Cell Therapy Program, Princess Margaret Hospital, 610 University Ave, Toronto, ON, Canada.
Haematologica. 2012 Jul;97(7):1020-8. doi: 10.3324/haematol.2011.054254. Epub 2012 Jan 22.
BACKGROUND: Novel therapies capable of targeting drug resistant clonogenic MM cells are required for more effective treatment of multiple myeloma. This study investigates the cytotoxicity of natural killer cell lines against bulk and clonogenic multiple myeloma and evaluates the tumor burden after NK cell therapy in a bioluminescent xenograft mouse model. DESIGN AND METHODS: The cytotoxicity of natural killer cell lines was evaluated against bulk multiple myeloma cell lines using chromium release and flow cytometry cytotoxicity assays. Selected activating receptors on natural killer cells were blocked to determine their role in multiple myeloma recognition. Growth inhibition of clonogenic multiple myeloma cells was assessed in a methylcellulose clonogenic assay in combination with secondary replating to evaluate the self-renewal of residual progenitors after natural killer cell treatment. A bioluminescent mouse model was developed using the human U266 cell line transduced to express green fluorescent protein and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow engraftment after intravenous NK-92 cell therapy. RESULTS: Three multiple myeloma cell lines were sensitive to NK-92 and KHYG-1 cytotoxicity mediated by NKp30, NKp46, NKG2D and DNAM-1 activating receptors. NK-92 and KHYG-1 demonstrated 2- to 3-fold greater inhibition of clonogenic multiple myeloma growth, compared with killing of the bulk tumor population. In addition, the residual colonies after treatment formed significantly fewer colonies compared to the control in a secondary replating for a cumulative clonogenic inhibition of 89-99% at the 20:1 effector to target ratio. Multiple myeloma tumor burden was reduced by NK-92 in a xenograft mouse model as measured by bioluminescence imaging and reduction in bone marrow engraftment of U266eGFPluc cells by flow cytometry. CONCLUSIONS: This study demonstrates that NK-92 and KHYG-1 are capable of killing clonogenic and bulk multiple myeloma cells. In addition, multiple myeloma tumor burden in a xenograft mouse model was reduced by intravenous NK-92 cell therapy. Since multiple myeloma colony frequency correlates with survival, our observations have important clinical implications and suggest that clinical studies of NK cell lines to treat MM are warranted.
背景：需要新型治疗方法来靶向耐药克隆性骨髓瘤细胞，以更有效地治疗多发性骨髓瘤。本研究旨在调查自然杀伤细胞系对多发性骨髓瘤细胞系的细胞毒性，并在生物发光异种移植小鼠模型中评估 NK 细胞治疗后的肿瘤负担。
设计与方法：使用铬释放和流式细胞术细胞毒性测定法评估自然杀伤细胞系对多发性骨髓瘤细胞系的细胞毒性。阻断自然杀伤细胞上的选定激活受体以确定它们在多发性骨髓瘤识别中的作用。在甲基纤维素集落形成测定中评估克隆性多发性骨髓瘤细胞的生长抑制作用，结合二次再种植以评估 NK 细胞治疗后残留祖细胞的自我更新。使用转导表达绿色荧光蛋白和荧光素酶的人 U266 细胞系（U266eGFPluc）开发生物发光小鼠模型，以监测体内疾病进展并评估静脉注射 NK-92 细胞治疗后的骨髓植入。
结果：三种多发性骨髓瘤细胞系对 NK-92 和 KHYG-1 介导的 NKp30、NKp46、NKG2D 和 DNAM-1 激活受体的细胞毒性敏感。与杀伤肿瘤群体相比，NK-92 和 KHYG-1 对克隆性多发性骨髓瘤生长的抑制作用要强 2-3 倍。此外，与对照组相比，在二次再种植中形成的剩余集落数量明显减少，在 20:1 的效应器与靶标比时，累积集落抑制率为 89-99%。通过生物发光成像和流式细胞术测量 U266eGFPluc 细胞在骨髓中的植入减少，NK-92 在异种移植小鼠模型中减少多发性骨髓瘤肿瘤负担。
结论：本研究表明，NK-92 和 KHYG-1 能够杀死克隆性和多发性骨髓瘤细胞。此外，静脉注射 NK-92 细胞治疗可减少异种移植小鼠模型中的多发性骨髓瘤肿瘤负担。由于多发性骨髓瘤集落频率与生存相关，我们的观察结果具有重要的临床意义，并表明 NK 细胞系治疗 MM 的临床研究是合理的。
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