肿瘤细胞治疗研究
英文原题：A multimodal imaging workflow for monitoring CAR T cell therapy against solid tumor from whole-body to single-cell level.
方法：携带皮下 AsPC1 异种移植瘤的 NSG 小鼠接受 EGFR CAR T 细胞（IL-2）或对照 BDCA-2 CAR T 细胞（IL-2）治疗（每组 n = 7）。
实体瘤CAR-T研究常缺少时空信息，因此需要分子断层成像，以支持高通量临床前监测CAR-T细胞。此外，为更好评估治疗细胞在肿瘤内的浸润，宏观和微观成像数据之间也存在衔接不足。本研究结合三维计算机断层生物发光断层成像（CT/BLT）、光片荧光显微镜（LSFM）和循环免疫荧光（IF）染色来解决这一问题。 方法：将皮下携带AsPC1异种移植瘤的NSG小鼠分为两组，分别接受EGFR CAR-T细胞（IL-2）或对照BDCA-2 CAR-T细胞（IL-2）治疗，每组7只。治疗性T细胞经基因改造后共同表达目标CAR和荧光素酶CBR2opt。治疗开始后第1、3、5和7天，在异种移植瘤皮下注射IL-2，每只小鼠剂量25,000 IU。每3至4天使用二维生物发光成像（BLI）和三维CT/BLT测量CAR-T分布。第6天切除4个肿瘤进行循环IF，用25种抗体染色组织切片；第6及第13天从预先注射罗丹明凝集素的小鼠切除8个肿瘤，经通透处理、CD3染色后使用LSFM成像。 结果：三维CT/BLT显示，CAR-T药代动力学受抗原识别影响；与非靶标依赖性浸润相比，靶标依赖性浸润显著增加CAR-T在肿瘤中的积累，并延迟其在脾脏积累。LSFM支持了这些发现：第6天靶标阳性组肿瘤内T细胞积累更多，且浸润更深。有趣的是，多数CAR-T细胞聚集在肿瘤边缘及血管周围。值得注意的是，LSFM和循环IF显示，局部给予IL-2可在早期增加CAR-T增殖，但长期过度刺激会停止早期获得的治疗获益。 结论：我们证明三维CT/BLT是一种有价值的非同位素全身成像技术，可监测细胞治疗并研究CAR-T药代动力学。我们还展示了结合LSFM和多重循环免疫荧光（MICS）进行离体三维及二维显微组织分析的方法，可用于评估肿瘤内治疗细胞分布和状态。
CAR T cell research in solid tumors often lacks spatiotemporal information and therefore, there is a need for a molecular tomography to facilitate high-throughput preclinical monitoring of CAR T cells. Furthermore, a gap exists between macro- and microlevel imaging data to better assess intratumor infiltration of therapeutic cells. We addressed this challenge by combining 3D Computer tomography bioluminescence tomography ( CT/BLT), light-sheet fluorescence microscopy (LSFM) and cyclic immunofluorescence (IF) staining. Methods: NSG mice with subcutaneous AsPC1 xenograft tumors were treated with EGFR CAR T cell ( IL-2) or control BDCA-2 CAR T cell ( IL-2) (n = 7 each). Therapeutic T cells were genetically modified to co-express the CAR of interest and the luciferase CBR2opt. IL-2 was administered s.c. under the xenograft tumor on days 1, 3, 5 and 7 post-therapy-initiation at a dose of 25,000 IU/mouse. CAR T cell distribution was measured in 2D BLI and 3D CT/BLT every 3-4 days. On day 6, 4 tumors were excised for cyclic IF where tumor sections were stained with a panel of 25 antibodies. On day 6 and 13, 8 tumors were excised from rhodamine lectin-preinjected mice, permeabilized, stained for CD3 and imaged by LSFM. Results: 3D CT/BLT revealed that CAR T cells pharmacokinetics is affected by antigen recognition, where CAR T cell tumor accumulation based on target-dependent infiltration was significantly increased in comparison to target-independent infiltration, and spleen accumulation was delayed. LSFM supported these findings and revealed higher T cell accumulation in target-positive groups at day 6, which also infiltrated the tumor deeper. Interestingly, LSFM showed that most CAR T cells accumulate at the tumor periphery and around vessels. Surprisingly, LSFM and cyclic IF revealed that local IL-2 application resulted in early-phase increased proliferation, but long-term overstimulation of CAR T cells, which halted the early added therapeutic effect. Conclusion: Overall, we demonstrated that 3D CT/BLT is a valuable non-isotope-based technology for whole-body cell therapy monitoring and investigating CAR T cell pharmacokinetics. We also presented combining LSFM and MICS for ex vivo 3D- and 2D-microscopy tissue analysis to assess intratumoral therapeutic cell distribution and status.
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