Mitochondria can govern local concentrations of second messengers, such as reactive oxygen species (ROS), and mitochondrial translocation to discrete subcellular regions may contribute to this signaling function. Here, we report that exposure of pulmonary artery endothelial cells to hypoxia triggered a retrograde mitochondrial movement that required microtubules and the microtubule motor protein dynein and resulted in the perinuclear clustering of mitochondria. This subcellular redistribution of mitochondria was accompanied by the accumulation of ROS in the nucleus, which was attenuated by suppressing perinuclear clustering of mitochondria with nocodazole to destabilize microtubules or with small interfering RNA–mediated knockdown of dynein. Although suppression of perinuclear mitochondrial clustering did not affect the hypoxia-induced increase in the nuclear abundance of hypoxia-inducible factor 1α (HIF-1α) or the binding of HIF-1α to an oligonucleotide corresponding to a hypoxia response element (HRE), it eliminated oxidative modifications of the VEGF (vascular endothelial growth factor) promoter. Furthermore, suppression of perinuclear mitochondrial clustering reduced HIF-1α binding to the VEGF promoter and decreased VEGF mRNA accumulation. These findings support a model for hypoxia-induced transcriptional regulation in which perinuclear mitochondrial clustering results in ROS accumulation in the nucleus and causes oxidative base modifications in the VEGF HRE that are important for transcriptional complex assembly and VEGF mRNA expression.

Scattering of shorter-wavelength visible light limits the fluorescence imaging depth of thick specimens such as whole organs. In this study, we report the use of four newly synthesized near-infrared and far-red fluorescence probes (excitation/emission, in nm: 644/670; 683/707; 786/814; 824/834) to image tumor cells in the subpleural vasculature of the intact rat lungs. Transpelural imaging of tumor cells labeled with long-wavelength probes and expressing green fluorescent protein (GFP; excitation/emission 488/507 nm) was done in the intact rat lung after perfusate administration or intravenous injection. Our results show that the average optimum imaging depth for the long-wavelength probes is higher (27.8 ± 0.7  μm) than for GFP (20 ± 0.5  μm; p = 0.008; n = 50), corresponding to a 40% increase in the volume of tissue accessible for high-resolution imaging. The maximum depth of cell visualization was significantly improved with the novel dyes (36.4 ± 1  μm from the pleural surface) compared with GFP (30.1 ± 0.5  μm; p = 0.01; n = 50). Stable binding of the long-wavelength vital dyes to the plasma membrane also permitted in vivo tracking of injected tumor cells in the pulmonary vasculature. These probes offer a significant improvement in the imaging quality of in situ biological processes in the deeper regions of intact lungs.

Vascularization of solid tumors is thought to occur by sprouting or intussusceptive angiogenesis, co-option of existing vessels, and vasculogenic mimicry after the onset of tumor hypoxia, when the tumor radius exceeds the oxygen diffusion distance. In contrast, here we show that individual endothelial cells that are incorporated into pre-hypoxic tumors give rise to tumor blood vessels via vasculogenesis. Small metastatic lung tumor sections obtained after tail-vein injection of a syngeneic breast cancer cell line in the nude mice were labeled with antibodies against endothelial cell markers. Immunofluorescence showed the incorporation and mixed growth of CD31-, Tie-2-, and CD105-positive endothelial cells in tumors with radii less than oxygen diffusion distance and subsequent development of blood vessels from these early-incorporated endothelial cells. This observation lays the foundation of a novel vasculogenic paradigm of tumor vascularization, where incorporation of endothelial cells and their growth among tumor cells occur before the onset of core hypoxia in lung metastatic tumors.

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Although mitochondrial ATP-production deficiency has been historically implicated to account for the Warburg effect or “aerobic” glycolysis in cancer cells, recent observations indicate the presence of functionally competent mitochondria in cancer cells. We hypothesized that a lung-metastatic breast cancer cell line with intact mitochondrial membrane potential would exhibit mitochondria-dependent oxygen consumption. A murine breast cancer cell line (4T1) was used to evaluate mitochondrial membrane potential by JC-1 fluorescence microscopy. Oxygen consumption by cancer cells in suspension was determined using a dissolved oxygen meter. Fluorescence microscopy of JC-1 red aggregates indicated the presence of highly polarized mitochondria in 4T1 cells, suggesting that these cells are capable of oxidative phosphorylation. Oxygen consumption rate of 4T1 cells suspended in 10 mM glucose-containing medium equilibrated with 21% oxygen (258 µM dissolved oxygen) at 25°C was 2.98 nmol/min/million cells (Control). Oxygen consumption was significantly enhanced by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), a mitochondrial uncoupler (7.88 nmol/min/million cells, p<0.05 vs. Control). Myxothiazol (a respiratory chain complex III inhibitor) inhibited the oxygen consumption (0.75 nmol/min/million cells; p<0.05 vs. Control), indicating the presence of a functional electron transport chain. A nitric oxide donor also inhibited oxygen consumption (0.84 nmol/min/million cells; p<0.05 vs. Control). These data indicate the presence of oxidative phosphorylation-competent mitochondria in breast cancer cells. We conclude that cancer cells possess respiration-capable mitochondria and their “aerobic” glycolysis may not reflect a compensatory mechanism for ATP generation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 75.