Bénédicte F. Jordan

Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice

Tumors contain oxygenated and hypoxic regions, so the tumor cell population is heterogeneous. Hypoxic tumor cells primarily use glucose for glycolytic energy production and release lactic acid, creating a lactate gradient that mirrors the oxygen gradient in the tumor. By contrast, oxygenated tumor cells have been thought to primarily use glucose for oxidative energy production. Although lactate is generally considered a waste product, we now show that it is a prominent substrate that fuels the oxidative metabolism of oxygenated tumor cells. There is therefore a symbiosis in which glycolytic and oxidative tumor cells mutually regulate their access to energy metabolites. We identified monocarboxylate transporter 1 (MCT1) as the prominent path for lactate uptake by a human cervix squamous carcinoma cell line that preferentially utilized lactate for oxidative metabolism. Inhibiting MCT1 with alpha-cyano-4-hydroxycinnamate (CHC) or siRNA in these cells induced a switch from lactate-fueled respiration to glycolysis. A similar switch from lactate-fueled respiration to glycolysis by oxygenated tumor cells in both a mouse model of lung carcinoma and xenotransplanted human colorectal adenocarcinoma cells was observed after administration of CHC. This retarded tumor growth, as the hypoxic/glycolytic tumor cells died from glucose starvation, and rendered the remaining cells sensitive to irradiation. As MCT1 was found to be expressed by an array of primary human tumors, we suggest that MCT1 inhibition has clinical antitumor potential.

Heterozygous Deficiency of PHD2 Restores Tumor Oxygenation and Inhibits Metastasis via Endothelial Normalization

A key function of blood vessels, to supply oxygen, is impaired in tumors because of abnormalities in their endothelial lining. PHD proteins serve as oxygen sensors and may regulate oxygen delivery. We therefore studied the role of endothelial PHD2 in vessel shaping by implanting tumors in PHD2(+/-) mice. Haplodeficiency of PHD2 did not affect tumor vessel density or lumen size, but normalized the endothelial lining and vessel maturation. This resulted in improved tumor perfusion and oxygenation and inhibited tumor cell invasion, intravasation, and metastasis. Haplodeficiency of PHD2 redirected the specification of endothelial tip cells to a more quiescent cell type, lacking filopodia and arrayed in a phalanx formation. This transition relied on HIF-driven upregulation of (soluble) VEGFR-1 and VE-cadherin. Thus, decreased activity of an oxygen sensor in hypoxic conditions prompts endothelial cells to readjust their shape and phenotype to restore oxygen supply. Inhibition of PHD2 may offer alternative therapeutic opportunities for anticancer therapy.

Deficiency or inhibition of oxygen sensor Phd1 induces hypoxia tolerance by reprogramming basal metabolism

HIF prolyl hydroxylases (PHD1–3) are oxygen sensors that regulate the stability of the hypoxia-inducible factors (HIFs) in an oxygen-dependent manner. Here, we show that loss of Phd1 lowers oxygen consumption in skeletal muscle by reprogramming glucose metabolism from oxidative to more anaerobic ATP production through activation of a Pparα pathway. This metabolic adaptation to oxygen conservation impairs oxidative muscle performance in healthy conditions, but it provides acute protection of myofibers against lethal ischemia. Hypoxia tolerance is not due to HIF-dependent angiogenesis, erythropoiesis or vasodilation, but rather to reduced generation of oxidative stress, which allows Phd1-deficient myofibers to preserve mitochondrial respiration. Hypoxia tolerance relies primarily on Hif-2α and was not observed in heterozygous Phd2-deficient or homozygous Phd3-deficient mice. Of medical importance, conditional knockdown of Phd1 also rapidly induces hypoxia tolerance. These findings delineate a new role of Phd1 in hypoxia tolerance and offer new treatment perspectives for disorders characterized by oxidative stress.

Macrophage skewing by Phd2 haplodeficiency prevents ischaemia by inducing arteriogenesis

PHD2 serves as an oxygen sensor that rescues blood supply by regulating vessel formation and shape in case of oxygen shortage. However, it is unknown whether PHD2 can influence arteriogenesis. Here we studied the role of PHD2 in collateral artery growth by using hindlimb ischaemia as a model, a process that compensates for the lack of blood flow in case of major arterial occlusion. We show that Phd2 (also known as Egln1) haplodeficient (Phd2+/−) mice displayed preformed collateral arteries that preserved limb perfusion and prevented tissue necrosis in ischaemia. Improved arteriogenesis in Phd2+/− mice was due to an expansion of tissue-resident, M2-like macrophages and their increased release of arteriogenic factors, leading to enhanced smooth muscle cell (SMC) recruitment and growth. Both chronic and acute deletion of one Phd2 allele in macrophages was sufficient to skew their polarization towards a pro-arteriogenic phenotype. Mechanistically, collateral vessel preconditioning relied on the activation of canonical NF-κB pathway in Phd2+/− macrophages. These results unravel how PHD2 regulates arteriogenesis and artery homeostasis by controlling a specific differentiation state in macrophages and suggest new treatment options for ischaemic disorders.

Tumor Hypoxia Does Not Drive Differentiation of Tumor-Associated Macrophages but Rather Fine-Tunes the M2-like Macrophage Population

Tumor-associated macrophages (TAM) are exposed to multiple microenvironmental cues in tumors, which collaborate to endow these cells with protumoral activities. Hypoxia, caused by an imbalance in oxygen supply and demand because of a poorly organized vasculature, is often a prominent feature in solid tumors. However, to what extent tumor hypoxia regulates the TAM phenotype in vivo is unknown. Here, we show that the myeloid infiltrate in mouse lung carcinoma tumors encompasses two morphologically distinct CD11b(hi)F4/80(hi)Ly6C(lo) TAM subsets, designated as MHC-II(lo) and MHC-II(hi) TAM, both of which were derived from tumor-infiltrating Ly6C(hi) monocytes. MHC-II(lo) TAM express higher levels of prototypical M2 markers and reside in more hypoxic regions. Consequently, MHC-II(lo) TAM contain higher mRNA levels for hypoxia-regulated genes than their MHC-II(hi) counterparts. To assess the in vivo role of hypoxia on these TAM features, cancer cells were inoculated in prolyl hydroxylase domain 2 (PHD2)-haplodeficient mice, resulting in better-oxygenated tumors. Interestingly, reduced tumor hypoxia did not alter the relative abundance of TAM subsets nor their M2 marker expression, but specifically lowered hypoxia-sensitive gene expression and angiogenic activity in the MHC-II(lo) TAM subset. The same observation in PHD2(+/+) → PHD2(+/-) bone marrow chimeras also suggests organization of a better-oxygenized microenvironment. Together, our results show that hypoxia is not a major driver of TAM subset differentiation, but rather specifically fine-tunes the phenotype of M2-like MHC-II(lo) TAM.

Changes in tumor oxygenation/perfusion induced by the no donor, isosorbide dinitrate, in comparison with carbogen: monitoring by EPR and MRI

PURPOSE In an effort to improve radiotherapy treatments, methods aimed at increasing the quantity of oxygen delivered to tumors were investigated. The aim of this study was to evaluate the effect of one nitric oxide (NO) donor (isosorbide dinitrate) on pO(2) and blood flow in a murine tumor model. The effect was compared to carbogen, used as a reference treatment. METHODS AND MATERIALS Thirty-six liver tumors implanted in mouse thighs were imaged using magnetic resonance imaging (MRI) at 4.7 Tesla with dynamic Gd-DTPA and blood oxygen level-dependent (BOLD) contrast-enhanced imaging after administration of isosorbide dinitrate or carbogen. The effect on the pO(2) was also tested by EPR oximetry (1.1 GHz) on 52 mice. RESULTS A significant increase in MRI intensity was observed for both treatments in comparison with the control group. EPR oximetry showed a dose-dependant increase in tumor pO(2) for isosorbide dinitrate (by 5.9 mmHg at 0.2 mg/kg) and a substantially greater change for carbogen breathing (by 23 mmHg). CONCLUSION Both tumor blood flow and pO(2) were increased by isosorbide dinitrate and carbogen. Carbogen is more efficient than isosorbide dinitrate in increasing the BOLD image intensity, as well as the tumor pO(2), but as efficient as isosorbide dinitrate in the Gd-DTPA contrast-enhanced imaging. We conclude that the effects of carbogen on improving tumor pO(2) involve both improved blood flow and improved hemoglobin oxygenation, whereas the effects of isosorbide dinitrate are predominantly mediated by improved blood flow alone.

Physiological noise in murine solid tumours using T2*-weighted gradient-echo imaging: a marker of tumour acute hypoxia?

T2*-weighted gradient-echo magnetic resonance imaging (T2*-weighted GRE MRI) was used to investigate spontaneous fluctuations in tumour vasculature non-invasively. FSa fibrosarcomas, implanted intramuscularly (i.m.) in the legs of mice, were imaged at 4.7 T, over a 30 min or 1 h sampling period. On a voxel-by-voxel basis, time courses of signal intensity were analysed using a power spectrum density (PSD) analysis to isolate voxels for which signal changes did not originate from Gaussian white noise or linear drift. Under baseline conditions, the tumours exhibited spontaneous signal fluctuations showing spatial and temporal heterogeneity over the tumour. Statistically significant fluctuations occurred at frequencies ranging from 1 cycle/3 min to 1 cycle/h. The fluctuations were independent of the scanner instabilities. Two categories of signal fluctuations were reported: (i) true fluctuations (TFV), i.e., sequential signal increase and decrease, and (ii) profound drop in signal intensity with no apparent signal recovery (SDV). No temporal correlation between tumour and contralateral muscle fluctuations was observed. Furthermore, treatments aimed at decreasing perfusion-limited hypoxia, such as carbogen combined with nicotinamide and flunarizine, decreased the incidence of tumour T2*-weighted GRE fluctuations. We also tracked dynamic changes in T2* using multiple GRE imaging. Fluctuations of T2* were observed; however, fluctuation maps using PSD analysis could not be generated reliably. An echo-time dependency of the signal fluctuations was observed, which is typical to physiological noise. Finally, at the end of T2*-weighted GRE MRI acquisition, a dynamic contrast-enhanced MRI was performed to characterize the microenvironment in which tumour signal fluctuations occurred in terms of vessel functionality, vascularity and microvascular permeability. Our data showed that TFV were predominantly located in regions with functional vessels, whereas SDV occurred in regions with no contrast enhancement as the result of vessel functional impairment. Furthermore, transient fluctuations appeared to occur preferentially in neoangiogenic hyperpermeable vessels. The present study suggests that spontaneous T2*-weighted GRE fluctuations are very likely to be related to the spontaneous fluctuations in blood flow and oxygenation associated with the pathophysiology of acute hypoxia in tumours. The disadvantage of the T2*-weighted GRE MRI technique is the complexity of signal interpretation with regard to pO2 changes. Compared to established techniques such as intravital microscopy or histological assessments, the major advantage of the MRI technique lies in its capacity to provide simultaneously both temporal and detailed spatial information on spontaneous fluctuations throughout the tumour.

Loss or Silencing of the PHD1 Prolyl Hydroxylase Protects Livers of Mice Against Ischemia/Reperfusion Injury

BACKGROUND & AIMS Liver ischemia/reperfusion (I/R) injury is a frequent cause of organ dysfunction. Loss of the oxygen sensor prolyl hydroxylase domain enzyme 1 (PHD1) causes tolerance of skeletal muscle to hypoxia. We assessed whether loss or short-term silencing of PHD1 could likewise induce hypoxia tolerance in hepatocytes and protect them against hepatic I/R damage. METHODS Hepatic ischemia was induced in mice by clamping of the portal vessels of the left lateral liver lobe; 90 minutes later livers were reperfused for 8 hours for I/R experiments. Hepatocyte damage following ischemia or I/R was investigated in PHD1-deficient (PHD1(-/-)) and wild-type mice or following short hairpin RNA-mediated short-term inhibition of PHD1 in vivo. RESULTS PHD1(-/-) livers were largely protected against acute ischemia or I/R injury. Among mice subjected to hepatic I/R followed by surgical resection of all nonischemic liver lobes, more than half of wild-type mice succumbed, whereas all PHD1(-/-) mice survived. Also, short-term inhibition of PHD1 through RNA interference-mediated silencing provided protection against I/R. Knockdown of PHD1 also induced hypoxia tolerance of hepatocytes in vitro. Mechanistically, loss of PHD1 decreased production of oxidative stress, which likely relates to a decrease in oxygen consumption as a result of a reprogramming of hepatocellular metabolism. CONCLUSIONS Loss of PHD1 provided tolerance of hepatocytes to acute hypoxia and protected them against I/R-damage. Short-term inhibition of PHD1 is a novel therapeutic approach to reducing or preventing I/R-induced liver injury.

Electron paramagnetic resonance as a tool to evaluate human ovarian tissue reoxygenation after xenografting.

OBJECTIVE To develop electron paramagnetic resonance (EPR) oximetry as a tool to characterize the oxygen environment in human ovarian xenografts in the early postgrafting period. DESIGN Prospective experimental study. SETTING Gynecology research unit in a university hospital. PATIENT(S) Biopsies were obtained from 6 women aged 22-35 years. INTERVENTION(S) Frozen-thawed human ovarian tissue fragments were grafted to an intraperitoneal site in nude mice. Before grafting, lithium phthalocyanine, an oxygen reporter, was implanted inside the fragments. MAIN OUTCOME MEASURE(S) To monitor partial pressure of oxygen (pO(2)) by EPR on postgrafting days 3, 5, 7, 10, 14, 17, and 21 and validate the technique by histologic assessment. RESULT(S) A period of hypoxia was identified before day 5, followed by gradual but significant oxygenation over the next 5 days, suggesting an active process of graft revascularization. Reoxygenation kinetics in human ovarian xenotransplants were quantified. CONCLUSION(S) Our data validated the EPR oximetry technique as a tool to monitor pO(2) in ovarian grafting. The critical early period of hypoxia was identified, and the first steps of reoxygenation were characterized. In the future, our model may be used to evaluate new freezing and grafting protocols with the aim of reducing potential cryoinjury and initial ischemia-reperfusion damage.

Pharmacological modifications of the partial pressure of oxygen in murine tumors: evaluation using in vivo EPR oximetry

EPR oximetry using an implantable paramagnetic probe was used to quantify the partial pressure of oxygen (pO2) in tissues in a transplantable mouse tumor model (TLT) after administration of 34 different vasodilators belonging to one of the following classes: angiotensin‐converting enzyme inhibitors, calcium antagonists, alpha antagonists, potassium channel openers, beta‐blockers, NO donors, and peripheral vasoactive agents. Twenty‐four compounds were efficient in significantly increasing the local pO2 in a majority of tumors. The increase of local pO2 using pharmacological treatments was lower than that achieved by using oxygen or carbogen breathing. This technique offers an unprecedented tool for rapidly and accurately measuring treatment‐induced modifications of pO2 in tumors. Magn Reson Med 42:627–630, 1999.