TY - JOUR
T1 - Cancer cells metabolically "fertilize" the tumor microenvironment with hydrogen peroxide, driving the Warburg effect
T2 - Implications for PET imaging of human tumors
AU - Martinez-Outschoorn, Ubaldo E.
AU - Lin, Zhao
AU - Trimmer, Casey
AU - Flomenberg, Neal
AU - Wang, Chenguang
AU - Pavlides, Stephanos
AU - Pestell, Richard G.
AU - Howell, Anthony
AU - Sotgia, Federica
AU - Lisanti, Michael P.
N1 - Funding Information:
significant.t-test. p values lower than 0.05 were consi%derPed sOtatPistiUcalEly JTSo(BUcCSietAJy (), aCAnVCSd a RU)F. Funds were also contributed by the Margaret esearch Scholar Grant from the American Cancer Immunocytochemistry. Cells were fixed after 5 d of culture Q. Landenberger Research Foundation (to M.P.L.). R.G.P. and immunostained as we have previously described, with minor was supported by grants from the NIH/NCI (R01-CA-70896, modifications.34 Briefly, cells were fixed for 30 min at room tem-R01-CA-75503, R01-CA-86072 and R01-CA-107382) and the perature in 2% paraformaldehyde diluted in PBS, after which Dr. Ralph and Marian C. Falk Medical Research Trust. The they were permeabilized with cold methanol at -20°C for 5 Kimmel Cancer Center was supported by the NIH/NCI Cancer min. All subsequent steps were performed at room temperature. Center Core grant P30-CA-56036 (to R.G.P.). After rinsing with PBS with 0.1 mM Calcium chloride and 1 This project is funded, in part, under a grant with the mM Magnesium chloride (PBS/CM), cells were incubated with Pennsylvania Department of Health (to M.P.L. and F.S.). The NH4Cl in PBS to quench free aldehyde groups. Cells were then Department specifically disclaims responsibility for any analyses, blocked with immunofluorescence (IF) buffer (PBS, 1% BSA, interpretations or conclusions. This work was also supported, in 0.1% Tween 20) for 1 h. Anti-GLUT1 antibodies (AB1340, part, by a Centre grant in Manchester from Breakthrough Breast Chemicon) were incubated in IF buffer for 1 h. After washing Cancer in the UK (to A.H.) and an Advanced ERC Grant from with IF buffer (3x, 10 min each), cells were incubated for 30 min the European Research Council. with fluorochrome-conjugated secondary antibodies diluted in
Funding Information:
detector with excitation wavelength of 496 nm and emission of M.P.L. and his laboratory were supported by grants from 615 (to detect PI) and an APC signal detector with excitation the NIH/NCI (R01-CA-080250; R01-CA-098779; wavelength of 650 nm and emission of 660 nm. Apoptosis analy-R01-CA-120876; R01-AR-055660), and the Susan G. Komen
Funding Information:
Statisticalanalysis.Data were asis was performed using FlowJo 8.8 sÜnalyzed using thoftware.-e SBtudOeEnt’Fs Tthe Bre#Wast CJ.WP. anTSmceDitr FJh FCounOhardatDitaioFble Trust, the Breast Cancer Alliance n. F.S. was supported by grants from
PY - 2011/8/1
Y1 - 2011/8/1
N2 - Previously, we proposed that cancer cells behave as metabolic parasites, as they use targeted oxidative stress as a "weapon" to extract recycled nutrients from adjacent stromal cells. Oxidative stress in cancer-associated fibroblasts triggers autophagy and mitophagy, resulting in compartmentalized cellular catabolism, loss of mitochondrial function, and the onset of aerobic glycolysis, in the tumor stroma. As such, cancer-associated fibroblasts produce high-energy nutrients (such as lactate and ketones) that fuel mitochondrial biogenesis and oxidative metabolism in cancer cells. We have termed this new energy-transfer mechanism the "Reverse Warburg Effect." To further test the validity of this hypothesis, here we used an in vitro MCF7-fibroblast co-culture system and quantitatively measured a variety of metabolic parameters by FACS analysis (analogous to laser-capture micro-dissection). Mitochondrial activity, glucose uptake and ROS production were measured with highly-sensitive fluorescent probes (MitoTracker, NBD-2-deoxy-glucose and DCF-DA). Interestingly, using this approach, we directly show that cancer cells initially secrete hydrogen peroxide that then triggers oxidative stress in neighboring fibroblasts. Thus, oxidative stress is contagious (spreads like a virus) and is propagated laterally and vectorially from cancer cells to adjacent fibroblasts. Experimentally, we show that oxidative stress in cancer-associated fibroblasts quantitatively reduces mitochondrial activity and increases glucose uptake, as the fibroblasts become more dependent on aerobic glycolysis. Conversely, co-cultured cancer cells show significant increases in mitochondrial activity and corresponding reductions in both glucose uptake and GLUT1 expression. Pretreatment of co-cultures with extracellular catalase (an anti-oxidant enzyme that detoxifies hydrogen peroxide) blocks the onset of oxidative stress and potently induces the death of cancer cells, likely via starvation. Given that cancer-associated fibroblasts show the largest increases in glucose uptake, we suggest that PET imaging of human tumors, with Fluoro-2-deoxy-D-glucose (F-2-DG), may be specifically detecting the tumor stroma, rather than epithelial cancer cells.
AB - Previously, we proposed that cancer cells behave as metabolic parasites, as they use targeted oxidative stress as a "weapon" to extract recycled nutrients from adjacent stromal cells. Oxidative stress in cancer-associated fibroblasts triggers autophagy and mitophagy, resulting in compartmentalized cellular catabolism, loss of mitochondrial function, and the onset of aerobic glycolysis, in the tumor stroma. As such, cancer-associated fibroblasts produce high-energy nutrients (such as lactate and ketones) that fuel mitochondrial biogenesis and oxidative metabolism in cancer cells. We have termed this new energy-transfer mechanism the "Reverse Warburg Effect." To further test the validity of this hypothesis, here we used an in vitro MCF7-fibroblast co-culture system and quantitatively measured a variety of metabolic parameters by FACS analysis (analogous to laser-capture micro-dissection). Mitochondrial activity, glucose uptake and ROS production were measured with highly-sensitive fluorescent probes (MitoTracker, NBD-2-deoxy-glucose and DCF-DA). Interestingly, using this approach, we directly show that cancer cells initially secrete hydrogen peroxide that then triggers oxidative stress in neighboring fibroblasts. Thus, oxidative stress is contagious (spreads like a virus) and is propagated laterally and vectorially from cancer cells to adjacent fibroblasts. Experimentally, we show that oxidative stress in cancer-associated fibroblasts quantitatively reduces mitochondrial activity and increases glucose uptake, as the fibroblasts become more dependent on aerobic glycolysis. Conversely, co-cultured cancer cells show significant increases in mitochondrial activity and corresponding reductions in both glucose uptake and GLUT1 expression. Pretreatment of co-cultures with extracellular catalase (an anti-oxidant enzyme that detoxifies hydrogen peroxide) blocks the onset of oxidative stress and potently induces the death of cancer cells, likely via starvation. Given that cancer-associated fibroblasts show the largest increases in glucose uptake, we suggest that PET imaging of human tumors, with Fluoro-2-deoxy-D-glucose (F-2-DG), may be specifically detecting the tumor stroma, rather than epithelial cancer cells.
KW - Aerobic glycolysis
KW - Cancer associated fibroblasts
KW - Caveolin-1
KW - Glucose uptake
KW - Hydrogen peroxide
KW - Microenvironment
KW - Mitochondrial oxidative phosphorylation
KW - Oxidative stress
KW - PET imaging
KW - Reactive oxygen species (ROS)
KW - The field effect
KW - Tumor stroma
UR - http://www.scopus.com/inward/record.url?scp=79961101955&partnerID=8YFLogxK
U2 - 10.4161/cc.10.15.16585
DO - 10.4161/cc.10.15.16585
M3 - Article
AN - SCOPUS:79961101955
SN - 1538-4101
VL - 10
SP - 2504
EP - 2520
JO - Cell Cycle
JF - Cell Cycle
IS - 15
ER -