycle arrest. This arrest in NCI-H1373 cells was associated with the induction of senescence and increased mRNA expression of senescence associated secretory phenotype (SASP) genes. To evaluate the in vivo MOA, we performed an NCI-H1373 xenograft study and collected tumors after 7 days of RBN-2397 treatment. PARP7 inhibition led to decreased expression of Ki67, and increased expression of P21 and cleaved caspase-3, suggesting decreased proliferation and increased apoptosis. Increased expression of SASP genes was also observed in RBN-2397 treated tumors. Finally, we investigated transcriptional changes after RBN-2397 treatment by RNA sequencing. In addition to the effects observed in Type I IFN signaling, we also observed differential expression of genes associated with other pathways including autophagy and energy metabolism. Further evaluation of key autophagy proteins revealed that RBN-2397 affects autophagy flux and leads to a decrease in the oxygen consumption rate of cells and reduced ATP production from the mitochondria, suggesting that a change in energy metabolism may be related to the tumor intrinsic effect of RBN-2397. In summary, we show treatment of cancer cells with RBN-2397 not only leads to activation of tumor cell IFN signaling, but also causes G1 arrest and senescence, and changes in cancer cell autophagy and energy metabolism. In vivo, RBN-2397 treatment leads to complete tumor regressions in xenografts accompanied by decreased proliferation and increased apoptosis of tumor cells. RBN-2397 is currently being evaluated in the clinic as single agent in selected cancer types (NCT04053673) and in combination with anti-PD-1 therapies. Citation Format: Jennifer R. Molina, Joseph M. Gozgit, Melissa M. Vasbinder, Ryan P. Abo, Kaiko kunii, Kristy G. Kuplast-Barr, Bin Gui, Sunaina P. Nayak, Elena Minissale, Kerren K. Swinger, Tim J. Wigle, Alvin Z. Lu, Danielle J. Blackwell, Christina R. Majer, Yue Ren, Ellen Bamberg, Mario Niepel, Jan-Rung Mo, William D. Church, Ahmed S. Mady, Jeff Song, Zacharenia A. Varsamis, Luke Utley, Patricia E. Rao, Timoty J. Mitchison, Kevin W. Kuntz, Victoria M. Richon, Kristen McEachern, Heike Keilhack. PARP7 inhibitor RBN-2397 increases tumoral IFN signaling leading to various tumor cell intrinsic effects and tumor regressions in mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2154.

Targeted Inhibition of HK-II Reversed the Warburg Effect to Improve the Radiosensitivity of Laryngeal Carcinoma.

PurposeHexokinase-II (HK-II) is the key enzyme in the first rate-limiting step of glycolysis that catalyzes the conversion of glucose to glucose-6-phosphate. Here, we examined the association between HK-II expression and radioresistance in laryngeal carcinoma and whether the inhibition of HK-II expression can enhance the radiosensitivity of these tumors.MethodsThe effects of HK-II small interfering RNA (siRNA) on the radiosensitivity of Tu212 cells were examined in vitro and in vivo in a mouse model. Cells were irradiated using a 6-MV linear accelerator. The cell viability, cell survival, proliferation, apoptosis, and cell cycle of Tu212 cells were evaluated using trypan blue staining, colony formation assays, CCK-8 assays, and flow cytometry, respectively. Oxygen consumption, lactic acid production, glucose consumption, and the ATP level of Tu212 cells were also examined. The expression of glycolytic and regulatory enzymes involved in the tricarboxylic acid cycle was assessed using Western blotting.ResultsThe HK-II siRNA and X-ray combination treatment led to a significantly greater reduction of cell viability, inhibition of cell survival and proliferation, increased apoptosis, and increased G2 phase arrest compared to either treatment alone (all, P<0.01). HK-II siRNA increased the oxygen consumption rate of cells, significantly inhibited lactic acid production and glucose consumption, and significantly suppressed the upregulation of HK-II, pyruvate kinase M2 (PKM2), pyruvate dehydrogenase (PDH), phosphofructokinase platelet (PFKP), lactate dehydrogenase (LD), and citrate synthase (CS) (all, P<0.01).ConclusionThe inhibition of HK-II by siRNA enhances the radiosensitivity of laryngeal carcinoma Tu212 cells by inhibiting glycolysis and partially inhibiting oxidative phosphorylation.

The Neurogenic Compound P7C3 Regulates the Aerobic Glycolysis by Targeting Phosphoglycerate Kinase 1 in Glioma.

BackgroundP7C3 is a neurogenic compound that exhibits neuroprotective properties in neural cells. However, its target proteins and effects in glioma are unknown.MethodsThe candidate P7C3 target proteins were analyzed using a human protein microarray containing 23136 human proteins. A streptavidin agarose affinity assay was used to verify the direct interaction between P7C3 and phosphoglycerate kinase 1 (PGK1). Mass spectrometry was used to identify the binding sites of PGK1 for P7C3 binding. Seahorse XF96 extracellular flux analyzer was used to measure the cell oxygen consumption rate and extracellular acidification rate. Glycolytic metabolites were measured using the related kits. Protein level was detected by western blotting and immunohistochemical staining. Autophagy was analyzed using a transmission electron microscope and western blotting. The malignancy of tumor progression in vitro and in vivo was analyzed based on cell viability, apoptosis and proliferation, migration and invasion, and xenograft model. Glial cells were marked by antibodies via immunohistochemical staining.ResultsThe human protein microarray identified 577 candidate P7C3 target proteins. The global profile of P7C3 target proteins indicated that P7C3 regulates glycolysis. Metabolic experiments confirmed that P7C3 regulates aerobic glycolysis in glioma cells. The underlying mechanism of P7C3 was found to be direct targeting PGK1 at lysine residues and asparagine residues, and the specific P7C3-PGK1 interaction led to decreased protein level and total intracellular kinase activity of PGK1. The Cancer Genome Atlas and Chinese Glioma Genome Atlas databases indicated that the mRNA level of PGK1 is significantly increased in high-grade glioma, and the abnormally high mRNA level of PGK1 is associated with a poor prognosis in patients with glioma, suggesting that PGK1 is a promising target for glioma therapy. The inhibition of PGK1 and the subsequent suppression of aerobic glycolysis caused by P7C3 inhibited the malignant growth of glioma in vitro and in vivo. Furthermore, P7C3 did not damage normal glial cells under concentration, which exhibit an inhibitory effect on gliomas.ConclusionsThis study revealed that P7C3 suppresses glioma by regulating aerobic glycolysis via directly targeting PGK1. Furthermore, we identified the P7C3 target proteins for the first time which is expected to provide scientific clues for future studies.

Pathogenicity evaluation and the genotype-phenotype analysis of OPA1 variants.

Autosomal dominant optic atrophy (ADOA) is an important cause of irreversible visual impairment in children and adolescents. About 60-90% of ADOA is caused by the pathogenic variants of OPA1 gene. By evaluating the pathogenicity of OPA1 variants and summarizing the relationship between the genotype and phenotype, this study aimed to provide a reference for clinical genetic test involving OPA1. Variants in OPA1 were selected from the exome sequencing results in 7092 cases of hereditary eye diseases and control groups from our in-house data. At the same time, the urine cells of some optic atrophy patients with OPA1 variants as well as their family members were collected and oxygen consumption rates (OCR) were measured in these cells to evaluate the pathogenicity of variants. As a result, 97 variants were detected, including 94 rare variants and 3 polymorphisms. And the 94 rare variants were classified into three groups: pathogenic (33), variants of uncertain significance (19), and likely benign (42). Our results indicated that the frameshift variants at the 3' terminus might be pathogenic, while the variants in exon 7 and intron 4 might be benign. The penetrance of the missense variants was higher than that of truncation variants. The OCR of cells with pathogenic OPA1 variants were significantly lower than those without pathogenic variants. In conclusion, some variants might be benign although predicted pathogenic in previous studies while some might have unknown pathogenesis. Measuring the OCR in urine cells could be used as a method to evaluate the pathogenicity of some OPA1 variants.

Dual-emissive, oxygen-sensing boron nanoparticles quantify oxygen consumption rate in breast cancer cells.

.Significance: Decreasing the oxygen consumption rate (OCR) of tumor cells is a powerful method for ameliorating tumor hypoxia. However, quantifying the change in OCR is challenging in complex experimental systems.Aim: We present a method for quantifying the OCR of two tumor cell lines using oxygen-sensitive dual-emissive boron nanoparticles (BNPs). We hypothesize that our BNP results are equivalent to the standard Seahorse assay.Approach: We quantified the spectral emissions of the BNP and accounted for external oxygen diffusion to quantify OCR over 24 h. The BNP-computed OCR of two breast cancer cell lines, E0771 and 4T07, were compared with their respective Seahorse assays. Both cell lines were also irradiated to quantify radiation-induced changes in the OCR.Results: Using a Bland–Altman analysis, our BNPs OCR was equivalent to the standard Seahorse assay. Moreover, in an additional experiment in which we irradiated the cells at their 50% survival fraction, the BNPs were sensitive enough to quantify 24% reduction in OCR after irradiation.Conclusions: Our results conclude that the BNPs are a viable alternative to the Seahorse assay for quantifying the OCR in cells. The Bland–Altman analysis showed that these two methods result in equivalent OCR measurements. Future studies will extend the OCR measurements to complex systems including 3D cultures and in vivo models, in which OCR measurements cannot currently be made.

The Role Of Radiation Dose-Dependent Enhancement Of Fatty Acid Oxidation On Radiation Surviving/Resistant Lung Cancer Cells

Radiotherapy plays a critical role in the integrated management of lung cancer. However, radioresistance limits the long-term control. Exploring the dynamic changes of metabolic reprogramming in radiation surviving/resistant (S/R) lung cancer cells is helpful to clarify the metabolic mechanism of radiation resistance and to develop new targets for intervention and early detection. Cell lines were irradiated with different doses (2Gy × 20F, 2Gy × 30F, 2Gy × 40F) in conventional dose fractionation. The cellular radiosensitivity was verified by colony formation assay and neutral comet assay. Cell proliferation ability was determined by EdU assay. Metabonomic analysis was used to identify the differentially expressed metabolites between high-dose radiation-resistant cells and their parent cells. Lipid droplet content was detected by Oil Red O (ORO)staining. Cell oxygen consumption rate (OCR) was measured by Seahorse XF24e analyzer. Western blot was used to detect the expression of metabolic enzymes. The growth of xenograft tumors from these cell lines in BALB/c nude mice were measured after the treatment of radiation (2Gy×5F), Etomoxir, or radiation combined with Etomoxir. Compared with parent cells, the radioresistance of S/R lung cancer cells after different doses of radiation was significantly increased with the increase of radiation exposure. ORO staining showed that fatty deposition of radiation S/R cells was obviously higher than their parent cells, and more fatty deposition in cells received higher dose of radiation. The ketone body metabolism-related substances, including acetoacetic acid, a metabolite of FAO, were significantly enriched in high-dose radiation-resistant cells. The expression of carnitine palmitoyltransferase1 (CPT1) and the OCR in radiation S/R cells were also radiation-dose dependently increased. Etomoxir, an inhibitor of fatty acid oxidation, significantly enhanced the radiosensitivity and decreased the OCR and DNA repair ability of various S/R cells exposed to radiation. We further confirmed that Etomoxir could significantly inhibit proliferation of radiation S/R cells in vivo, which also presented with radiation-dose dependent model. The enhancement of radiation dose-dependent FAO promotes radiation surviving/resistance of lung cancer cells. CPT1A, a key metabolic enzyme mediating FAO, may be a potential target for treatment of radiation resistant lung cancer. Funding: 81972853, 81572279, 2016J004, LC2019ZD009, 2018CR033.

Abstract 6180: The role of radiation dose-dependent enhancement of fatty acid oxidation on radiation surviving/resistant lung cancer cells

Abstract Objective: Radiotherapy plays a critical role in the integrated management of lung cancer. However, radioresistance limits the long-term control. Exploring the dynamic changes of metabolic reprogramming in radiation surviving/resistance lung cancer cells is helpful to clarify the metabolic mechanism of radiation resistance and to develop new targets for intervention and early detection. Methods: Multiple lung cancer cell lines were irradiated with different doses (2Gy × 20F, 2Gy × 30F, 2Gy × 40F) in conventional dose fractionation. The cellular radiosensitivity was verified by colony formation assay and neutral comet assay. Cell proliferation ability was determined by EdU assay. Metabonomic analysis was used to identify the differentially expressed metabolites between high-dose radiation-resistant cells and their parent cells. Lipid droplet content was detected by Oil Red O staining. Cell oxygen consumption rate (OCR) was measured by Seahorse XF24e analyzer. Western blot was used to detect the expression of metabolic enzymes. The growth of xenograft tumors from these cell lines in BALB/c nude mice were measured after the treatment of radiation (2Gy × 5F), Etomoxir, or radiation combined with Etomoxir. Results: Compared with parent cells, the radioresitance of surviving/resistant lung cancer cells after different doses of radiation was significantly increased with the increase of radiation exposure. Oil Red O staining showed that fatty deposition of radiation surviving/resistant cells was obviously higher than their parent cells, and more fatty deposition in cells received higher dose of radiation. The ketone body metabolism-related substances, including acetoacetic acid, a metabolite of FAO, were significantly enriched in high-dose radiation-resistant lung cancer cells. The expression of carnitine palmitoyltransferase1 (CPT1) and the OCR in radiation surviving/resistant lung cancer cells were also radiation-dose dependently increased. Etomoxir, an inhibitor of fatty acid oxidation, significantly enhanced the radiosensitivity and decreased the OCR and DNA repair ability of various surviving/resistant lung cancer cells exposed to radiation. We further confirmed that Etomoxir could significantly inhibit proliferation of radiation surviving/resistant cells in vivo, which also presented with radiation-dose dependent model. Conclusions: The establishment of equidifferent dose radiation surviving/resistance lung cancer cell lines in this study provides a preclinical research model for exploring the mechanism of dose-dependent radiation resistance. The enhancement of radiation dose-dependent FAO promotes radiation surviving/resistance of lung cancer cells. CPT1A, a key metabolic enzyme mediating FAO, may be a potential target for treatment of radiation resistant lung cancer. Funding: 81972853, 81572279, 2016J004, LC2019ZD009, 2018CR033. Citation Format: Xiaoxia Zhu, Shangbiao Li, Lijuan Wang, Zhihao Zheng. The role of radiation dose-dependent enhancement of fatty acid oxidation on radiation surviving/resistant lung cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6180.

The role of radiation dose-dependent enhancement of fatty acid oxidation in radiation surviving/resistant lung cancer cells.

e21724 Background: Radiotherapy plays a critical role in the integrated management of lung cancer. However, radioresistance limits the long-term control. Exploring the dynamic changes of metabolic reprogramming in radiation surviving/resistant (S/R) lung cancer cells is helpful to clarify the metabolic mechanism of radiation resistance and to develop new targets for intervention and early detection. Methods: Cell lines were irradiated with different doses (2Gy × 20F, 2Gy × 30F, 2Gy × 40F) in conventional dose fractionation. The cellular radiosensitivity was verified by colony formation assay and neutral comet assay. Cell proliferation ability was determined by EdU assay. Metabonomic analysis was used to identify the differentially expressed metabolites between high-dose radiation-resistant cells and their parent cells. Lipid droplet content was detected by Oil Red O (ORO) staining. Cell oxygen consumption rate (OCR) was measured by Seahorse XF24e analyzer. Western blot was used to detect the expression of metabolic enzymes. The growth of xenograft tumors from these cell lines in BALB/c nude mice were measured after the treatment of radiation (2Gy×5F), Etomoxir, or radiation combined with Etomoxir. Results: Compared with parent cells, the radioresitance of S/R lung cancer cells after different doses of radiationwas significantly increased with the increase of radiation exposure. ORO staining showed that fatty deposition of radiation S/R cells was obviously higher than their parent cells, and more fatty deposition in cells received higher dose of radiation. The ketone body metabolism-related substances, including acetoacetic acid, a metabolite of FAO, were significantly enriched in high-dose radiation-resistant cells. The expression of carnitine palmitoyltransferase1 (CPT1) and the OCR in radiation S/R cells were also radiation-dose dependently increased. Etomoxir, an inhibitor of fatty acid oxidation, significantly enhanced the radiosensitivity and decreased the OCR and DNA repair ability of various S/R cells exposed to radiation. We further confirmed that Etomoxir could significantly inhibit proliferation of radiation S/R cells in vivo, which also presented with radiation-dose dependent model. Conclusions: The enhancement of radiation dose-dependent FAO promotes radiation surviving/resistance of lung cancer cells. CPT1A, a key metabolic enzyme mediating FAO, may be a potential target for treatment of radiation resistant lung cancer. Funding: 81972853, 81572279, 2016J004, LC2019ZD009, 2018CR033.

Effects of Flow Rate on Mesenchymal Stem Cell Oxygen Consumption Rates in 3D Bone-Tissue-Engineered Constructs Cultured in Perfusion Bioreactor Systems

Bone grafts represent a multibillion-dollar industry, with over a million grafts occurring each year. Common graft types are associated with issues such as donor site morbidity in autologous grafts and immunological response in allogenic grafts. Bone-tissue-engineered constructs are a logical approach to combat the issues commonly encountered with these bone grafting techniques. When creating bone-tissue-engineered constructs, monitoring systems are required to determine construct characteristics, such as cellularity and cell type. This study aims to expand on the current predictive metrics for these characteristics, specifically analyzing the effects of media flow rate on oxygen uptake rates (OURs) of mesenchymal stem cells seeded on poly(L-lactic acid) (PLLA) scaffolds cultured in a flow perfusion bioreactor. To do this, oxygen consumption rates were measured for cell/scaffold constructs at varying flow rates ranging from 150 to 750 microliters per minute. Residence time analyses were performed for this bioreactor at these flow rates. Average observed oxygen uptake rates of stem cells in perfusion bioreactors were shown to increase with increased oxygen availability at higher flow rates. The residence time analysis helped identify potential pitfalls in current bioreactor designs, such as the presence of channeling. Furthermore, this analysis shows that oxygen uptake rates have a strong linear correlation with residence times of media in the bioreactor setup, where cells were seen to exhibit a maximum oxygen uptake rate of 3 picomoles O2/hr/cell.

Abstract 701: Mitochondrial mass is a critical determinant of cisPt-induced cell death in ovarian cancer

Abstract Platinum compounds are an inherent part of chemotherapy for patients with high grade serous ovarian cancer (HGSC). Platinum based therapeutics, such as cisPt, induce DNA-damage by directly binding to nuclear DNA. Interestingly, cisPt also critically affects mitochondrial DNA. Elucidating the role of mitochondria in cisplatin induced cell death of HGSC cells likely identifies new efficient therapy approaches to overcome resistance. Flow cytometric analysis of cisPt sensitive and resistant HGSC cell lines from the NCI60 panel revealed higher mitochondrial mass and higher levels of mitochondrial ROS (mtROS) in cells that are sensitive to cisPt-induced cell death. In clonal sub-lines derived from OVCAR3 the mitochondrial mass correlates with basal oxygen consumption rate and survival after cisPt exposure. The correlation of mitochondrial mass and sensitivity to cisPt-induced apoptosis is corroborated by data from the human protein atlas (www.proteinatlas.org) that indicates high expression of the mitochondrial transcription factor TFAM and the mitochondrial inner membrane protein TIMM23 as favorable prognostic factors for the survival of ovarian cancer patients. Together, this shows the importance of mitochondrial mass in cellular response to cisPt treatment. Furthermore, exposure to cisPt enhances TFAM expression and increases mitochondrial mass as well as mitochondrial ROS. Incubation with cisPt in the presence of the ROS scavenger glutathione (GSH) or pan-caspase inhibitor zVAD-fmk reduces cisPt induced apoptosis while mitochondrial mass is not affected. However, the oxygen consumption rate in cells exposed to cisPt and GSH shows that increased ROS levels induce mitochondrial dysfunction. In line with a sensitivity-determining role of mitochondrial mass, knock-down of key-regulators of mitochondrial biogenesis, i.e. PGC-1α or TFAM, blocks mitochondrial ROS and protects cells from cisPt-induced apoptosis. Mitochondrial ROS is also reduced in an autoregulatory feedback loop by so-called uncoupling proteins (UCPs). Inhibition of UCP2 by Genipin results in increased induction of mitochondrial ROS by cisPt and consequently results in enhanced apoptosis. A comparable sensitization to cisPt-induced apoptosis and ROS production is evident in cells incubated with the iron chelator VLX600.Mitochondria are a critical element in cell response to cisPt since mitochondrial mass correlates with induction of ROS and apoptosis. We show the relevance of cellular mitochondrial content by manipulation of mitochondrial mass and function. We propose the relative mitochondrial content as a biomarker that indicates the response of ovarian cancer cells to cisPt-induced apoptosis. Furthermore, our data provides evidence that increasing mitochondrial mass or induction of mtROS enhances sensitivity to cisPt-induced apoptosis and therefore is a reliable strategy to overcome resistance of ovarian cancer. Citation Format: Markus Kleih, Kathrin Böpple, Andrea Gaißler, Meng Dong, Walter E. Aulitzky, Frank Essmann. Mitochondrial mass is a critical determinant of cisPt-induced cell death in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 701.

Mitochondrial Toxicity of Selected Micropollutants, Their Mixtures, and Surface Water Samples Measured by the Oxygen Consumption Rate in Cells.

Some environmental pollutants impair mitochondria, which are of vital importance as energy factories in eukaryotic cells. Mitochondrial toxicity was quantified by measuring the change of the oxygen consumption rate (OCR) of HepG2 cells with the Agilent Seahorse XFe 96 Analyzer. Various mechanisms of mitochondrial toxicity, including inhibition of the electron transport chain or adenosine triphosphate (ATP) synthase as well as uncoupling of oxidative phosphorylation, were differentiated by dosing the sample in parallel with reference compounds following the OCR over time. These time-OCR traces were used to derive effect concentrations for 10% inhibition of the electron transport chain or 10% of uncoupling. The low effect level of 10% was necessary because environmental mixtures contain thousands of chemicals; only few of them interfere with mitochondria, but the others cause cytotoxicity. The OCR bioassay was validated with environmental pollutants of known mechanism of mitochondrial toxicity. Binary mixtures of uncouplers or inhibitors acted according to the mixture model of concentration addition. Uncoupling and/or inhibitory effects were detected in extracts of river water samples without apparent cytotoxicity. Uncoupling effects could only be quantified in water samples if inhibitory effects occurred at lower concentrations because no uncoupling can be detected without an appreciable membrane potential built up. The OCR bioassay can thus complement chemical analysis and in vitro bioassays for monitoring micropollutants in water. Environ Toxicol Chem 2019;00:1-12. © 2019 SETAC.

Enhanced Hepatic Differentiation of Human Induced Pluripotent Stem Cells Using Gas-Permeable Membrane.

Although oxygen is a vital nutrient for the hepatocytes in vitro, few reports have focused on its effect during hepatic differentiation of induced pluripotent stem cells (iPSCs). In this report, we performed the hepatic differentiation of human iPSCs (hiPSCs) under different atmospheric oxygen concentrations and oxygen supply fluxes to investigate the effects of oxygen in terms of both the concentration and the supply flux. Results demonstrate that direct oxygenation through a polydimethylsiloxane (PDMS) membrane enhances the maturation and efficient production of hiPSC-derived hepatocyte-like cells (iHeps). Thus, direct oxygenation through a PDMS membrane is a better alternative culture method over conventional tissue culture-treated polystyrene (TCPS) plates for the maturation of hiPSC-derived hepatocytes in vitro.

Oxygenation in cell culture: Critical parameters for reproducibility are routinely not reported.

Mammalian cell culture is foundational to biomedical research, and the reproducibility of research findings across the sciences is drawing increasing attention. While many components contribute to reproducibility, the reporting of factors that impact oxygen delivery in the general biomedical literature has the potential for both significant impact, and immediate improvement. The relationship between the oxygen consumption rate of cells and the diffusive delivery of oxygen through the overlying medium layer means parameters such as medium depth and cell type can cause significant differences in oxygenation for cultures nominally maintained under the same conditions. While oxygenation levels are widely understood to significantly impact the phenotype of cultured cells in the abstract, in practise the importance of the above parameters does not appear to be well recognized in the non-specialist research community. On analyzing two hundred articles from high-impact journals we find a large majority missing at least one key piece of information necessary to ensure consistency in replication. We propose that explicitly reporting these values should be a requirement for publication.

Effects of laser stimulation with different power on oxygen consumption rate of C2C12 myoblasts in mice

To investigate the effects of 650 nm laser irradiation on cell oxygen consumption rate in C2C12 myoblasts following different doses. C2C12 cells were irradiated with 650 nm laser(λ=650 nm, p=5 mW) with energy densities of 0, 0.4, and 0.8 J/cm2. Cell oxidative function was measured by oxygen consumption rate kit. Protein expression of myogenic determination factor (MyoD), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), mammalian target of rapamycin (mTOR) and its phosphorylation were detected by Western blot. Compared to the control group, the expression levels of MyoD, PGC-1α protein were increased and cell oxygen consumption rate was promoted in the low dose group(P<0.05). MyoD and PGC-1α protein expressions were also increased(P<0.05), the ratio of mTOR and its phosphorylationwere decreased significantly in the high dose group(P<0.05). 650 nm laser irradiation that dose is 0.4 J/cm2 enhances cell oxidative function, it related to that proper dose laser irradiation promoted the expression of PGC-1a protein.

Comparison of Oxygen Consumption Rates of Nondegenerate and Degenerate Human Intervertebral Disc Cells.

In vitro measurements of the oxygen consumption rates (OCR) of human intervertebral disc (IVD) cells. The aim of this study was to determine the differences in the OCR of nondegenerate and degenerate human annulus fibrosus (AF), nucleus pulposus (NP), and cartilage endplate (CEP) cells at different glucose concentrations. The avascular nature of the IVD creates a delicate balance between rate of nutrient transport through the matrix and rate of disc cell consumption necessary to maintain tissue health. Previous studies have shown a dependence of OCR for animal (e.g., bovine and porcine) IVD cells on oxygen level and glucose concentration. However, the OCR of nondegenerate human IVD cells compared to degenerate human IVD cells at different glucose concentrations has not been investigated. IVD cells were isolated from the AF, NP, and CEP regions of human cadaver spines and surgical samples. The changes in oxygen concentration were recorded when cells were sealed in a metabolic chamber. The OCR of cells was determined by curve fitting using the Michaelis-Menton equation. Under identical cell culture conditions, the OCR of degenerate human IVD cells was three to five times greater than that of nondegenerate human IVD cells. The degenerate IVD cells cultured in low-glucose medium (1 mmol/L) exhibited the highest OCR compared to degenerate cells cultured at higher glucose levels (i.e., 5 mmol/L, 25 mmol/L), whereas no significant differences in OCR were found among the nondegenerate IVD cells for all glucose levels. Considering the significantly higher OCR and unique response to glucose of degenerate human IVD cells, the degeneration of the IVD is associated with a cell phenotypic change related to OCR. The OCR of IVD cells reported in this study will be valuable for understanding human IVD cellular behavior and tissue nutrition in response to disc degeneration. N/A.