Miltenyi Biotec B.V. & Co. KG, R&D Reagents, Bergisch Gladbach, North Rhine-Westphalia, Germany.
University Medical Center Göttingen, Translational Molecular Imaging, Institute for Diagnostic and Interventional Radiology & Clinic for Haematology and Medical Oncology, Göttingen, Lower Saxony, Germany.
Theranostics. 2022 Jun 13;12(11):4834-4850. doi: 10.7150/thno.68966. eCollection 2022.
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 µComputer tomography bioluminescence tomography (µCT/BLT), light-sheet fluorescence microscopy (LSFM) and cyclic immunofluorescence (IF) staining. 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 µCT/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. 3D µCT/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. Overall, we demonstrated that 3D µCT/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 3D- and 2D-microscopy tissue analysis to assess intratumoral therapeutic cell distribution and status.
实体瘤 CAR T 细胞研究通常缺乏时空信息，因此需要一种分子层析技术来促进 CAR T 细胞的高通量临床前监测。此外，宏观和微观成像数据之间存在差距，无法更好地评估治疗细胞在肿瘤内的浸润情况。我们通过结合三维μ计算机断层扫描生物发光断层扫描（μ CT/BLT）、光片荧光显微镜（LSFM）和循环免疫荧光（IF）染色来解决这一挑战。将皮下接种 AsPC1 异种移植肿瘤的 NSG 小鼠用 EGFR CAR T 细胞（± IL-2）或对照 BDCA-2 CAR T 细胞（± IL-2）治疗（每组 n = 7）。治疗性 T 细胞经过基因修饰，共同表达感兴趣的 CAR 和荧光素酶 CBR2opt。IL-2 于治疗开始后第 1、3、5 和 7 天每天经皮下注射到异种移植肿瘤下，剂量为 25000 IU/只。每隔 3-4 天通过 2D BLI 和 3D μ CT/BLT 测量 CAR T 细胞的分布。第 6 天，取出 4 个肿瘤进行循环 IF，其中肿瘤切片用一组 25 种抗体进行染色。第 6 天和第 13 天，从罗丹明凝集素预处理的小鼠中取出 8 个肿瘤，进行透化、CD3 染色并通过 LSFM 成像。3D μ CT/BLT 显示，CAR T 细胞药代动力学受抗原识别的影响，基于靶依赖性浸润的 CAR T 细胞肿瘤积累与靶非依赖性浸润相比显著增加，脾脏积累延迟。LSFM 支持这些发现，并显示出在第 6 天靶阳性组中 T 细胞的积累更高，并且也更深地浸润肿瘤。有趣的是，LSFM 显示大多数 CAR T 细胞聚集在肿瘤边缘和血管周围。令人惊讶的是，LSFM 和循环 IF 显示局部 IL-2 应用导致早期增殖增加，但长期过度刺激 CAR T 细胞，从而停止了早期的附加治疗效果。总体而言，我们证明了 3D μ CT/BLT 是一种有价值的非同位素基技术，可用于全身细胞治疗监测和研究 CAR T 细胞药代动力学。我们还介绍了将 LSFM 和 MICS 结合用于 3D 和 2D 显微镜组织分析，以评估肿瘤内治疗性细胞的分布和状态。
