利用双标记抗体在预临床小鼠模型中进行人类胶质母细胞瘤的术前PET成像及荧光引导手术：一种治疗-诊断结合策略

基础：胶质母细胞瘤（GBM）由于其异质性和侵袭性，导致完全切除面临巨大挑战，强调了有效治疗方案的必要性。在过去二十年中，荧光引导手术（FGS）使用如5-氨基乙酰丙酸（5-ALA）等荧光染料以增强肿瘤界限的能力，受到神经外科医生的关注。然而，一些低级别肿瘤未能显示示踪剂的积累，患者分层的缺失成为一个重要限制。自2000年以来，血管紧张素轴在癌症进展中的作用被广泛研究。特别是，我们团队已鉴定出在胶质母细胞瘤干细胞中过度表达的血管紧张素A受体（ETA），作为GBM成像的潜在靶点。本研究旨在评价一种新型双模态成像剂[89Zr]Zr-axiRA63-MOMIP的疗诊结合效果，用于：i）检测人类GBM正位模型中的ETA+细胞；ii）实现完整的肿瘤切除。方法：含有荧光染料（IRDye800CW）和正电子发射放射金属配体（脱铁血红素酶B，DFO）的单分子多模态成像平台（MOMIP）被偶联到靶向ETA受体的axiRA63抗体，该受体在GBM干细胞表面过度表达。在接受正位放射性核素[89Zr]Zr-axiRA63-MOMIP的小鼠模型中，经过48小时的成像，包括正电子发射断层扫描（PET）和光学成像。随后进行了死后证实的FGS及脑组织的离体分析（H&E染色、自动放射成像、连续块面成像），以评估PET与荧光信号的相关性。结果：[89Zr]Zr-axiRA63-MOMIP的PET成像成功检测到正位模型中ETA+细胞。术中光学成像实现了几乎完全的肿瘤切除，并通过H&E染色确认残留肿瘤细胞伴随弱荧光信号（长时间曝光后观察到）。两种成像信号之间表现出良好的定性相关性。结论：使用[89Zr]Zr-axiRA63-MOMIP提供了一种有效的治疗-诊断结合策略，用于在预临床小鼠模型中检测和切除GBM。PET与荧光信号的高度相关性有助于患者分层，该双模态试剂具有很大的临床转化潜力，优于已在临床使用的非靶向性荧光剂。

Rationale: Glioblastoma (GBM) poses significant challenges regarding complete tumor removal due to its heterogeneity and invasiveness, emphasizing the need for effective therapeutic options. In the last two decades, fluorescence-guided surgery (FGS), employing fluorophores such as 5-aminolevulinic acid (5-ALA) to enhance tumor delineation, has gained attraction among neurosurgeons. However, some low-grade tumors do not show any accumulation of the tracers, and the lack of patient stratification represents an important limitation. Since 2000, endothelin axis has been extensively investigated for its role in cancer progression. More specifically, our team has identified endothelin A receptors (ETA), overexpressed in glioblastoma cancer stem cells, as a target of interest for GBM imaging. This study aims to evaluate the efficacy of a novel preclinical bimodal imaging agent, [89Zr]Zr-axiRA63-MOMIP, as a theranostic approach to: i) detect ETA + cells in an orthotopic model of human GBM, ii) achieve complete tumoral resection. Methods: Monomolecular multimodal imaging platform (MOMIP) - containing both a fluorophore (IRDye800CW) and a chelator for a positron-emitting radiometal (desferroxamine B, DFO) - was conjugated to the axiRA63 antibody targeting ETA receptors, overexpressed on the surface of GBM stem cells. Mice bearing orthotopic human GBM were imaged 48 h post injection of [89Zr]Zr-axiRA63-MOMIP via positron emission tomography (PET) and optical imaging. Subsequently, post-mortem proof-of-concept FGS was implemented as well as ex vivo analyses (H&E staining, autoradiography, serial block face imaging) on brains with resected or unresected tumor to assess the correlation between PET and fluorescence signals. Results: PET imaging of [89Zr]Zr-axiRA63-MOMIP enabled a clear detection of ETA + cells in an orthotopic model of human GBM. Intraoperative optical imaging allowed a near-complete tumor resection together with the visualization of a weak fluorescence signal, after a prolonged exposure time, that was attributed to residual tumor cells via H&E staining. Besides, a qualitative correlation between the signals of both modalities was observed. Conclusions: The use of [89Zr]Zr-axiRA63-MOMIP provides an effective theranostic approach to detect and treat GBM by surgery in a preclinical mouse model. Thanks to the high correlation between PET and fluorescence signal allowing patients stratification, this bimodal agent should have a great potential for clinical translation and should present a significant advantage over non-targeted fluorophores already used in the clinic.