Glucose uptake assay

Seventy-five thousand MSCs, CAFs, or MDA-MB-231 cells per well were seeded into 12-well plates. After attachment and growth for 24 h (Fig. 1C) to 48 h (Fig. 2C and ​and6E),6E), cells were washed once with 1 mL PBS and incubated for 1 h with 0.025 μCi [U-¹⁴C]-D-glucose in 200 μL glucose uptake buffer (GUB: 120 mM NaCl, 25 mM NaHCO3, 4 mM KCl, 1.2 mM KH2PO4, 2.5 mM MgSO4-7 hydrate, 70 μM CaCl₂-dihydrate, pH 7.4) per well at 37°C and 5% CO₂. For experiments, studying the effects of a flavonoid on glucose uptake (Fig. 6E), 100 μM phloretin was added to GUB. All cells were subsequently washed with 500 μL ice-cold GUB to stop transport. Cells were then exposed to 200 μL 0.25% Trypsin / 0.53 mM EDTA per well for 15 minutes at 37°C and 5% CO₂. The entire content of each well was placed into scintillation liquid and counted in a scintillation counter to generate counts per minute (CPM). Where appropriate (Fig. 1C), CPM was normalized to 100,000 cells. In co-culture experiments (Fig. 4B), 5•10⁴ MDA-MB-231 cells were seeded into the chambers of a Boyden chamber plate and 1•10⁵ MDA-MB-231 cells or CAFs were seeded into the inserts. Cells were co-cultured for 2 weeks, after which MDA-MB-231 cells in chambers were assayed to determine glucose uptake as described above.

Lactate oxidation metabolite (LOM)-mediated growth of MDA-MB-231 cells – Modulation of MDA-MB-231 cell growth in the absence or presence of 2.5 mM glucose (Gluc) by 2.5 mM of (A) α-alanine (α-Ala), β-alanine (β-Ala), GABA, acetate (Acet) or (B) by 2.5 mM pyruvate (sodium pyruvate). Compared to control cells exposed to culture medium without glucose and added nutrients (Dep), neither GABA, alanine or acetate produced a significant growth benefit for MDA-MB-231 cells; While α-alanine appears to increase cell growth in the presence of 2.5 mM glucose, GABA, β-alanine, and acetate abrogate the growth benefit of 2.5 mM glucose, as seen in (A, B). Glucose alone (B) and pyruvate alone (B) enhanced MDA-MB-231 cell growth similarly, while both together enhanced growth synergistically (B). (C) Pyruvate does not modulate glucose uptake in MDA-MB-231 cells (over a range of 1.25 mM to 5 mM). Data are displayed as mean±SD (n = 3 for A, B and n = 4 for C); * p<0.05, ** p<0.005, *** p<0.0005 by two-tailed, unpaired, unequal variance Student’s T-test (compared to Dep or as indicated by lines between subgroups).

Conditioned medium from CAFs (CCM) influences ¹⁴C-pyruvate (A) and ¹⁴C-glucose uptake (B) in MDA-MB-231 cells (MDAs). Data are displayed as mean±SD (n=4 for A and n=3 for B); ** p=0.005, *** p<0.0005 by two-tailed, unpaired, unequal variance Student’s T-test (compared to MDAs).

Phloretin as a potential inhibitor of tumor-TME metabolic coupling – (A) Left panel: Electrostatic potential map of solvent accessible surface area of MCT1. Site A = positive electrostatic potential, Site B = negative electrostatic potential; Right panel: 3D model of Phloretin binding to Site B (Eint = −50.7 kcal/mol). (B) Growth inhibitory profile of flavonoids in MDA-MB-231 cells in response to 24 h exposure to 100 μM of indicated drug. (C) Phloretin inhibits ¹⁴C-lactate uptake in MSC and CAFs. (D) Phloretin inhibits ¹⁴C-pyruvate uptake in MDA-MB-231 cells. (E) Phloretin inhibits ¹⁴C-D-glucose uptake in MDA-MB-231 cells. ¹⁴C-L-glucose was used as a negative control. (F) Phloretin decreases ROS accumulation in MDA-MB-231 cells and protects from the ROS accumulation effects of H₂O₂exposure. (G) Phloretin attenuates MDA-MB-231 cell growth in CCM. The glucose concentrations at the start of the experiments were 21 mM and 11 mM for complete medium (Comp Media) and CCM, respectively. In B (n=3), C (n=4), D (n=4), E (n=4), F (n=6), and G (n=3) data are displayed as mean±SD; * p<0.05, ** p≤0.005, *** p<0.0005, **** p<0.0001 by two-tailed, unpaired, unequal variance Student’s T-test for the comparison to corresponding controls (“Untreated”, no line) and comparisons as indicated by line.