Effect of tumor progression on hypoxia and expression of E-cadherin in the BM microenvironment. (A) MNCs isolated from the BM of mice injected with MM1s. Different stages were analyzed and the hypoxic state of BM microenvironment was determined by flow cytometry as the MFI of PIM in GFP⁻ cells. The correlation between hypoxia in the BM microenvironment and tumor progression (detected by BLI) demonstrates increased hypoxia in the microenvironment with tumor progression. (B) Correlation between expression of E-cadherin and level of hypoxia in BM microenvironment showing decreased expression of E-cadherin with hypoxia. (C) Effect of incubation of BMSCs (isolated from 3 different MM patients) under hypoxic conditions for 24 hours in vitro on expression of HIF1α, HIF2α, SNAIL, and E-cadherin in MM cells detected by immunoblotting. This shows increased expression of HIF1α, HIF2α, and SNAIL and decreased expression of E-cadherin in hypoxia. (D) The effect of incubation of BMSCs (isolated from 3 different MM patients) under hypoxic conditions for 24 hours in vitro on adhesion of MM cells to a monolayer of BMSCs. This shows decreased adhesion of MM cells to hypoxic BMSCs. The effect of treating stromal cells with E-cadherin–blocking Ab on adhesion of MM cells to stroma under normoxic (E) or hypoxic (F) conditions is shown. The blocking Ab decreased adhesion to normoxic but not to hypoxic stroma. (G) Effect of hypoxia on the secretion of SDF1α from MM stroma showing decreased secretion of SDF1α under hypoxic conditions. (H) The migration of MM cells to medium from normoxic and hypoxic stroma showing decreased migration of MM cells toward the medium from hypoxic stroma.