Effect of hypoxia on expression of E-cadherin and adhesion to BMSCs. (A) MM cells isolated from the BM of mice injected with MM1s cells at different tumor burdens were analyzed for the expression of cadherins by flow cytometry and compared with the hypoxic state in these cells indicated as the MFI of PIM in the MM cells. The expression of cadherins was decreased significantly with the increase in hypoxia in the MM cells. (B) The expression of E-cadherin in circulating MM cells compared with MM cells in the BM, showing that circulating MM cells maintained a low expression of E-cadherin. (C) IHC images of specimens taken from the femurs of mice with different tumor burdens stained with Ab for E-cadherin, which shows that the expression of E-cadherin decreased with tumor progression. (D) Gene-expression analysis of E-cadherin and EMT-related genes (including SNAIL, FOXC2, and TGFβ1) in plasma cells isolated from normal subjects and MM patients using published datasets from the Gene Expression Omnibus by Chng et al (series number GSE 6477²⁶ ). This shows decreased E-cadherin expression and increased expression of SNAIL, FOXC2, and TGFβ1 in MM. (E) The effect of incubation of MM cells (MM1s and H929) under hypoxic conditions for 24 hours in vitro on expression of HIF1α and HIF2α GSK3β, SNAIL, and E-cadherin in MM cells detected by immunoblotting. (F) The effect of incubation of MM cells (MM1s and H929) under hypoxic conditions for 24 hours in vitro on adhesion to a monolayer of BMSCs isolated from MM patients. This shows decreased adhesion of hypoxic MM cells. The effect of treating MM cells with E-cadherin–blocking Ab on adhesion of MM cells to stromal cells under normoxic (G) or hypoxic (H) conditions. This shows that the blocking Ab significantly decreased adhesion in normoxic conditions but not in hypoxic conditions. Effect of inhibition of GSK3β by a small-molecule inhibitor under normoxic conditions on expression of SNAIL and E-cadherin detected by immunoblotting (I) and on adhesion of MM cells to a monolayer of BMSCs (J). This shows increased expression of SNAIL, decreased expression of E-cadherin, and decreased adhesion to BMSCs.