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 inhibitior 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.

Cell detachment is a procedure routinely performed in cell culture and a necessary step in many biochemical assays including the determination of oxygen consumption rates (OCR) in vitro. In vivo, cell detachment has been shown to exert profound metabolic influences notably in cancer but also in other pathologies, such as retinal detachment for example. In the present study, we developed and validated a new technique combining electron paramagnetic resonance (EPR) oximetry and the use of cytodex 1 and collagen-coated cytodex 3 dextran microbeads, which allowed the unprecedented comparison of the OCR of adherent and detached cells with high sensitivity. Hence, we demonstrated that both B16F10 melanoma cells and human umbilical vein endothelial cells (HUVEC) experience strong OCR decrease upon trypsin or collagenase treatments. The reduction of cell oxygen consumption was more pronounced with a trypsin compared to a collagenase treatment. Cells remaining in suspension also encounter a marked intracellular ATP depletion and an increase in the lactate production/glucose uptake ratio. These findings highlight the important influence exerted by cell adhesion/detachment on cell respiration, which can be probed with the unprecedented experimental assay that was developed and validated in this study.

Introduction The ability to track the migration of metastatic cells when leaving the seeding tumor should become an invaluable tool to understand this phenomenon and propose new therapeutic anti-cancer strategies. Magnetic cell labeling is a promising technique to detect metastatic cancer cells with magnetic resonance imaging. Usually, cells are incubated with iron oxides (T 2 contrast agent) in order to uptake the particles before being injected in vivo. In addition to MRI protocols, there is generally a need for complementary techniques able to confirm and quantify the cells that have migrated inside a host tissue. We propose here to implement Electron Paramagnetic Resonance (EPR) as a very sensitive method to quantify iron oxide concentration (in cells and tissues). Iron oxide particles exhibit an EPR spectrum, which directly reflects the number of iron oxide particles in a sample. EPR spectroscopy has already been proposed as a method of quantifying the accumulation of iron oxide inside tissues (1). In order to compare EPR with existing methods (Perl's Prussian blue reaction, and fluorimetry), we labeled tumor cells (Melanoma B16F10-luc, fibrosarcoma KHT-luc) and fibroblasts (3T3) with fluorescent iron oxide particles, and defined the limit of detection of the different techniques.

MRI cell tracking is a promising technique for tracking various cell types in living animals. Usually, cells are incubated with iron oxides so that the particles are taken up before the cells are injected in vivo. In the present study, we aimed to monitor migration of luciferase-expressing mouse renal cancer cells (RENCA-luc) after intrarenal or intrasplenic injection. These cells were labelled using Molday Ion Rhodamine B (MIRB) fluorescent superparamagnetic iron oxide particles. Their fate after injection was first assessed using ex vivo X-band electron paramagnetic resonance (EPR) spectroscopy. This biodistribution study showed that RENCA-luc cells quickly colonized the lungs and the liver after intrarenal and intrasplenic injection, respectively. Bioluminescence imaging (BLI) studies confirmed that this cell line preferentially metastasized to these organs. Early tracking of labelled RENCA-luc cells in the liver using high-field MRI (11.7 T) was not feasible because of a lack of sensitivity. MRI of MIRB-labelled RENCA-luc cells after injection in the left kidney was then performed. T 2 -and T 2 *-weighted images showed that the labelled cells induced hypointense signals at the injection site. Nevertheless, the hypointense regions tended to disappear after several days, mainly owing to dilution of the MIRB iron oxides with cell proliferation. In conclusion, EPR is well adapted to ex vivo analysis of tissues after cell tracking experiments and allows short-term monitoring of metastasizing cells. MRI is a suitable tool for checking labelled cells at their injection site, but dilution of the iron oxides owing to cell division remains a major limitation. BLI remains the most suitable technique for long-term monitoring of metastatic cells.

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.