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, 11 (7), 1445-54

Mitochondrial Metabolism in Cancer Metastasis: Visualizing Tumor Cell Mitochondria and the "Reverse Warburg Effect" in Positive Lymph Node Tissue

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Mitochondrial Metabolism in Cancer Metastasis: Visualizing Tumor Cell Mitochondria and the "Reverse Warburg Effect" in Positive Lymph Node Tissue

Federica Sotgia et al. Cell Cycle.

Abstract

We have recently proposed a new two-compartment model for understanding the Warburg effect in tumor metabolism. In this model, glycolytic stromal cells produce mitochondrial fuels (L-lactate and ketone bodies) that are then transferred to oxidative epithelial cancer cells, driving OXPHOS and mitochondrial metabolism. Thus, stromal catabolism fuels anabolic tumor growth via energy transfer. We have termed this new cancer paradigm the "reverse Warburg effect," because stromal cells undergo aerobic glycolysis, rather than tumor cells. To assess whether this mechanism also applies during cancer cell metastasis, we analyzed the bioenergetic status of breast cancer lymph node metastases, by employing a series of metabolic protein markers. For this purpose, we used MCT4 to identify glycolytic cells. Similarly, we used TO MM20 and COX staining as markers of mitochondrial mass and OXPHOS activity, respectively. Consistent with the "reverse Warburg effect," our results indicate that metastatic breast cancer cells amplify oxidative mitochondrial metabolism (OXPHOS) and that adjacent stromal cells are glycolytic and lack detectable mitochondria. Glycolytic stromal cells included cancer-associated fibroblasts, adipocytes and inflammatory cells. Double labeling experiments with glycolytic (MCT4) and oxidative (TO MM20 or COX) markers directly shows that at least two different metabolic compartments co-exist, side-by-side, within primary tumors and their metastases. Since cancer-associated immune cells appeared glycolytic, this observation may also explain how inflammation literally "fuels" tumor progression and metastatic dissemination, by "feeding" mitochondrial metabolism in cancer cells. Finally, MCT4(+) and TO MM20(-) "glycolytic" cancer cells were rarely observed, indicating that the conventional "Warburg effect" does not frequently occur in cancer-positive lymph node metastases.

Figures

Figure 1
Figure 1
Metastatic breast cancer cells have increased mitochondrial mass. Paraffin-embedded sections of human breast cancer-positive lymph nodes were immunostained with antibodies directed against TOMM20 (brown color). Slides were then counterstained with hematoxylin (blue color). Note that TOMM20 is highly expressed in metastatic breast cancer cells. Two representative images are shown. Original magnification, 40x.
Figure 2
Figure 2
Metastatic breast cancer cells show increased mitochondrial activity. Frozen sections of human breast cancer-positive lymph nodes were subjected to COX activity staining (brown color). Slides were then counterstained with hematoxylin (blue color). Note that epithelial tumor cells are intensely stained, as compared with adjacent stromal cells, which show little or no COX activity. Two representative images are shown. Original magnification, 40x.
Figure 3
Figure 3
Mitochondrial activity staining is ablated with metabolic inhibitors. Frozen sections of human breast cancer-positive lymph nodes were subjected to COX activity staining (brown color). Slides were then counterstained with hematoxylin (blue color). Note that sodium azide (+AZ; 1 mM; a Complex IV inhibitor) effectively abolished the COX activity staining.
Figure 4
Figure 4
Lymph node associated stromal cells are glycolytic. Paraffin-embedded sections of human breast cancer-positive lymph nodes were immunostained with antibodies directed against MCT4. Slides were then counterstained with hematoxylin. Note that MCT4 is highly expressed in the stromal compartment of positive lymph nodes. Two representative images are shown. Original magnification, 40x and 60x, as indicated.
Figure 5
Figure 5
Cav-1 immunostaining of breast cancer-positive lymph nodes. Paraffin-embedded sections of human breast cancer-positive lymph nodes were immunostained with antibodies directed against Cav-1. Slides were then counterstained with hematoxylin. Note that Cav-1 is largely absent from the stromal compartment of positive lymph nodes, with the exception of blood vessels (indicated by red arrowheads). Two representative images are shown. Original magnification, 40x and 60x, as indicated.
Figure 6
Figure 6
Visualizing the “reverse Warburg effect” by double labeling with MCT4 and COX activity staining. Frozen sections of human breast cancer-positive lymph nodes were subjected to COX activity staining (brown color) and immunostaining with MCT4 antibodies (red color). Slides were then counterstained with hematoxylin (blue color). Note that MCT4 staining is predominantly localized to the cancer-associated lymph node stroma. In contrast, COX activity is strongly associated with the metastatic breast cancer cells. Two representative images are shown. Original magnification, 40x.
Figure 7
Figure 7
Visualizing the “reverse Warburg effect” by double labeling with MCT4 and TOMM20: Lymph node metastasis. Paraffin-embedded sections of human breast cancer-positive lymph nodes were immunostained with antibodies directed against MCT4 (red color) and TOMM20 (brown color). Slides were then counterstained with hematoxylin (blue color). Note that MCT4 staining is predominantly localized to the cancer-associated lymph node stroma. In contrast, TOMM20 is strongly associated with the metastatic breast cancer cells. Original magnification, 60x.
Figure 8
Figure 8
Lymph node-associated adipocytes and inflammatory cells are glycolytic. Paraffin-embedded sections of human breast cancer-positive lymph nodes were immunostained with antibodies directed against MCT4 (red color) and TOMM20 (brown color). Slides were then counterstained with hematoxylin (blue color). Note that lymph-node associated adipocytes (asterisks) and inflammatory cells (red arrowhead) are MCT4(+) and TOMM20(-), consistent with a glycolytic phenotype. In contrast, TOMM20 is strongly associated with the metastatic breast cancer cells. Rarely, MCT4(+) and TOMM20(-) epithelial cancer cells were also observed (black arrowhead). Original magnification, 60x.
Figure 9
Figure 9
Visualizing the “reverse Warburg effect” by double labeling with MCT4 and TOMM20: Primary tumor tissue. Paraffin-embedded sections of human breast cancer primary tumors were immunostained with antibodies directed against MCT4 (red color) and TOMM20 (brown color). Slides were then counterstained with hematoxylin (blue color). Note that MCT4 staining is largely confined to the cancer-associated tumor stroma. In contrast, TOMM20 is specifically associated with the epithelial breast cancer cells. Two representative images are shown. Original magnification, 40x.
Figure 10
Figure 10
Tumor-associated inflammatory cells are glycolytic. Paraffin-embedded sections of human breast cancer primary tumors were immunostained with antibodies directed against MCT4 (red color) and TOMM20 (brown color). Slides were then counterstained with hematoxylin (blue color). Note that tumor-associated inflammatory cells (red arrowheads) are MCT4(+) and TOMM20(-), consistent with a glycolytic phenotype. In contrast, TOMM20 is strongly associated with the primary breast cancer cells. Two representative images are shown. Original magnification, 60x.
Figure 11
Figure 11
Mitochondrial metabolism and the “reverse Warburg effect” in cancer metastasis. Metastatic cancer cells secrete ROS, such as hydrogen peroxide, which induces oxidative stress in neighboring stromal cells. Oxidative stress, in turn, initiates the autophagic destruction of mitochondria (mitophagy), resulting in the onset of aerobic glycolysis. As a consequence, mitochondrial dysfunction in stromal cells leads to the production of high-energy mitochondrial fuels (L-lactate and ketone bodies) which are effluxed to the extracellular environment via MCT4. These fuels are then taken up by metastatic cancer cells and converted to Acetyl-CoA, which is “burned” in the TCA cycle and OXPHOS, via mitochondrial metabolism. Hence, metastatic cancer cells are mitochondria-rich and are positive for markers of mitochondrial mass (TOMM20) and OXPHOS activity (COX). Experimentally, glycolytic stromal cells included cancer-associated fibroblasts, adipocytes and inflammatory cells, which were MCT4(+) and TOMM20(-). LN, lymph node. ROS, reactive oxygen species, OXPHOS, oxidative mitochondrial metabolism.

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