Mechanism of bortezomib-induced immunogenic cell death: role of cell surface hsp90 on the immunogenicity of bortezomib-induced cell death. (A) Phenotype of dendritic cells after interaction with bortezomib-killed U266 cells. Day 5 immature DCs were cultured for 24 hours with U266 myeloma cells killed by γ irradiation, dexamethasone, or bortezomib. Second set of cocultures was set up in transwells to prevent DCs/tumor cells contact. After 24 hours, expression of CD80 and CD86 on DCs was analyzed by flow cytometry. Mean fluorescence intensity is shown. *P value for comparison with γ-irradiated tumor cell–loaded DCs, P < .05. (B) Induction of inducible HSP70 (iHPS70) and HSP90 expression on U266 treated with bortezomib. Apoptosis of U266 myeloma cells was induced by γ irradiation or bortezomib and surface expression of HSP70 and HSP90 analyzed 24 hours later by flow cytometry. Preincubation of tumor cells with geldanamycin 1 hour prior to the addition of bortezomib inhibits the increase in HSP90 expression. (C) HSP90 expression 24 hours after bortezomib treatment on myeloma (U266 and cag), mantle cell lymphoma (NCEB1), and breast cancer (MCF-7) cell lines. (D) Kinetics of HSP90 expression on U266 cells after dexamethasone or bortezomib treatment analyzed by flow cytometry. (E) Phenotype of dendritic cells after interaction with dexamethasone or bortezomib-killed U266 cells at different stages of apoptosis. U266 cells were killed by bortezomib or dexamethasone, and cocultures with day 5 immature DCs were set up at 6, 12, and 24 hours after induction of apoptosis. After 24 hours, expression of CD86 on DCs was analyzed by flow cytometry. Mean fluorescence intensity is shown. *P value for comparison with immature DCs, P < .05. (F) Kinetics of U266 myeloma cell apoptosis after γ irradiation, bortezomib treatment, or combined bortezomib + geldanamycin (GDC) treatment. Percentage of live cells was followed for 24 hours by flow cytometry. Live cells were defined as annexin V negative/To-Pro3 negative. (G) Inhibition of increase in CD86 expression on DCs after interaction with bortezomib-killed myeloma cells by GDC or anti-HSP90 antibody. Immature DCs were cultured for 24 hours with U266 myeloma cells killed by bortezomib and expression of CD86 analyzed by flow cytometry. Preincubation of tumor cells with geldanamycin or anti-HSP90 mAb prior to the induction of apoptosis by bortezomib abrogates increase in CD86 expression. Staining with isotype control (gray line) and anti-HPS90 mAb (black line) is shown. Error bars represent mean ± SD.