Monitoring of In Vivo Function of Superparamagnetic Iron Oxide Labelled Murine Dendritic Cells during Anti-Tumour Vaccination

Dendritic cells (DCs) generated in vitro to present tumour antigens have been injected in cancer patients to boost in vivo anti-tumour immune responses. This approach to cancer immunotherapy has had limited success. For anti-tumour therapy, delivery and subsequent migration of DCs to lymph nodes leading to effective stimulation of effector T cells is thought to be essential. The ability to non-invasively monitor the fate of adoptively transferred DCs in vivo using magnetic resonance imaging (MRI) is an important clinical tool to correlate their in vivo behavior with response to treatment. Previous reports of superparamagnetic iron oxides (SPIOs) labelling of different cell types, including DCs, have indicated varying detrimental effects on cell viability, migration, differentiation and immune function. Here we describe an optimised labelling procedure using a short incubation time and low concentration of clinically used SPIO Endorem to successfully track murine DC migration in vivo using MRI in a mouse tumour model. First, intracellular labelling of bone marrow derived DCs was monitored in vitro using electron microscopy and MRI relaxometry. Second, the in vitro characterisation of SPIO labelled DCs demonstrated that viability, phenotype and functions were comparable to unlabelled DCs. Third, ex vivo SPIO labelled DCs, when injected subcutaneously, allowed for the longitudinal monitoring by MR imaging of their migration in vivo. Fourth, the SPIO DCs induced the proliferation of adoptively transferred CD4+ T cells but, most importantly, they primed cytotoxic CD8+ T cell responses to protect against a B16-Ova tumour challenge. Finally, using anatomical information from the MR images, the immigration of DCs was confirmed by the increase in lymph node size post-DC injection. These results demonstrate that the SPIO labelling protocol developed in this study is not detrimental for DC function in vitro and in vivo has potential clinical application in monitoring therapeutic DCs in patients with cancer.树突状细胞（Dendritic cells, DCs）经体外诱导以呈递肿瘤抗原后，已被注射入癌症患者体内以激活体内抗肿瘤免疫应答。该癌症免疫治疗策略的临床疗效较为有限。在抗肿瘤治疗中，树突状细胞的递送及后续迁移至淋巴结以有效激活效应T细胞，被认为是治疗成功的关键环节。利用磁共振成像（magnetic resonance imaging, MRI）无创监测过继转移树突状细胞的体内命运的能力，是将其体内行为与治疗应答相关联的重要临床手段。既往采用超顺磁性氧化铁（superparamagnetic iron oxides, SPIOs）标记包括树突状细胞在内的多种细胞的研究显示，其对细胞活力、迁移能力、分化状态及免疫功能存在不同程度的负面影响。本文描述了一种优化的标记方案，即采用短孵育时间与低浓度的临床用超顺磁性氧化铁制剂Endorem，可在小鼠肿瘤模型中通过MRI成功追踪鼠源树突状细胞的体内迁移过程。首先，我们通过电子显微镜与MRI弛豫测定法，在体外监测了骨髓衍生树突状细胞的胞内标记情况。其次，对超顺磁性氧化铁标记树突状细胞的体外表征结果显示，其活力、表型与功能均与未标记树突状细胞无显著差异。第三，将体外制备的超顺磁性氧化铁标记树突状细胞皮下注射后，可通过MRI成像纵向追踪其体内迁移过程。第四，该超顺磁性氧化铁标记的树突状细胞可诱导过继转移的CD4+ T细胞增殖，而最为关键的是，其可激活细胞毒性CD8+ T细胞应答，从而抵御B16-Ova肿瘤的攻击。最后，借助MRI图像的解剖学信息，树突状细胞注射后淋巴结体积增大的现象证实了其迁移定植过程。本研究开发的超顺磁性氧化铁标记方案对树突状细胞的体外与体内功能均无不良影响，在癌症患者治疗性树突状细胞的临床监测中具有潜在应用价值。