Department of Surgery, School of Medicine, Keio University, Tokyo, Japan.
Department of Surgery, School of Medicine, Keio University, Tokyo, Japan.
J Surg Res. 2014 Jul;190(1):134-43. doi: 10.1016/j.jss.2014.02.047. Epub 2014 Feb 28.
Mesenchymal stem cells (MSCs) are being developed as a new clinically relevant stem cell type to be recruited into and to repair injured tissue. A number of studies have focused on the therapeutic potential of MSCs by virtue of their immunomodulatory properties. Systemically administered MSCs can also migrate to sites of malignancies. Because of this latter phenomenon, we transfected human MSCs to secrete anti-high mobility group box (HMGB) 1 proteins. They were then injected into mice bearing human colon cancer to evaluate their efficacy as an antineoplastic agent.
The ABOX gene was used in this model, which encodes part of the HMGB1 protein and acts as an HMGB1 antagonist. It was cotransduced by electroporation with a FLAG-tag to visualize the secreted ABOX protein, levels of which in supernatants from cultured transfected MSCs were quantified by immunofluorescence imaging using an anti-FLAG antibody. Antiangiogenic effects were evaluated in vitro using a novel optical assay device for the quantitative measurement of cellular chemotaxis assessing the velocity and direction of endothelial cell movement stimulated by supernatant from tumor cells. We found that ABOX proteins released from transfected MSCs suppressed migration in this assay. Finally, MSCs were injected subcutaneously into Nonobese diabetic/severe combined immunodeficiency mice bearing human colon cancer from a cell line, which secreted large amounts of HMGB1. Ten days after MSC injection, mice were sacrificed and tumors evaluated by immunohistochemistry.
From 12 ho through 7 d after gene transfection, ABOX proteins secreted from MSCs could be detected by immunofluorescence and enzyme-linked immunosorbent assay. Quantitative measurement of cellular chemotaxis demonstrated that ABOX proteins secreted from transfected MSCs decreased the velocity and interfered with the direction of movement of vascular endothelial cells. Moreover, in an in vivo human colon cancer xenograft model, injection of anti-HMGB1-transfected MSCs resulted in a decreased tumor volume due to the antiangiogenic properties of the secreted ABOX proteins.
MSC modified to secrete HMGB1 antagonist proteins have therapeutic antineoplastic potential. These findings may contribute to future novel targeting strategies using autologous bone marrow-derived cells as gene delivery vectors.
间充质干细胞（MSCs）正在被开发成为一种新的临床相关的干细胞类型，以募集和修复受损组织。许多研究都集中在 MSC 的治疗潜力上，因为它们具有免疫调节特性。全身给药的 MSC 也可以迁移到恶性肿瘤部位。由于后一种现象，我们转染了人 MSC 分泌抗高迁移率族蛋白（HMGB）1 蛋白。然后将它们注射到患有人类结肠癌的小鼠体内，以评估它们作为抗肿瘤剂的疗效。
该模型使用 ABOX 基因，其编码 HMGB1 蛋白的一部分，并且作为 HMGB1 拮抗剂。它通过电穿孔与 FLAG 标签共转导，以可视化分泌的 ABOX 蛋白，并用抗 FLAG 抗体通过免疫荧光成像定量测量培养转染 MSC 上清液中的 ABOX 蛋白水平。在体外使用一种新的光学测定装置评估抗血管生成作用，该装置用于定量测量细胞趋化性，评估由肿瘤细胞上清液刺激的内皮细胞运动的速度和方向。我们发现，从转染 MSC 释放的 ABOX 蛋白抑制了该测定中的迁移。最后，将从大量分泌 HMGB1 的细胞系中患有人类结肠癌的非肥胖型糖尿病/严重联合免疫缺陷小鼠的皮下注射 MSC。MSC 注射后 10 天，处死小鼠并通过免疫组织化学评估肿瘤。
从基因转染后的 12 小时到 7 天，通过免疫荧光和酶联免疫吸附试验可以检测到 MSC 分泌的 ABOX 蛋白。细胞趋化性的定量测量表明，从转染 MSC 分泌的 ABOX 蛋白降低了血管内皮细胞的速度并干扰了其运动方向。此外，在体内人类结肠癌异种移植模型中，由于分泌的 ABOX 蛋白的抗血管生成特性，注射抗 HMGB1 转染的 MSC 导致肿瘤体积减小。
修饰以分泌 HMGB1 拮抗剂蛋白的 MSC 具有治疗抗肿瘤潜力。这些发现可能为未来使用自体骨髓来源细胞作为基因传递载体的新型靶向策略做出贡献。
