目的:切除肿瘤引流淋巴结(TDLN)是一种标准的方法来确定转移的几种恶性肿瘤。有趣的是,最近的临床前研究表明,TDLN切除会降低基于免疫检查点抑制剂的癌症免疫疗法的疗效.因此,TDLNs的准确临床前鉴定对于揭示潜在的免疫学机制至关重要。因此,我们进行了临床前验证,和临床上可用的非侵入性体内成像方法,用于精确的TDLN识别。
方法:为了通过非侵入性体内光学成像可视化淋巴引流到TDLNs中,我们注入了光学成像造影剂专利BlueV(582.7gmol-1)和IRDye®800CW聚乙二醇(PEG;25,000-60,000gmol-1),皮下(s.c.)靠近实验小鼠右侧的MC38腺癌。为了通过非侵入性体内PET/磁共振成像(PET/MRI)确定TDLN中的淋巴引流和葡萄糖代谢,我们以类似的方式注射了正电子发射断层扫描(PET)示踪剂(2-脱氧-2[18F]氟-D-葡萄糖(18F-FDG)[181.1gmol-1])。对于离体互相关,我们分离了TDLNs和对侧非肿瘤引流淋巴结(NTDLNs)并进行了光学成像,生物分布,和放射自显影分析。
结果:与IRDye®800CWPEG相比,临床上公认的专利BlueV在TDLN的术中宏观鉴定方面优于IRDye®800CWPEG,但对于通过光学成像进行非侵入性体内检测不够灵敏。离体专利蓝V生物分布分析清楚地确定了正确的副腋窝和正确的腋窝淋巴结(LN)为TDLN,而体外IRDye®800CWPEG完全失败。相比之下,功能性非侵入性体内18F-FDGPET/MRI发现,仅在实验小鼠的同侧副腋窝TDLN内,摄取显着升高,并且能够区分副腋窝和适当的LN。离体生物分布和放射自显影证实了我们的体内18F-FDGPET/MRI结果。
结论:放在一起,我们的结果证明了18F-FDG-PET/MRI作为一种有效的方法的可行性,术中,和离体鉴定TDLN内的淋巴引流和葡萄糖代谢。此外,使用专利蓝V为目视和离体光学成像分析的淋巴引流的宏观定位提供了附加价值。因此,这两种方法都很有价值,易于实现,以及临床前鉴定TDLN的成本效益。
Resection of the tumor-draining lymph -node (TDLN) represents a standard method to identify metastasis for several malignancies. Interestingly, recent preclinical studies indicate that TDLN resection diminishes the efficacy of immune checkpoint inhibitor-based cancer immunotherapies. Thus, accurate preclinical identification of TDLNs is pivotal to uncovering the underlying immunological mechanisms. Therefore, we validated preclinically, and clinically available non-invasive in vivo imaging approaches for precise TDLN identification.
For visualization of the lymphatic drainage into the TDLNs by non-invasive in vivo optical imaging, we injected the optical imaging contrast agents Patent Blue V (582.7 g mol-1) and IRDye® 800CW polyethylene glycol (PEG; 25,000-60,000 g mol-1), subcutaneously (s.c.) in close proximity to MC38 adenocarcinomas at the right flank of experimental mice. For determination of the lymphatic drainage and the glucose metabolism in TDLNs by non-invasive in vivo PET/magnetic resonance imaging (PET/MRI), we injected the positron emission tomography (PET) tracer (2-deoxy-2[18F]fluoro-D-glucose (18F-FDG) [181.1 g mol-1]) in a similar manner. For ex vivo cross-correlation, we isolated TDLNs and contralateral nontumor-draining lymph nodes (NTDLNs) and performed optical imaging, biodistribution, and autoradiography analysis.
The clinically well-established Patent Blue V was superior for intraoperative macroscopic identification of the TDLNs compared with IRDye® 800CW PEG but was not sensitive enough for non-invasive in vivo detection by optical imaging. Ex vivo Patent Blue V biodistribution analysis clearly identified the right accessory axillary and the proper axillary lymph node (LN) as TDLNs, whereas ex vivo IRDye® 800CW PEG completely failed. In contrast, functional non-invasive in vivo 18F-FDG PET/MRI identified a significantly elevated uptake exclusively within the ipsilateral accessory axillary TDLN of experimental mice and was able to differentiate between the accessory axillary and the proper LN. Ex vivo biodistribution and autoradiography confirmed our in vivo 18F-FDG PET/MRI results.
When taken together, our results demonstrate the feasibility of 18F-FDG-PET/MRI as a valid method for non-invasive in vivo, intraoperative, and ex vivo identification of the lymphatic drainage and glucose metabolism within the TDLNs. In addition, using Patent Blue V provides additive value for the macroscopic localization of the lymphatic drainage both visually and by ex vivo optical imaging analysis. Thus, both methods are valuable, easy to implement, and cost-effective for preclinical identification of the TDLN.