Fig. 1.
IL-6/PGE2 feedback loop between IRISOE TNBC cells and activated MSCs. a Normalized IL-6 level detected using ELISA in CM of HME, IRIS291, and IRIS293 cells expressing siLuc or siHIF-1α exposed 24 h to normoxic or hypoxic conditions. Experiments were done three separate times in triplicates. b Expression of indicated proteins in MSCs exposed to CM from IRIS291, IRIS292, or IRIS293 cells grown under normoxic or hypoxic condition added plus vehicle or anti-IL-6 NeuAb. Fold change in black compares normoxic and hypoxic conditions, and in red compares hypoxia plus vehicle vs. plus IL-6 NeuAb conditions. Experiments were done at least twice. c Top: effects of 10 μM of STAT3 inhibitor; VII, PI3’K/AKT inhibitor; LY294002 and ERK inhibitor; PD98059 on their cognate kinase. Bottom: MTS assay measuring proliferation of MSCs exposed to CM from normoxic or hypoxic IRIS291 or IRIS293 in the presence of vehicle, VII, LY294002, or PD98059. Experiments were done three separate times in triplicates. The level of p-STAT3Y705 in MSCs exposed to vehicle [−] or 30 ng/ml rIL-6 or CM from HME, IRIS291, IRIS292, or IRIS293 cells for 30 min (d), or CM from MDA-231 or MDA-468 expressing si/shCtrl or si/shIRIS or MCF7 or T47D expressing vector or IRIS cDNA for 30 min (e). d, e Experiments were done at least twice. f The level of p-STAT3Y705, total STAT3 or Cyclin D1 in cytoplasm (C) or nuclear/chromatin (N + Ch) fractions of MSCs exposed to vehicle [−] or 30 ng/ml rIL-6 or CM from MDA-231, MDA-468, or MDA-453 expressing si/shCtrl or si/shIRIS for 30 min. Experiments were done at least twice. g Fluorescence IHC for p-STAT3Y705 and CD105 on sections from the core (1–4) or periphery (5–8) of IRIS291 tumors. Scale bar = 100 μm. h Migration in Boyden chambers of MSCs layered in inserts and exposed to HME (1) or IRISOE cells CM plus vehicle (2) or plus IL-6 NeuAb (3). Experiments were done in triplicates three separate times. i Migration in Boyden chambers of MSCs layered in the inserts exposed to vehicle [−], IRIS291 or IRIS293 cell CM containing vehicle, VII, LY294002, or PD98059 (at 10 μM each). Experiments were done three separate times in triplicates. j Inset: normalized PGE2 level secreted from naïve MSCs, HME, IRIS291, or IRIS293 cells as detected using ELISA. Experiments were done three separate times in triplicates. Normalized PGE2 levels secreted from naïve MSCs or MSCs exposed to CM from HME, IRIS291, or IRIS293 cells expressing (48 h) siLuc or siIL-6 and exposed (24 h) to normoxia or hypoxia as detected using ELISA. Experiments were done three separate times in triplicates. k The level of EP2 and EP4 in HME, IRIS291, IRIS292, or IRIS293 cells. Experiments were done at least twice. l Schematic representation of the data represented above