BDNF promotes MM-cell survival. (A) Survival of primary MM cultured with BM stroma. CD138-immunoselected primary MM plasma cells were cultured in 1% serum on HS5 stromal cells. BDNF (50 ng/mL) and NGF (100 ng/mL) were added every 3 days. Viable cells were counted at 0 (□),3(▧), and 6 weeks (▪). The results using aspirates taken from 5 patients are presented as mean ± standard deviation. (B) Survival of primary MM cultured alone. CD138-immunoselected primary MM plasma cells (5 × 10⁴) were cultured without stroma in 1% FBS without (▨) or with BDNF (50 ng/mL; ▪) or NGF (100 ng/mL; ▤). 10% FBS (□) was used as comparator. Viable cells were counted at days 6, 8, and 13. The results using aspirates taken from 5 patients are presented as mean ± standard deviation. (C) BDNF protects HMCL RPMI from dexamethasone-induced death. RPMI 8226 cells in 1% FBS were exposed to increasing concentrations of dexamethasone ± BDNF (50 ng/mL) or NGF (100 ng/mL) or OPG (500 ng/mL). Viable cell numbers were assayed at 5 days using trypan blue exclusion. Each condition was assessed in triplicate, and the results are expressed as the mean ± standard deviation. (D-E) BDNF delays bortezomib-induced death of HMCL JJN3. JJN3 cells in 1% FBS were exposed to bortezomib (10 nM) for 24 hours, stained with annexin V–FITC, and analyzed by FACS. BDNF (50 ng/mL), IGF (100 ng/mL), or LY294002 (5 μM) was added 12 hours prior to bortezomib. (D) FACS profiles of a representative experiment. After 24-hour exposure to bortezomib, 39% of untreated, 21% of BDNF-treated, and 29% of BDNF/LY294002-treated JJN3 cells bind annexin V. Each condition was assessed in triplicate, and the results are presented in panel E as the mean ± standard deviation. ▤ indicates bortezomib alone; ▪, plus IGF; □ plus BDNF; and ▨, plus BDNF and LY294002. Student t test was used to determine significance (P < .05).