A versatile EPR toolbox for the simultaneous measurement of oxygen consumption and superoxide production.

In this paper, we describe an assay to analyze simultaneously the oxygen consumption rate (OCR) and superoxide production in a biological system. The analytical set-up uses electron paramagnetic resonance (EPR) spectroscopy with two different isotopically-labelled sensors: 15N-PDT (4-oxo-2,2,6,6-tetramethylpiperidine-d16-15N-1-oxyl) as oxygen-sensing probe and 14N-CMH (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine, a cyclic hydroxylamine, as sensor of reactive oxygen species (ROS). The superoxide contribution to CMH oxidation is assessed using SOD or PEGSOD as controls. Because the EPR spectra are not superimposable, the variation of EPR linewidth of 15N-PDT (linked to OCR) and the formation of the nitroxide from 14N-CMH (linked to superoxide production) can be recorded simultaneously over time on a single preparation. The EPR toolbox was qualified in biological systems of increasing complexity. First, we used an enzymatic assay based on the hypoxanthine (HX)/xanthine oxidase (XO) which is a well described model of oxygen consumption and superoxide production. Second, we used a cellular model of superoxide production using macrophages exposed to phorbol 12-myristate 13-acetate (PMA) which stimulates the NADPH oxidase (NOX) to consume oxygen and produce superoxide. Finally, we exposed isolated mitochondria to established inhibitors of the electron transport chain (rotenone and metformin) in order to assess their impact on OCR and superoxide production. This EPR toolbox has the potential to screen the effect of intoxicants or drugs targeting the mitochondrial function.

Fitter Mitochondria Are Associated With Radioresistance in Human Head and Neck SQD9 Cancer Cells.

The clinical management of head and neck squamous cell carcinoma (HNSCC) commonly involves chemoradiotherapy, but recurrences often occur that are associated with radioresistance. Using human SQD9 laryngeal squamous cell carcinoma cancer cells as a model, we aimed to identify metabolic changes associated with acquired radioresistance. In a top-down approach, matched radiosensitive and radioresistant SQD9 cells were generated and metabolically compared, focusing on glycolysis, oxidative phosphorylation (OXPHOS) and ROS production. The cell cycle, clonogenicity, tumor growth in mice and DNA damage-repair were assessed. Mitochondrial DNA (mtDNA) was sequenced. In a bottom-up approach, matched glycolytic and oxidative SQD9 cells were generated using FACS-sorting, and tested for their radiosensitivity/radioresistance. We found that acquired radioresistance is associated with a shift from a glycolytic to a more oxidative metabolism in SQD9 cells. The opposite was also true, as the most oxidative fraction isolated from SQD9 wild-type cells was also more radioresistant than the most glycolytic fraction. However, neither reduced hexokinase expression nor OXPHOS were directly responsible for the radioresistant phenotype. Radiosensitive and radioresistant cells had similar proliferation rates and were equally efficient for ATP production. They were equally sensitive to redox stress and had similar DNA damage repair, but radioresistant cells had an increased number of mitochondria and a higher mtDNA content. Thus, an oxidative switch is associated with but is not responsible for acquired radioresistance in human SQD9 cells. In radioresistant cells, more abundant and fitter mitochondria could help to preserve mitochondrial functions upon irradiation.

Targeting tumor cells and neovascularization using RGD-functionalized magnetoliposomes.

PURPOSE: Magnetoliposomes (MLs) have shown great potential as magnetic resonance imaging contrast agents and as delivery vehicles for cancer therapy. Targeting the MLs towards the tumor cells or neovascularization could ensure delivery of drugs at the tumor site. In this study, we evaluated the potential of MLs targeting the αvß3 integrin overexpressed on tumor neovascularization and different tumor cell types, including glioma and ovarian cancer. METHODS: MLs functionalized with a Texas Red fluorophore (anionic MLs), and with the fluorophore and the cyclic Arginine-Glycine-Aspartate (cRGD; cRGD-MLs) targeting the αvß3 integrin, were produced in-house. Swiss nude mice were subcutaneously injected with 107 human ovarian cancer SKOV-3 cells. Tumors were allowed to grow for 3 weeks before injection of anionic or cRGD-MLs. Biodistribution of MLs was followed up with a 7T preclinical magnetic resonance imaging (MRI) scanner and fluorescence imaging (FLI) right after injection, 2h, 4h, 24h and 48h post injection. Ex vivo intratumoral ML uptake was confirmed using FLI, electron paramagnetic resonance spectroscopy (EPR) and histology at different time points post injection. RESULTS: In vivo, we visualized a higher uptake of cRGD-MLs in SKOV-3 xenografts compared to control, anionic MLs with both MRI and FLI. Highest ML uptake was seen after 4h using MRI, but only after 24h using FLI indicating the lower sensitivity of this technique. Furthermore, ex vivo EPR and FLI confirmed the highest tumoral ML uptake at 4 h. Last, a Perl's stain supported the presence of our iron-based particles in SKOV-3 xenografts. CONCLUSION: Uptake of cRGD-MLs can be visualized using both MRI and FLI, even though the latter was less sensitive due to lower depth penetration. Furthermore, our results indicate that cRGD-MLs can be used to target SKOV-3 xenograft in Swiss nude mice. Therefore, the further development of this particles into theranostics would be of interest.

Towards in vivo melanin radicals detection in melanomas by electron paramagnetic resonance (EPR) spectroscopy: a proof-of-concept study.

Melanoma is the most aggressive skin tumour type. Although complete cure can be achieved when the whole tumour is resected, prognostic dramatically drops when melanoma cells reach deeper tissues and lymph nodes. Hence, there is an urgent need to develop accurate tools allowing (i) discriminating benign naevi from malignant tumours and (ii) being able to characterise melanoma infiltration. For that purpose, we exploited the paramagnetic properties of melanin by using electron paramagnetic resonance (EPR) spectroscopy to measure the melanin content in pigmented (B16F10 cancer cells) and non-pigmented melanomas (WM2664 cancer cells) inoculated intradermally in nude mice. Specifically, we took advantage of a new clinical EPR device (1 GHz), which provides sensitive measurements of radical species in vivo. Results showed that the melanin-specific EPR signal increased with tumour growth in pigmented tumours, whereas no EPR signal could be detected in achromic melanomas. These data plead for the development of new EPR spectrometers/imagers with an improved in-depth resolution for the detection of invasive melanomas.

Hypoxia Patterns in Primary and Metastatic Prostate Cancer Environments.

Metastatic dissemination continues to be a major cause of prostate cancer (PCa) mortality, creating a compelling need to understand factors that play a role in the metastatic cascade. Since hypoxia plays an important role in PCa aggressiveness, we characterized patterns of hypoxia in the primary tumor and metastatic environments of a human PCa xenograft. We previously developed and characterized an imaging strategy based on the hypoxia response element (HRE)-driven expression of long-lived enhanced green fluorescent protein (EGFP) and short-lived luciferase (luc) fused to the oxygen-dependent degradation domain in human PCa PC-3 cells. Both reporter proteins were placed under the transcriptional control of a five-tandem repeat HRE sequence. PC-3 cells also constitutively expressed the tdTomato red fluorescent protein, allowing cancer cell detection in vivo. This "timer" strategy can provide information on the temporal evolution of HIF activity and hypoxia in tumors. Here, for the first time, we performed in vivo and ex vivo imaging of this dual HIF reporter system in PC-3 metastatic tumors implanted orthotopically in the prostate and PC-3 nonmetastatic tumors implanted subcutaneously. We observed distinct patterns of EGFP and luc expression in subcutaneous and orthotopic tumors, and in metastatic nodules, that provide new insights into the presence of hypoxia at primary and metastatic tumor sites, and of the role of hypoxia in metastasis.

Characterization of a clinically used charcoal suspension for in vivo EPR oximetry.

OBJECTIVES: Electron paramagnetic resonance (EPR) oximetry using particulate materials allows repeatable measurements of oxygen in tissues. However, the materials identified so far are not medical devices, thus precluding their immediate use in clinical studies. The aim of this study was to assess the magnetic properties of Carbo-Rep®, a charcoal suspension used as a liquid marker for preoperative tumor localization. MATERIALS AND METHODS: Calibration curves (EPR linewidth as a function of pO2) were built using 9-GHz EPR spectrometry. The feasibility of performing oxygen measurements was examined in vivo by using a low-frequency (1 GHz) EPR spectrometer and by inducing ischemia in the gastrocnemius muscle of mice or by submitting rats bearing tumors to different oxygen-breathing challenges. RESULTS: Paramagnetic centers presenting a high oxygen sensitivity were identified in Carbo-Rep®. At 1 GHz, the EPR linewidth varied from 98 to 426 µT in L-band in nitrogen and air, respectively. The sensor allowed repeated measurements of oxygen over 6 months in muscles of mice. Subtle variations of tumor oxygenation were monitored in rats when switching gas breathing from air to carbogen. DISCUSSION: The magnetic properties of Carbo-Rep® are promising for its future use as oxygen sensor in clinical EPR oximetry.

Optimized acriflavine-loaded lipid nanocapsules as a safe and effective delivery system to treat breast cancer.

Acriflavine (ACF) hydrochloride is currently repurposed as multimodal drug, inhibiting hypoxia-inducible factors (HIF) pathways and exerting cytotoxic properties. The aim of this study was to encapsulate ACF in reverse micelles and to incorporate this suspension in lipid nanocapsules (LNC). Designs of experiments were used to work under quality by design conditions. LNC were formulated using a phase-inversion temperature method, leading to an encapsulation efficiency around 80%. In vitro, the encapsulated drug presented similar cytotoxic activity and decrease in HIF activity in 4T1 cells compared to the free drug. In vivo, ACF-loaded nanoparticles (ACF dose of 5 mg/kg) demonstrated a higher antitumor efficacy compared to free ACF on an orthotopic model of murine breast cancer (4T1 cells). Moreover, the use of LNC allowed to drastically decrease the number of administrations compared to the free drug (2 versus 12 injections), suppressing the ACF-induced toxicity.

Magnetic targeting of paclitaxel-loaded poly(lactic-co-glycolic acid)-based nanoparticles for the treatment of glioblastoma.

INTRODUCTION: Glioblastoma (GBM) therapy is highly challenging, as the tumors are very aggressive due to infiltration into the surrounding normal brain tissue. Even a combination of the available therapeutic regimens may not debulk the tumor completely. GBM tumors are also known for recurrence, resulting in survival rates averaging <18 months. In addition, systemic chemotherapy for GBM has been challenged for its minimal desired therapeutic effects and unwanted side effects. PURPOSE: We hypothesized that paclitaxel (PTX) and superparamagnetic iron oxide (SPIO)-loaded PEGylated poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs; PTX/SPIO-NPs) can serve as an effective nanocarrier system for magnetic targeting purposes, and we aimed to demonstrate the therapeutic efficacy of this system in an orthotopic murine GBM model. MATERIALS AND METHODS: PTX/SPIO-NPs were prepared by emulsion-diffusion-evaporation method and characterized for physicochemical properties. In vitro cellular uptake of PTX/SPIO-NPs was evaluated by fluorescence microscopy and Prussian blue staining. Orthotopic U87MG tumor model was used to evaluate blood-brain barrier disruption using T1 contrast agent, ex vivo biodistribution, in vivo toxicity and in vivo antitumor efficacy of PTX/SPIO-NPs. RESULTS: PTX/SPIO-NPs were in the size of 250 nm with negative zeta potential. Qualitative cellular uptake studies showed that the internalization of NPs was concentration dependent. Through magnetic resonance imaging, we observed that the blood-brain barrier was disrupted in the GBM area. An ex vivo biodistribution study showed enhanced accumulation of NPs in the brain of GBM-bearing mice with magnetic targeting. Short-term in vivo safety evaluation showed that the NPs did not induce any systemic toxicity compared with Taxol® (PTX). When tested for in vivo efficacy, the magnetic targeting treatment significantly prolonged the median survival time compared with the passive targeting and control treatments. CONCLUSION: Overall, PTX/SPIO-NPs with magnetic targeting could be considered as an effective anticancer targeting strategy for GBM chemotherapy.

Hypoxia-Induced Reporter Genes with Different Half-Lives.

The utility of reporter genes has gained significant momentum over the last three decades. Reporter genes are used to understand the transcriptional activity of a gene both in vitro and in vivo, and in pathway analysis and drug screening for diseases involving protozoan parasites, and in anti-cancer drug developments. Here, using a human prostate cancer xenograft model (PC3), we describe a method to construct and validate hypoxia reporter genes with different half-lives. Using molecular biology and optical imaging techniques, we have validated the expression of long half-life enhanced green fluorescence protein (EGFP) expression and short half-life luciferase gene expression to report on the spatial and temporal evolution of hypoxia in vivo.

(+)-Catechin in a 1:2 Complex with Lysine Inhibits Cancer Cell Migration and Metastatic Take in Mice.

Metastasis is of dismal prognosis for cancer patients, but recent evidence in mouse models of cancer shows that metastasis prevention is a reachable clinical objective. These experiments indicate that altered mitochondrial activities are associated with the metastatic phenotype. Mitochondrial transfer from metastatic to non-metastatic cells can indeed transfer the metastatic phenotype, and metastatic progenitor cells differ from other cancer cells by a higher sublethal production of mitochondrial reactive oxygen species (ROS). Moreover, mitochondria-targeted antioxidants can prevent metastatic dissemination in mouse models of cancer. Comparatively, general antioxidants have unpredictable effects on cancer metastasis, most probably because they affect several cell types, several subcellular ROS production sites and, often, several endogenous oxidant species. Thus, targeting antioxidants to mitochondria could improve their antimetastatic activities, as previously exemplified with mitochondria-targeted mitoTEMPO and mitoQ that can prevent metastatic dissemination in cancer-bearing mice. Our objective in this study was to identify whether catechins, which are known to be potent antioxidants, can inhibit cancer cell migration in vitro and metastatic take in vivo. Comparative analysis of the response to epigallocatechin-3-gallate, (+)-catechin and (+)-catechin:lysine complexes revealed that, whereas all compounds had similar general antioxidant properties, (+)-catechin:lysine 1:2, but not epigallocatechin-3-gallate, can prevent metastatic take of melanoma cells to the lungs of mice. (+)-Catechin:lysine 1:2 possesses two net positive charges provided by lysines at physiological pH, which could provide high affinity for the negatively charged mitochondrial matrix. While this study reveals that (+)-catechin:lysine 1:2 has interesting antimetastatic effects, future experiments are needed to formally demonstrate the stability of the complex, its effective tropism for mitochondria and whether or not its activity can be globally attributed to its antioxidant activity at this precise subcellular location.

Contribution of macrophages in the contrast loss in iron oxide-based MRI cancer cell tracking studies.

Magnetic resonance imaging (MRI) cell tracking of cancer cells labeled with superparamagnetic iron oxides (SPIO) allows visualizing metastatic cells in preclinical models. However, previous works showed that the signal void induced by SPIO on T2(*)-weighted images decreased over time. Here, we aim at characterizing the fate of iron oxide nanoparticles used in cell tracking studies and the role of macrophages in SPIO metabolism.In vivo MRI cell tracking of SPIO positive 4T1 breast cancer cells revealed a quick loss of T2* contrast after injection. We next took advantage of electron paramagnetic resonance (EPR) spectroscopy and inductively coupled plasma mass spectroscopy (ICP-MS) for characterizing the evolution of superparamagnetic and non-superparamagnetic iron pools in 4T1 breast cancer cells and J774 macrophages after SPIO labeling. These in vitro experiments and histology studies performed on 4T1 tumors highlighted the quick degradation of iron oxides by macrophages in SPIO-based cell tracking experiments.In conclusion, the release of SPIO by dying cancer cells and the subsequent uptake of iron oxides by tumor macrophages are limiting factors in MRI cell tracking experiments that plead for the use of (MR) reporter-gene based imaging methods for the long-term tracking of metastatic cells.

Cancer metabolism in space and time: Beyond the Warburg effect.

Altered metabolism in cancer cells is pivotal for tumor growth, most notably by providing energy, reducing equivalents and building blocks while several metabolites exert a signaling function promoting tumor growth and progression. A cancer tissue cannot be simply reduced to a bulk of proliferating cells. Tumors are indeed complex and dynamic structures where single cells can heterogeneously perform various biological activities with different metabolic requirements. Because tumors are composed of different types of cells with metabolic activities affected by different spatial and temporal contexts, it is important to address metabolism taking into account cellular and biological heterogeneity. In this review, we describe this heterogeneity also in metabolic fluxes, thus showing the relative contribution of different metabolic activities to tumor progression according to the cellular context. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux.

Manipulation of tumor oxygenation and radiosensitivity through modification of cell respiration. A critical review of approaches and imaging biomarkers for therapeutic guidance.

Tumor hypoxia has long been considered as a detrimental factor for the response to irradiation. In order to improve the sensitivity of tumors cells to radiation therapy, tumor hypoxia may theoretically be alleviated by increasing the oxygen delivery or by decreasing the oxygen consumption by tumor cells. Mathematical modelling suggested that decreasing the oxygen consumption should be more efficient than increasing oxygen delivery in order to alleviate tumor hypoxia. In this paper, we review several promising strategies targeting the mitochondrial respiration for which alleviation of tumor hypoxia and increase in sensitivity to irradiation have been demonstrated. Because the translation of these approaches into the clinical arena requires the use of pharmacodynamics biomarkers able to identify shift in oxygen consumption and tumor oxygenation, we also discuss the relative merits of imaging biomarkers (Positron Emission Tomography and Magnetic Resonance) that may be used for therapeutic guidance. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux.

Lactate Dehydrogenase B Controls Lysosome Activity and Autophagy in Cancer.

Metabolic adaptability is essential for tumor progression and includes cooperation between cancer cells with different metabolic phenotypes. Optimal glucose supply to glycolytic cancer cells occurs when oxidative cancer cells use lactate preferentially to glucose. However, using lactate instead of glucose mimics glucose deprivation, and glucose starvation induces autophagy. We report that lactate sustains autophagy in cancer. In cancer cells preferentially to normal cells, lactate dehydrogenase B (LDHB), catalyzing the conversion of lactate and NAD(+) to pyruvate, NADH and H(+), controls lysosomal acidification, vesicle maturation, and intracellular proteolysis. LDHB activity is necessary for basal autophagy and cancer cell proliferation not only in oxidative cancer cells but also in glycolytic cancer cells.

Direct Evidence of the Link Between Energetic Metabolism and Proliferation Capacity of Cancer Cells In Vitro.

The aim of the study was to assess the link between the metabolic profile and the proliferation capacity of a range of human and murine cancer cell lines. First, the combination of mitochondrial respiration and glycolytic efficiency measurements allowed the determination of different metabolic profiles among the cell lines, ranging from a mostly oxidative to a mostly glycolytic phenotype. Second, the study revealed that cell proliferation, evaluated by DNA synthesis measurements, was statistically correlated to glycolytic efficiency. This indicated that glycolysis is the key energetic pathway linked to cell proliferation rate. Third, to validate this hypothesis and exclude non-metabolic factors, mitochondria-depleted were compared to wild-type cancer cells, and the data showed that enhanced glycolysis observed in mitochondria-depleted cells is also associated with an increase in proliferation capacity.

A Fast Hydrogen Sulfide-Releasing Donor Increases the Tumor Response to Radiotherapy.

Hydrogen sulfide (H2S) is the last gaseous transmitter identified in mammals, and previous studies have reported disparate conclusions regarding the implication of H2S in cancer progression. In the present study, we hypothesized that sodium hydrosulfide (NaHS), a fast H2S-releasing donor, might interfere with the mitochondrial respiratory chain of tumor cells, increase tumor oxygenation, and potentiate the response to irradiation. Using electron paramagnetic resonance (EPR) oximetry, we found a rapid increase in tumor pO2 after NaHS administration (0.1 mmol/kg) in two human tumor models (breast MDA-MB-231 and cervix SiHa), an effect that was due to a decreased oxygen consumption and an increased tumor perfusion. Tumors irradiated 15 minutes after a single NaHS administration were more sensitive to irradiation compared with those that received irradiation alone (increase in growth delay by 50%). This radiosensitization was due to the oxygen effect, as the increased growth delay was abolished when temporarily clamped tumors were irradiated. In contrast, daily NaHS injection (0.1 mmol/kg/day for 14 days) did not provide any effect on tumor growth in vivo. To understand these paradoxical data, we analyzed the impact of external factors on the cellular response to NaHS. We found that extracellular pH had a dramatic effect on the cell response to NaHS, as the proliferation rate (measured in vitro by BrdU incorporation) was increased at pH = 7.4, but decreased at pH = 6.5. Overall, our study highlights the complex role of environmental components in the response of cancer cells to H2S and suggests a new approach for the use of H2S donors in combination with radiotherapy.

Inhibition of the pentose phosphate pathway by dichloroacetate unravels a missing link between aerobic glycolysis and cancer cell proliferation.

Glucose fermentation through glycolysis even in the presence of oxygen (Warburg effect) is a common feature of cancer cells increasingly considered as an enticing target in clinical development. This study aimed to analyze the link between metabolism, energy stores and proliferation rates in cancer cells. We found that cell proliferation, evaluated by DNA synthesis quantification, is correlated to glycolytic efficiency in six cancer cell lines as well as in isogenic cancer cell lines. To further investigate the link between glycolysis and proliferation, a pharmacological inhibitor of the pentose phosphate pathway (PPP) was used. We demonstrated that reduction of PPP activity decreases cancer cells proliferation, with a profound effect in Warburg-phenotype cancer cells. The crucial role of the PPP in sustaining cancer cells proliferation was confirmed using siRNAs against glucose-6-phosphate dehydrogenase, the first and rate-limiting enzyme of the PPP. In addition, we found that dichloroacetate (DCA), a new clinically tested compound, induced a switch of glycolytic cancer cells to a more oxidative phenotype and decreased proliferation. By demonstrating that DCA decreased the activity of the PPP, we provide a new mechanism by which DCA controls cancer cells proliferation.

Non-invasive in vivo imaging of early metabolic tumor response to therapies targeting choline metabolism.

The cholinic phenotype, characterized by elevated phosphocholine and a high production of total-choline (tCho)-containing metabolites, is a metabolic hallmark of cancer. It can be exploited for targeted therapy. Non-invasive imaging biomarkers are required to evaluate an individual's response to targeted anticancer agents that usually do not rapidly cause tumor shrinkage. Because metabolic changes can manifest at earlier stages of therapy than changes in tumor size, the aim of the current study was to evaluate (1)H-MRS and diffusion-weighted MRI (DW-MRI) as markers of tumor response to the modulation of the choline pathway in mammary tumor xenografts. Inhibition of choline kinase activity was achieved with the direct pharmacological inhibitor H-89, indirect inhibitor sorafenib and down-regulation of choline-kinase α (ChKA) expression using specific short-hairpin RNA (shRNA). While all three strategies significantly decreased tCho tumor content in vivo, only sorafenib and anti-ChKA shRNA significantly repressed tumor growth. The increase of apparent-diffusion-coefficient of water (ADCw) measured by DW-MRI, was predictive of the induced necrosis and inhibition of the tumor growth in sorafenib treated mice, while the absence of change in ADC values in H89 treated mice predicted the absence of effect in terms of tumor necrosis and tumor growth. In conclusion, (1)H-choline spectroscopy can be useful as a pharmacodynamic biomarker for choline targeted agents, while DW-MRI can be used as an early marker of effective tumor response to choline targeted therapies. DW-MRI combined to choline spectroscopy may provide a useful non-invasive marker for the early clinical assessment of tumor response to therapies targeting choline signaling.

Combining Optical Reporter Proteins with Different Half-lives to Detect Temporal Evolution of Hypoxia and Reoxygenation in Tumors.

Here we have developed a hypoxia response element driven imaging strategy that combined the hypoxia-driven expression of two optical reporters with different half-lives to detect temporal changes in hypoxia and hypoxia inducible factor (HIF) activity. For this purpose, human prostate cancer PC3 cells were transfected with the luciferase gene fused with an oxygen-dependent degradation domain (ODD-luc) and a variant of the enhanced green fluorescent protein (EGFP). Both ODD-luciferase and EGFP were under the promotion of a poly-hypoxia-response element sequence (5xHRE). The cells constitutively expressed tdTomato red fluorescent protein. For validating the imaging strategy, cells were incubated under hypoxia (1% O2) for 48 hours and then reoxygenated. The luciferase activity of PC3-HRE-EGFP/HRE-ODD-luc/tdtomato cells detected by bioluminescent imaging rapidly decreased after reoxygenation, whereas EGFP levels in these cells remained stable for several hours. After in vitro validation, PC3-HRE-EGFP/HRE-ODD-luc/tdtomato tumors were implanted subcutaneously and orthotopically in nude male mice and imaged in vivo and ex vivo using optical imaging in proof-of-principle studies to demonstrate differences in optical patterns between EGFP expression and bioluminescence. This novel "timer" imaging strategy of combining the short-lived ODD-luciferase and the long-lived EGFP can provide a time frame of HRE activation in PC3 prostate cancer cells and will be useful to understand the temporal changes in hypoxia and HIF activity during cancer progression and following treatments including HIF targeting strategies.

In vivo visualization and ex vivo quantification of murine breast cancer cells in the mouse brain using MRI cell tracking and electron paramagnetic resonance.

Cell tracking could be useful to elucidate fundamental processes of cancer biology such as metastasis. The aim of this study was to visualize, using MRI, and to quantify, using electron paramagnetic resonance (EPR), the entrapment of murine breast cancer cells labeled with superparamagnetic iron oxide particles (SPIOs) in the mouse brain after intracardiac injection. For this purpose, luciferase-expressing murine 4 T1-luc breast cancer cells were labeled with fluorescent Molday ION Rhodamine B SPIOs. Following intracardiac injection, SPIO-labeled 4 T1-luc cells were imaged using multiple gradient-echo sequences. Ex vivo iron oxide quantification in the mouse brain was performed using EPR (9 GHz). The long-term fate of 4 T1-luc cells after injection was characterized using bioluminescence imaging (BLI), brain MRI and immunofluorescence. We observed hypointense spots due to SPIO-labeled cells in the mouse brain 4 h after injection on T2 *-weighted images. Histology studies showed that SPIO-labeled cancer cells were localized within blood vessels shortly after delivery. Ex vivo quantification of SPIOs showed that less than 1% of the injected cells were taken up by the mouse brain after injection. MRI experiments did not reveal the development of macrometastases in the mouse brain several days after injection, but immunofluorescence studies demonstrated that these cells found in the brain established micrometastases. Concerning the metastatic patterns of 4 T1-luc cells, an EPR biodistribution study demonstrated that SPIO-labeled 4 T1-luc cells were also entrapped in the lungs of mice after intracardiac injection. BLI performed 6 days after injection of 4 T1-luc cells showed that this cell line formed macrometastases in the lungs and in the bones. Conclusively, EPR and MRI were found to be complementary for cell tracking applications. MRI cell tracking at 11.7 T allowed sensitive detection of isolated SPIO-labeled cells in the mouse brain, whereas EPR allowed the assessment of the number of SPIO-labeled cells in organs shortly after injection.