Supplementary Materials1. (C) Constant state 12C metabolites in TYK-nu cells stimulated with control CM or CAF CM for 6 hr (n=3/group). Important metabolites are shown along with a schematic depicting the central pathways Regorafenib (BAY 73-4506) (* p 0.05, **p 0.01, ***p 0.001). (D) ROS level in GFP-labeled SKOV3ip1 malignancy cells with or without CAF co-culture and CP-91149. test (* Goat polyclonal to IgG (H+L)(FITC) p 0.05, **p 0.01). (E) Proliferation of TYK-nu cells exposed to CAF CM with or without the glycogen phosphorylase inhibitor CP-91149. Values are mean + SEM from 4 impartial experiments (n=6/group). Comparisons were made to CAF CM using a Two-way ANOVA (* p 0.05, **p 0.01). (F) Invasion of TYK-nu cells exposed to CAF CM with or without the glycogen phosphorylase inhibitor CP-91149. Data is usually representative Regorafenib (BAY 73-4506) of 3 impartial experiments. Values are mean + SEM (n=3/group). ***p 0.001. In order to understand further the metabolic changes induced by CAFs, we performed targeted metabolic profiling of OvCa cells stimulated with control or CAF conditioned media using high-performance liquid chromatography (HPLC) coupled to high-resolution mass spectrometry. Consistent with our previous data, we observed after 6 hr increased glycolysis in malignancy cells, indicated by increased glucose-6-phosphate/fructose-6-phosphate, 2/3-phospho-glycerate, and lactate production with CAF conditioned media (Physique 3C). The levels of UDP-glucose/UDP-galactose, intermediates for glycogen synthesis, remained constant. A higher level of ribose-5-phosphate was also detected in the malignancy cells stimulated with CAF conditioned media (Physique 3C) suggesting an increase in the pentose phosphate pathway. As shown by a quantitative assay, glucose-1-phosphate was also increased in OvCa cells following activation with CAF CM, which is consistent with an increased breakdown of glycogen (Physique S4H). Since the pentose phosphate pathway contributes to both increased ribose-5-phosphate and an increased availability of reducing equivalents, we next asked if CAF co-culture affects reactive oxygen species (ROS) levels in malignancy cells. Indeed, CAFs decreased the production of ROS in the malignancy cells, an effect that was rescued by treatment with the glycogen phosphorylase inhibitor (Physique 3D). This is consistent with a report showing that glycogen phosphorylase inhibition induces ROS under Regorafenib (BAY 73-4506) conditions of hypoxia (Favaro et al., 2012). Increased glycolysis was paralleled by increased proliferation and invasion in TYK-nu and SKOV3ip1 cell lines following activation by CAF conditioned media. Treatment with the glycogen phosphorylase inhibitor, CP-91149, blocked the increase in both proliferation and invasion (Physique 3E, F and Figure S5A, B). Direct CAF co-culture showed similar effects on proliferation in SKOV3ip1 and in one main OvCa cell clone (Physique S5C). Of notice, treatment with CP-91149 experienced no effect on the basal level of glycolysis, proliferation or invasion. We next asked if hydrolyzed glycogen contributes directly to the metabolite pool in malignancy cells. To accomplish this, we devised a new glycogen labeling strategy in which malignancy cells were fed U-13C-glucose for 48hr to label glycogen (Physique 4A) and then media was changed to 12C-glucose for 1 hour prior to the experiment. This would leave glycogen as the singular source for 13C-carbon in the cell. 13C-labeling patterns were analyzed following culture either with or without human-derived main CAFs in a transwell place. U-13C-glycogen was hydrolyzed and contributed to the levels of glycolytic metabolites, including glucose-6-phosphate/fructose-6-phosphate (m+6), dihydroxyacetone phosphate (DHAP) (m+3), and 2/3-phospho-glycerate (m+3), all of which were increased upon CAF co-culture (Physique 4B). Glycogen also contributed to increases in citrate/isocitrate (m+2) and -ketoglutarate (m+2), while there was no increase in additional TCA cycle intermediates such as fumarate and malate. These data suggest that CAFs induce malignancy cells to metabolize glycogen through glycolysis, which increases energy production and tumor aggressiveness. Open in a separate window Physique 4. Glycogen-derived metabolites feed glycolysis.(A) Percent of glycogen labeled with U-13C-glucose tracer at 24 and 48hr. 13C-glucose (m+6) was measured using mass spectrometry following hydrolysis of U-13C-glycogen to glucose. TYK-nu cells were cultured in U-13C-glucose made up of DMEM for 24hr or 48hr to synthesize U-13C-glycogen. (B) Glycogen tracing analysis. 13C metabolite tracing analysis of TYK-nu cells cultured with or without CAFs in.