Tumor Cell Oxygen Consumption Research Articles

Mitochondria-targeted nanoplatforms for enhanced photodynamic therapy against hypoxia tumor

BackgroundPhotodynamic therapy (PDT) is a promising therapeutic modality that can convert oxygen into cytotoxic reactive oxygen species (ROS) via photosensitizers to halt tumor growth. However, hypoxia and the unsatisfactory accumulation of photosensitizers in tumors severely diminish the therapeutic effect of PDT. In this study, a multistage nanoplatform is demonstrated to overcome these limitations by encapsulating photosensitizer IR780 and oxygen regulator 3-bromopyruvate (3BP) in poly (lactic-co-glycolic acid) (PLGA) nanocarriers.ResultsThe as-synthesized nanoplatforms penetrated deeply into the interior region of tumors and preferentially remained in mitochondria due to the intrinsic characteristics of IR780. Meanwhile, 3BP could efficiently suppress oxygen consumption of tumor cells by inhibiting mitochondrial respiratory chain to further improve the generation of ROS. Furthermore, 3BP could abolish the excessive glycolytic capacity of tumor cells and lead to the collapse of ATP production, rendering tumor cells more susceptible to PDT. Successful tumor inhibition in animal models confirmed the therapeutic precision and efficiency. In addition, these nanoplatforms could act as fluorescence (FL) and photoacoustic (PA) imaging contrast agents, effectuating imaging-guided cancer treatment.ConclusionsThis study provides an ideal strategy for cancer therapy by concurrent oxygen consumption reduction, oxygen-augmented PDT, energy supply reduction, mitochondria-targeted/deep-penetrated nanoplatforms and PA/FL dual-modal imaging guidance/monitoring. It is expected that such strategy will provide a promising alternative to maximize the performance of PDT in preclinical/clinical cancer treatment.Graphical

Chemoradiation With or Without Metformin in Locally Advanced Cervical Cancer: Phase II Randomized Trial

Tumor hypoxia is associated with poor response to radiation (RT) and chemotherapy, and worse treatment outcome. We previously discovered a novel mechanism of metformin in a xenograft model: enhancing tumor RT response by inhibiting tumor cell oxygen consumption. Our population-based study showed that cumulative dose of metformin after cervical cancer diagnosis was independently associated with a decreased risk of cervical cancer-specific mortality in diabetic women. Based on the pre-clinical and retrospective data, we hypothesized that metformin would decrease tumor hypoxia and improve tumor response to RT in locally advanced cervical cancer.A window-of-opportunity, phase II randomized trial was performed in women with stage IB-IVA cervical adenocarcinoma, squamous cell or adenosquamous carcinoma. Patients underwent screening positron emission tomography (PET) imaging with hypoxia tracer fluoroazomycin arabinoside (FAZA). Those with non-hypoxic tumor (no FAZA uptake) were excluded. Patients with FAZA uptake were randomized centrally in a 2:1 ratio (in favor of metformin) to receive either metformin in combination with standard chemoRT or standard chemoRT alone. Metformin was started at 850mg once daily x 3 days, followed by 850mg twice daily throughout the entire duration of external radiotherapy. A second FAZA-PET/CT scan was performed after 1 week of metformin or no intervention in control group, just before start of chemoRT. The hypoxic volume was defined as all voxels within a tumor with standardized uptake values (SUVs) greater than 3 standard deviations (SD) from the mean gluteus maximus muscle SUV value. The hypoxic fraction (HF) was defined as the ratio of the number of hypoxic voxels to the total number of tumor voxels. The primary endpoint was absolute mean change in HF between the two FAZA-PET scans, compared using the Wilcoxon sign rank test. Disease-free survival (DFS) was defined as the duration of time from randomization to the time of relapse or death and compared using the log-rank test. Target accrual was 48 patients; the study was closed early to accrual due to FAZA availability and the COVID-19 pandemic.Of the twenty patients who consented, 6 were excluded due to no FAZA uptake and 1 withdrew. The median age of the 13 enrolled patients was 52; 8 (62%) had squamous cell carcinoma and 8 had stage IIB disease. HF of the 10 patients in the metformin arm decreased by an average of 10.2% (from 44.4% to 34.2%) ± SD 16.9% after 1 week of metformin, compared to an average increase of 4.7% (from 29.1% to 33.8%) ± 11.5% for the 3 patients in the control arm (P = 0.027). With a median follow-up of 2.8 years, the 2-year DFS was 67% for the metformin arm vs 33% for control (P = 0.09).Metformin decreases cervical tumor hypoxia with a trend towards improved DFS in this trial. A larger confirmatory trial is warranted.

Repurposing Atovaquone as a Tumor Hypoxia Modifier: A Window of Opportunity Study in Patients with Resectable Non-small Cell Lung Cancer

Tumor hypoxia confers profound resistance to radiation. An emerging strategy to tackle hypoxia is by reprogramming tumor metabolism. The antimalarial drug atovaquone inhibits oxidative phosphorylation (OXPHOS) and preclinically has been shown to reduce tumor cell oxygen consumption and alleviate tumor hypoxia resulting in radiosensitization. We conducted a translational clinical trial to determine whether atovaquone reduces tumor hypoxia in patients with non-small cell lung cancer (NSCLC). Patients with NSCLC scheduled for surgical resection were recruited sequentially into two cohorts. Cohort 1 received oral atovaquone 750mg bd until the day before surgery. Cohort 2 received no treatment. Tumor hypoxia imaging with 18F-FMISO or 18F-FAZA PET-CT was undertaken at baseline and repeated just prior to resection. Tumor hypoxic volume (HV) was defined using tumor-to-blood tracer uptake ratio ≥1.4, with ≥10% reduction in HV classed a response to treatment. Patients with <1.5 mL HV at baseline were deemed not evaluable for HV analysis and were replaced. Perfusion CT (pCT) and shMOLLI T1 map MRI were undertaken at the same timepoints to assess for changes in tumor perfusion and biochemistry, respectively. An inter-cohort comparison of hypoxia metagene expression signatures obtained by RNA sequencing was conducted from multiple regions per resected tumor. Adverse event monitoring (CTCAE v4.0) was performed until surgery. Forty-six patients were recruited with 30 (15 per cohort) evaluable for paired HV analysis. Patient and tumor characteristics were similar between cohorts. Median duration of atovaquone treatment between imaging timepoints was 12 days. The median change in HV in atovaquone-treated patients was -28% (95% CI: -58.2, -4.4), with 11 (73.3%) patients classed as responders. In contrast, the median change in untreated patients was +15.5% (95% CI: -6.5, +35.5). Using linear regression with pre-surgery HV as outcome adjusting for cohort and baseline HV and tumor volume (log-transformed), the expected geometric mean HV was 60% (95% CI: 30%, 70%) lower for treated patients (p = 0.004). Transcriptomic analysis revealed significantly decreased expression of multiple hypoxia metagene signatures in treated tumors. Altered expression of OXPHOS related pathways was also observed suggesting wider metabolic reprogramming. pCT and MRI results did not show altered tumor perfusion or suggest change in cellularity, respectively, in keeping with an OXPHOS-dependent mechanism of action. No adverse events related to atovaquone were reported. This the first clinical study to investigate whether an OXPHOS inhibitor reduces tumor hypoxia. The data presented supports our pre-clinical finding that atovaquone rapidly reduces tumor hypoxia through decreased oxygen consumption in NSCLC. We will shortly commence phase I clinical testing combining atovaquone with radical chemoradiotherapy in NSCLC.

Interfering with Lactate‐Fueled Respiration for Enhanced Photodynamic Tumor Therapy by a Porphyrinic MOF Nanoplatform

AbstractLactate is a prominent energy substrate for oxidative tumor cells. Interfering with the lactate‐fueled respiration of oxidative tumor cells would be a promising therapeutic strategy for cancer treatment. In this study, α‐cyano‐4‐hydroxycinnamate (CHC) is incorporated into a porous Zr (IV)‐based porphyrinic metal‐organic framework (PZM) nanoparticle, to reduce the lactate uptake by inhibiting the expression of lactate‐proton symporter, monocarboxylate transporter 1 (MCT1) in tumor cells, thus transform lactate‐fueled aerobic respiration to anaerobic glycolysis. The alteration in energy supply can also decrease the oxygen consumption in tumor cells, which would facilitate the photodynamic therapy (PDT) in cancer treatment. Moreover, hyaluronic acid (HA) is coated on the surface of PZM nanoparticles for CD44‐targeting and hyaluronidase‐induced intracellular drug releasing. Both in vitro and in vivo studies confirmed good biocompatibility and enhanced PDT efficacy of the HA‐coated PZM nanoparticles (CHC‐PZM@HA) in tumor cells. The CHC‐PZM@HA platform will provide a new perspective in cancer therapy.

Efficacy of PD-1 Blockade Is Potentiated by Metformin-Induced Reduction of Tumor Hypoxia.

Blockade of the coinhibitory checkpoint molecule PD-1 has emerged as an effective treatment for many cancers, resulting in remarkable responses. However, despite successes in the clinic, most patients do not respond to PD-1 blockade. Metabolic dysregulation is a common phenotype in cancer, but both patients and tumors are metabolically heterogeneous. We hypothesized that the deregulated oxidative energetics of tumor cells present a metabolic barrier to antitumor immunity through the generation of a hypoxic microenvironment and that normalization of tumor hypoxia might improve response to immunotherapy. We show that the murine tumor lines B16 and MC38 differed in their ability to consume oxygen and produce hypoxic environments, which correlated with their sensitivity to checkpoint blockade. Metformin, a broadly prescribed type II diabetes treatment, inhibited oxygen consumption in tumor cells in vitro and in vivo, resulting in reduced intratumoral hypoxia. Although metformin monotherapy had little therapeutic benefit in highly aggressive tumors, combination of metformin with PD-1 blockade resulted in improved intratumoral T-cell function and tumor clearance. Our data suggest tumor hypoxia acts as a barrier to immunotherapy and that remodeling the hypoxic tumor microenvironment has potential to convert patients resistant to immunotherapy into those that receive clinical benefit. Cancer Immunol Res; 5(1); 9-16. ©2016 AACR.

Anti-cancer effect of metformin and regulation on HIF-1α

Studies show that metformin inhibits the proliferation of several types of cancer cells. All evidences, therefore propose the anti-tumor effect of metformin. Metformin can repress the oxygen consumption of tumor cells, and suppress hypoxia-inducible factor-1α (HIF-1α) accumulation through the translational and posttranslational mechanisms, and suppress the expression of HIF-1α by activating AMP-activated protein kinase (AMPK). Key words: Metformin; Neoplasms; Hypoxia-inducible factor 1, alpha subunit

Abstract 1140: PI3K/Akt inhibition decreases oxygen consumption In tumor cells by phosphorylating and inactivating pyruvate dehydrogenase PDH E1α subunit

Abstract The PI3K/mTOR pathway plays a central role in coupling metabolic processes to the cellular proliferative state. Pharmacologic inhibitors of the PI3K/mTOR pathway or genetic inhibition of Akt/PI3K decreased the oxygen consumption rate (OCR) in transformed cell lines in vitro by 30-40%. Pharmacologic inhibition of this pathway increased phosphorylation of the E1α subunit of the pyruvate dehydrogenase (PDH) complex on Ser293, an inhibitory modification of this critical gatekeeper of mitochondrial respiration. Expressing wild type PTEN in a doxycycline-inducible manner in a glioblastoma cell line with mutant PTEN led to an increase in PDH E1α phosphorylation and a decrease in OCR. The decrease in OCR with PI3K/Akt/mTOR inhibition was recapitulated by knocking down PDH E1α expression using siRNA. Pre-treatment with dichloroacetate (DCA), a known inhibitor of the pyruvate dehydrogenase kinases (PDKs) that phosphorylate PDH E1α, prevented the upregulation in PDH E1α phosphorylation, induced by inhibition of the pathway and also blunted the decrease in OCR. Likewise, introduction of exogenous PDH-E1α that contains serine to alanine mutations, which can no longer be regulated by phosphorylation, also blunted the decrease in OCR seen with PI3K/mTOR inhibition. Our findings highlight an association between the PI3K/mTOR pathway and tumor cell oxygen consumption that is regulated in part by PDH phosphorylation. These results have important implications for understanding the effects PI3K pathway activation in tumor metabolism and also in designing cancer therapy trials that use inhibitors of this pathway. Citation Format: George John Cerniglia, Souvik Day, Shannon M. Gallagher-Colombo, Natalie Daurio, Stephen Tuttle, Theresa M. Busch, Theresa M. Busch, Alexander Lin, Tatiana V. Esipova, Sergei A. Vinogradov, Constantinos Koumenis, Amit Maity. PI3K/Akt inhibition decreases oxygen consumption In tumor cells by phosphorylating and inactivating pyruvate dehydrogenase PDH E1α subunit. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1140. doi:10.1158/1538-7445.AM2015-1140

The PI3K/Akt Pathway Regulates Oxygen Metabolism via Pyruvate Dehydrogenase (PDH)-E1α Phosphorylation.

Inhibition of the PI3K/Akt pathway decreases hypoxia within SQ20B human head and neck cancer xenografts. We set out to understand the molecular mechanism underlying this observation. We measured oxygen consumption using both a Clark electrode and an extracellular flux analyzer. We made these measurements after various pharmacologic and genetic manipulations. Pharmacologic inhibition of the PI3K/mTOR pathway or genetic inhibition of Akt/PI3K decreased the oxygen consumption rate (OCR) in vitro in SQ20B and other cell lines by 30% to 40%. Pharmacologic inhibition of this pathway increased phosphorylation of the E1α subunit of the pyruvate dehydrogenase (PDH) complex on Ser293, which inhibits activity of this critical gatekeeper of mitochondrial respiration. Expressing wild-type PTEN in a doxycycline-inducible manner in a cell line with mutant PTEN led to an increase in PDH-E1α phosphorylation and a decrease in OCR. Pretreatment of SQ20B cells with dichloroacetate (DCA), which inhibits PDH-E1α phosphorylation by inhibiting dehydrogenase kinases (PDK), reversed the decrease in OCR in response to PI3K/Akt/mTOR inhibition. Likewise, introduction of exogenous PDH-E1α that contains serine to alanine mutations, which can no longer be regulated by phosphorylation, also blunted the decrease in OCR seen with PI3K/mTOR inhibition. Our findings highlight an association between the PI3K/mTOR pathway and tumor cell oxygen consumption that is regulated in part by PDH phosphorylation. These results have important implications for understanding the effects of PI3K pathway activation in tumor metabolism and also in designing cancer therapy trials that use inhibitors of this pathway.

Abstract B05: PI3K/mTOR pathway-dependent regulation of oxygen metabolism via pyruvate dehydrogenase (PDH)-E1alpha phosphorylation

Abstract The PI3K/mTOR pathway plays a central role in coupling metabolic processes to the cellular proliferative state. In the current study we show that pharmacological inhibition of this pathway leads to a decrease in hypoxia within SQ20B human head and neck cancer xenografts. The mechanism underlying the effect appears in part to be due to reduced tumor cell oxygen consumption induced by the drug. Pharmacologic inhibitors of the PI3K/mTOR pathway or genetic inhibition of Akt/PI3K decreased the oxygen consumption rate (OCR) in transformed cell lines in vitro by 30-40%. Pharmacologic inhibition of this pathway increased phosphorylation of the E1alpha subunit of the pyruvate dehydrogenase (PDH) complex on Ser293, an inhibitory modification of this critical gatekeeper of mitochondrial respiration. Expressing wild type PTEN in a doxycycline-inducible manner in a glioblastoma cell line with mutant PTEN led to an increase in PDH-E1alpha phosphorylation and a decrease in OCR. The decrease in OCR with PI3K/Akt/mTOR inhibition was recapitulated by knocking down PDH-E1alpha expression using siRNA. Pre-treatment with dichloroacetate (DCA), a known inhibitor of the pyruvate dehydrogenase kinases (PDKs) that phosphorylate PDH-E1alpha prevented the upregulation in PDH-E1alpha phosphorylation, induced by inhibition of the pathway and also blunted the decrease in OCR. Our findings highlight an association between the PI3K/mTOR pathway and tumor cell oxygen consumption and reveal a novel link between this pathway and PDH phosphorylation. These results have important implications for understanding the effects PI3K pathway activation in tumor metabolism and also in the use of inhibitors of this pathway in cancer therapy Citation Format: George Cerniglia, Souvik Dey, Shannon M. Gallagher-Colombo, Natalie Daurio, Stephen Tuttle, Theresa M. Busch, Alexander Lin, Tatiana V. Esipova, Sergei Vinogradov, Constantinos Koumenis, Amit Maity. PI3K/mTOR pathway-dependent regulation of oxygen metabolism via pyruvate dehydrogenase (PDH)-E1alpha phosphorylation. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B05.

Abstract 4314: Metabolic manipulation of hypoxia and radiotherapy response by electron transport inhibitors

Abstract The presence of hypoxia in solid tumors is correlated with poor treatment outcome. Even small population of hypoxic cells within tumors can significantly decrease the efficacy of radiation therapy. One approach to treating hypoxia in tumours is to reduce overall tumor cell oxygen consumption by modulating mitochondrial respiration. This could reoxygenate hypoxic cells within the tumor and restore radiation sensitivity. To guide in vivo application we tested the effects of three respiratory chain inhibitors; the biguanides metformin and phenformin and rotenone on oxygen consumption in 3D tissue engineered discs. Experiments were carried out to evaluate the effect of respiratory inhibition on the hypoxic fraction of HCT116 and HT29 tissue discs using immunostaining with the hypoxic cell marker pimonidazole. An oxygen sensing phosphorescence lifetime probe assessed pO2 on the basal side of the disc as a function of time after inhibitors were exposed to the apical surface. Clonogenic assays were then performed to confirm the radiosensitization of hypoxic cells within the tissue discs. Pimonidazole staining and direct pO2 sensing confirmed that all three electron transfer inhibitors decrease the hypoxic fraction by reducing cellular oxygen consumption. Rotenone was found to completely eliminate the hypoxic fraction at nanomolar concentrations compared to metformin and phenformin which require milli to micromolar concentrations respectively. Furthermore, rotenone was found to have an effect on oxygen utilization within minutes of drug exposure compared to both metformin and phenformin, which took hours to be effective. In addition, clonogenics assays determined that all three electron transfer inhibitors radiosensitize tumor cells with rotenone being the most efficient compound on a molar basis. The mitochondrial inhibitors of complex I metformin, phenformin and rotenone decrease oxygen utilization by tumor cells in a concentration dependent manner resulting in reduction of the hypoxic fraction and increased radiosensitivity. Citation Format: Maria Jose Gandolfo, Alastair H. Kyle, Andrew I. Minchinton. Metabolic manipulation of hypoxia and radiotherapy response by electron transport inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4314. doi:10.1158/1538-7445.AM2014-4314

Pyruvate induces transient tumor hypoxia by enhancing mitochondrial oxygen consumption and potentiates the anti-tumor effect of a hypoxia-activated prodrug TH-302.

BackgroundTH-302 is a hypoxia-activated prodrug (HAP) of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302.Methodology/ResultsThe hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR) oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O2), with minimal effect under aerobic conditions (21% O2). Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500–1500 mm3. Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼550 mm3), significantly delayed tumor growth.Conclusions/SignificanceOur in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the appropriate tumor size and oxygen concentration.

PI3K/Akt Inhibition Decreases Oxygen Consumption in Tumor Cells by Phosphorylating and Inactivating Pyruvate Dehydrogenase (PDH) E1a Subunit

Hypoxia is associated with resistance to radiation. We have found that the dual PI3K/mTOR inhibitor NVP-BEZ235 reduces tumor hypoxia, which may contribute to its ability to increase radiation response. In vitro exposure of cells to the drug results in decreased O2 consumption rate (OCR). Our objective in this study was to determine the mechanism underlying this phenomenon.

Abstract 1708: Metabolic regulation of the ubiquitin+proteasome system.

Abstract The ubiquitin+proteasome system (UPS) is a highly complex network that maintains protein homeostasis and cell viability through the highly regulated and selective turnover of targeted proteins. The proteasome is the major intracellular protease in eukaryotes and serves as the catalytic core of the UPS. The cytotoxic effect of proteasome inhibition has been translated therapeutically to dramatically improve the overall survival of patients diagnosed with the plasma cell malignancy multiple myeloma (MM). Bortezomib has emerged as the standard-of-care therapy for MM to catapult the UPS into a position of prominence in cancer biology and drug development but significant obstacles remain since many patients do not respond to bortezomib, clinical responses are not sustained and drug resistance inevitably emerges through unknown mechanisms. Gene profiling was performed as an unbiased approach to address the precise molecular events that promote bortezomib-resistance. Results indicated that effectors of energy metabolism were differentially expressed in cells resistant to bortezomib. Subunits of the cellular energy sensor AMP-activated protein kinase (AMPK) were expressed at lower levels in resistant cells relative to the parental cells. Real-time, cell-based metabolic measurements demonstrated that bortezomib rapidly and dramatically reduced tumor cell oxygen-consumption while bortezomib-resistant cells were less sensitive to metabolic deregulation. Genetic ablation of the AMPKα1/α2 encoding-genes also reduced the effect of bortezomib to promote autophagosome formation as well as bortezomib-induced cytotoxicity. Similarly, knockout of the AMPK-substrate and autophagy-initiating kinase ULK1 also reduced bortezomib-induced autophagosome formation and cell death. Proteasome inhibition promoted ULK-1 association with the autophagosome initiators ATG13, FIP200 and beclin-1. The AMPK activator AICAR further enhanced bortezomib-induced cytotoxicity in myeloma, lymphoma, leukemic and solid tumor cells. Proteasome inhibitors induce AMPK+ULK-mediated autophagosome formation that is coupled to apoptosis and can be exploited as a novel anti-cancer therapeutic strategy. Citation Format: James J. Driscoll, Sajjeev Jagannathan. Metabolic regulation of the ubiquitin+proteasome system. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1708. doi:10.1158/1538-7445.AM2013-1708

Abstract B7: Hypoxia-inducible factor 1 mediates thrombosis-associated cancer progression

Abstract Background. Cancer patients are up to 50 times more likely to suffer from lethal venous thromboembolism compared with non-cancer patients, but mechanisms that regulate the positive correlation between these conditions are unclear. Our primary aim was to test whether hypoxia within cells contained in the thrombus and adjacent vein leads to upregulation of hypoxia-inducible factor 1 (HIF1), which in turn stimulates the production of factors that mediate tumor growth and metastasis. Methods. Systemic or endothelial and myeloid HIF1 levels were altered (via administration of HIF1 agonist L-mimosine or via cre-Tie2 driven HIF1 deletion respectively) in established mouse models of thrombosis and breast cancer (inferior vena cava stenosis or polyoma middle T mutation driven by mouse mammary tumour virus respectively). Protein arrays were used to quantify the expression of 25 factors that regulate coagulation, thrombosis, and cancer progression. Adhesion and migration assays were used to characterise tumor and endothelial cell behaviour on thrombotic surfaces; while endothelial and tumor cell oxygen consumption rate was measured before and after thrombin treatment. Image analysis was also used to quantify intra-tumor thrombus formation. Results. Thrombus formation or upregulation of HIF1 increased the circulating expression of 10 factors that mediate tumor growth and remodelling including vascular endothelial growth factor (VEGF, 2±1 vs 1±0.2pg/ml in controls, n=6/group, P&amp;lt;0.05), hepatocyte growth factor (HGF, 10±5 vs 7±2% in controls, n=8/group, P&amp;lt;0.05), and stromal cell-derived factor 1 (SDF1, 14±6 vs 2±1% in controls, n=8/group, P&amp;lt;0.05). Hypoxia (1% oxygen) tended to attenuate endothelial cell migration on fibrin (100±13 vs 64±10 cells in controls, n=6/group, P=0.05); while HIF1 deletion in Lewis lung cancer cells abolished their hypoxia-induced increase in adhesion onto fibrin (n=5/group, P&amp;lt;0.05). Thrombin administration reduced the oxygen consumption rate of Lewis lung cancer cells (59±13 vs 79±16 pMol/min in controls, n=5/group, P&amp;lt;0.005) but not endothelial cells (P=0.06). Deposition of fibrin in mammary tumours was reduced following endothelial and myeloid HIF1 deletion (5±1 vs 13±3% in controls, n=7/group, P&amp;lt;0.05) and this was associated with reduced tumour size (n=14, R=0.7, P&amp;lt;0.01). Conclusions. HIF1 regulates the progression of venous thrombosis and cancer, and could also be responsible for the positive association between these conditions. Cell and tissue-specific HIF1 is an attractive therapeutic target for treatments that aim to reduce thrombosis-associated cancer progression. Citation Format: Colin E. Evans, Cristina Branco-Price, Julia Humphries, Ashar Wadoodi, Prakash Saha, Jung-whan Kim, Mattias Belting, Alberto Smith, Randall S. Johnson. Hypoxia-inducible factor 1 mediates thrombosis-associated cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B7.

Decrease in Tumor Cell Oxygen Consumption after Treatment with Vandetanib (ZACTIMA™; ZD6474) and its Effect on Response to Radiotherapy

We investigated the early effects of vandetanib (ZACTIMA; ZD6474), an inhibitor of VEGFR-dependent angiogenesis, on tumor oxygenation and on the possible consequences of combining vandetanib with radiotherapy. Tumor oxygenation, perfusion, cellular consumption of oxygen, and radiation sensitivity were studied in transplantable liver tumors after daily doses of vandetanib (25 mg kg(-1) i.p.). Measurements of oxygenation (pO(2)) and tumor cell oxygen consumption were carried out using electron paramagnetic resonance (EPR), and perfusion parameters were assessed by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Regrowth delay assays were performed after treatment with vandetanib alone, radiation alone or a combination of both treatments. Vandetanib induced an early increase in tumor oxygenation that did not correlate with remodeling of the tumor vasculature or with changes in tumor perfusion. A decrease in tumor cell oxygen consumption was observed that could have been responsible for this increase in tumor oxygenation. Consistent with this increase in tumor oxygenation, we found that vandetanib potentiated the tumor response to radiotherapy. Our results confirm that treatment with an inhibitor of VEGFR signaling reduces oxygen consumption rate by tumor cells. The observation that vandetanib causes an early increase in tumor oxygenation has implications for the timing and sequencing of treatment with VEGF signaling inhibitors in combination with radiation.

Comparison of methods for measuring oxygen consumption in tumor cells in vitro

The oxygen consumption rate of tumor cells affects tumor oxygenation and response to therapies. Highly sensitive methods for determining cellular oxygen consumption are, therefore, needed to identify treatments that can modulate this parameter. We compared the performances of three different methods for measuring cellular oxygen consumption: electron paramagnetic resonance (EPR) oximetry, the Clark electrode, and the MitoXpress fluorescent assay. To compare the assays, we used K562 cells in the presence of rotenone and hydrocortisone, compounds that are known to inhibit the mitochondrial electron transport chain to different extents. The EPR method was the only one that could identify both rotenone and hydrocortisone as inhibitors of tumor cell oxygen consumption. The Clark electrode and the fluorescence assay demonstrated a significant decrease in cellular oxygen consumption after administration of the most potent inhibitor (rotenone) but failed to show any significant effect of hydrocortisone. EPR oximetry is, therefore, the most sensitive method for identifying inhibitors of oxygen consumption on cell assays, whereas the Clark electrode offers the unique opportunity to add external compounds during experiments and still shows great sensitivity in studying enzyme and chemical reactions that consume oxygen (non-cell assays). Finally, the MitoXpress fluorescent assay has the advantage of a high-sample throughput and low bulk requirements but at the cost of a lower sensitivity.

Noninvasive monitoring of carbogen-induced changes in tumor blood flow and oxygenation by functional magnetic resonance imaging

The response of tumors to radiotherapy can be enhanced if carbogen (95% O2, 5% CO2) is breathed. The timing of carbogen administration is critical, and a noninvasive method of monitoring the response of individual tumors would have obvious utility. Functional gradient recalled echo (GRE) magnetic resonance imaging (MRI) techniques are sensitive to changes in the concentrations of deoxyhemoglobin, which, thus, acts as an endogenous contrast agent for oxygenation status and blood flow. Subcutaneous GH3 prolactinomas in three rats were imaged at 4.7 Tesla with a GRE 1H sequence [echo time (TE) = 20 ms, repetition time (TR) = 80 ms, flip angle = 45 degrees, 1 mm slice, 256 phase encode steps, 4 cm field of view, in-plane resolution 0.08 x 0.08 mm, acquisition time = 4 min]. The rats breathed air or carbogen for four periods of 20 min; three control rats breathed only air. Carbogen breathing caused increases of up to 100% in the GRE image intensity of the tumors. Reversion of air breathing caused the image intensity to fall; essentially the same response was observed with the second cycle of carbogen and air breathing. Control rat tumors showed no significant change. The response of tumors to carbogen can be monitored noninvasively by GRE MRI. In principle, this could be due to an increase in oxygen content of the blood, a decrease in tumor cell oxygen consumption, or an increase in tumor blood flow. The very large changes in signal intensity suggest that a blood flow increase is the most probable explanation. If this technique can be successfully applied in man, it should be possible to optimize carbogen treatment for individual radiotherapy patients, and perhaps also to enhance tumor uptake of chemotherapeutic agents.

Oxygen content determined by acrylamide polymerization: screening of anticancer agents, generation of oxyhemoglobin dissociation curves, and potential applications.

Many investigators have endeavored to accurately measure oxygen content produced or consumed from substrates or tissues. Most of the techniques currently available are time consuming or exceedingly complex. Many require expensive apparatus and demand the skills of a highly trained technician. We have deveoloped a rapid and sensitive method of measuring oxygen content or consumption. This method is based upon the time for polymerization of acrylamide gels. The time of acrylamide polymerization is directly proportional to the oxygen content of the test medium. We have used this method to generate oxyhemoglobin dissociation curves which closely resemble those published by Severinghaus and Duvelleroy, as well as those we determined by the conventional Van Slyke analysis. We have also used this method to measure the oxygen consumption of tumor cells and to evaluate the effects of Actinomycin, cyclohexamide and cytosine arabinoside on Walker 256 ascites tumor. The results correlate with the known in vivo effects of these drugs on this tumor cell line. Therefore, because of its sensitivity and adaptability this method may be useful in a variety of clinical and biological situations.

Oxygen consumption by acrylamide polymerization: a method for rapid screening of anticancer agents.

We have developed a rapid and sensitive method to measure oxygen consumption of tumor cells by acrylamide polymerization. This method could be used as an in vitro screen for potential chemotherapeutic agents. Previous techniques have lacked either the sensitivity or the speed required for use as an effective clinical tool. This method was modified from a technique previously developed in our laboratory for measuring oxygen in blood. Standard anticancer agents were tested against Walker 256 ascites tumor. Ascites was harvested from 20 rats on each of 50 days, incubated briefly with drugs, and acrylamide polymerization time was measured hourly. Individual controls were established for each drug-treated group, and oxygen uptake measured after one hour of incubation. Actinomycin, cyclohexamide, and cytosine arabinoside inhibited tumor cell oxygen consumption by 80%, 40%, and 30%, respectively. These results correlated with the known in vivo effects of these drugs on Walker carcinosarcoma. Therefore, this sensitive method could potentially be used directly on tumor cells removed from biopsy material so that the testing of anticancer drugs could be completed on the day of biopsy.