Tumor progression increases hypoxia in MM cells in vivo. (A) Representative BLIs of 12 SCID mice with different levels of tumor burden after injection of MM1s-GFP-Luc cells. (B) Correlation between tumor burden as detected by BLI and hypoxia in MM cells shown as the MFI of allophycocyanin-PIM in the MM cells (GFP⁺). (C) IHC images of specimens taken from the femurs of mice injected with MM1s-GFP-Luc with different levels of BLI stained with Abs for CD138 and HIF1α. Red arrows show nuclear staining with HIF1α. (D) IHC images of specimens taken from mice at different time points after the injection of 5T33MM mouse cells stained with Abs for PIM and HIF1α showing that binding of PIM and expression of HIF1α were directly correlated with tumor burden. (E) Gene-expression analysis of hypoxia-induced genes (including HIF1β, HIF2β, CREBBP, HYOU1, and VEGF1) 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 ).