脑源性神经营养因子单克隆抗体在NOD/SCID小鼠骨髓瘤模型体内的抗瘤活性

Monoclonal antibody against brain derived neurotrophic factor inhibits myeloma growth and angio- genesis in the xerograft NOD/SCID animal model

目的以多发性骨髓瘤(MM)细胞株 RPMI 8226异体移植小鼠为动物模型,评估抗脑源性神经营养因子(BDNF)单克隆抗体(单抗)存体内的抗肿瘤活性。方法选择糖尿病抵抗/重症联合免疫缺陷(NOD/SCID)小鼠,皮下注射 RPMI 8226细胞建立骨髓瘤细胞异体移植动物模型。以20μg/只的 BDNF 单抗剂量于接种肿瘤细胞后第1、2、3天腹腔内注射或者以100 μg/只的剂量于检测到瘤体后每周1次腹腔内注射。采用组织学方法检测瘤细胞的形态特征,免疫化学染色法分析瘤组织的微血管密度(MVD)。用~3H-TdR 掺入法和 Matrigel 网状结构形成实验分别检测 BDNF 单抗对 RPMI8226细胞体外增殖和 PRMI 8226细胞诱导的内皮细胞血管新生的影响。结果在 NOD/SCID 小鼠体内建立的骨髓瘤细胞异体移植动物模型,其皮下种植的成瘤率高,具有多种与浆细胞瘤相似的病理学特征。未治疗组小鼠皮下注射 RPMI 8226细胞后(20±2)d 可检测到皮下肿瘤;接种肿瘤细胞后连续3 d 注射20 μg BDNF 单抗的治疗组,小鼠平均无肿瘤生存期延长为(30±6)d,而相应埘照抗体治疗组无肿瘤生存期为(22±3)d,与末治疗组相比差异无统计学意义;同时,其中位存活时间为(57±7)d,与相应的对照抗体治疗组[(48±4)d]相比明显延长(P<0.05);治疗组小鼠自然死亡时肿瘤体积为(157.9±21.6)mm^3,较对照抗体组[(405.5±35.2)mm^3]明显缩小(P<0.05)。对于接种肿瘤细胞后第27天起每周注射1次 BDNF 单抗(100 μg/次)的治疗组,肿瘤的生长亦受到部分抑制,相应对照抗体组与未治疗组相比肿瘤生长差异无统计学意义;同时在 BDNF 单抗治疗组瘤体的组织切片中,观察到部分瘤细胞出现凋亡的形态学表现,坏死区被纤维结缔组织浸润。未治疗组瘤块的 MVD 为38个/0.216 mm^2,BDNF 单抗治疗组(100 μg/次)MVD 为11个/0.216 mm^2,与未治疗组相比对照抗体治疗组瘤块的 MVD 没有明显下降(35个/0.216 mm^2)(P>0.05)。在体外,BDNF 单抗可显著抑制 RPMI8226细胞的增殖和其诱导的内皮细胞网状结构形成(抑制率为68.2%),而相应的对照抗体却无此效应。结论 BDNF 单抗可抑制皮下浆细胞瘤的生长和血管新生,BDNF 是治疗 MM 的潜在靶点。

Objective To evaluate the in vivo antitumor effect of anti-brain derived neurotrophic factor（BDNF） monoclonal antibody （ MoAb ） on a human myeloma xenograft animal model. Methods The xenograft tumor model was established in the nonobese diabetic/severe combined immunodeficiency （ NOD/ SCID） mice by subcutaneous injection of human myeloma cell line RPMI 8226. The antibodies were injected intraperitoneally at a dose of 20 μg/mouse at day 1, day 2, day 3 after tumor cell inoculation or at a dose of 100 μg/mouse once a week after tumors were developed. The histologic and cytologic examination were performed to confirm the development of plasmacytomas. The microvascular densities（MVD） in tumors were analyzed by immunohistochemistry. The effect of anti-BDNF MoAb on the proliferation of RPMI 8226 cells in vitro and on endothelial cell network formation in the co-culture system were determined by 3 H-thymidine incorporation assay and Matrigel network formation assay, respectively. Results The xerograft NOD/SCID animal model had high capacity for growth of RPMI 8226 subcutaneous tumors and presented pathologic features of plasmacytomas. After subcutaneous injection of RPMI 8226 cells, all mice developed localized tumors in （20 ±2） d. On 20 μg anti-BDNF MoAb 3 consecutive treatment, the mean tumor-free time was extended to （30 ±6） d and survival was significantly prolonged compared with IgG-treated group [ （57 ±7 ） d vs （48 ±4 ） d, （ P 〈 0. 05 ]. When mice died naturally, the tumors size in anti-BDNF MoAb treated ones was also reduced compared with control group [ （ 157.9 ± 21.6 ） mm^3 vs （405.5±35.2 ） mm^3, P 〈 0. 05 ]. When the antibody treatment （ 100 μg/mouse） underwent from 27 th to 60 day once a week after tumor inoculation, the local tumor growth was inhibited partially and necrosis and infiltration were observed in the tumors. The median MVD in the antibody-treated mice （ 100 μg/mouse） was 11 vessels/0. 216 mm^2. The IgG treated mice had no decrease in MVD of subcutaneous tumors compared with untreated mice. In vitro, anti-BDNF MoAb （ 1.5 μg/ml） significantly but partially inhibited HUVEC network formation induced by RPMI 8226 （68.2% reduction） and significantly inhibited RPMI 8226 proliferation, too. The IgG （ 1.5 μg/ml） treated mice had no significant effect on both of two assays. Conclusions Anti-BDNF monoclonal antibody could inhibit growth and angiogenesis in subcutaneous myeloma tumors. BDNF is a potential therapeutic target in MM.