手术切除是治疗神经母细胞瘤（儿童最常见的颅外实体恶性肿瘤）不可或缺的一部分。安全定位和切除原发肿瘤和远处的疾病沉积物仍然是一个重大挑战，导致高并发症率和不完整的手术，使结果恶化。术中分子成像 (IMI) 使用靶向放射性或荧光示踪剂在术中识别和可视化肿瘤。选择 GD2 作为 IMI 靶标，因为它在神经母细胞瘤中高度过表达，而在正常组织中表达极少。通过流式细胞术测量神经母细胞瘤细胞系中的 GD2 表达。 DTPA 和 IRDye® 800CW 与抗 GD2 抗体缀合，生成 DTPA-αGD2-IR800。然后通过 ELISA 测定测量抗体和非放射性标记示踪剂的结合亲和力 (Kd)。将人神经母细胞瘤SK-N-BE(2)细胞通过手术注射到3.5-5周龄裸鼠的左肾上腺中，原位异种移植肿瘤生长5周。通过尾静脉注射施用 111In-αGD2-IR800 或同种型对照示踪剂。 4天和6天后，对小鼠实施安乐死，使用伽马计数器测量伽马和荧光生物分布，并对切除器官（包括肿瘤、对侧肾上腺、肾脏、肝脏、肌肉、血液等）获得的 SPY-PHI 荧光图像进行 ImageJ 分析。 ）。通过单向方差分析（对每个示踪剂/天组合进行单独分析）比较器官的摄取，如果显着，则使用 Sidak 的多重比较检验来比较每个器官与肿瘤的摄取。手持式工具还用于原位检测和可视化肿瘤，并评估非引导切除后的残留疾病。每个抗体使用 0.75-2.0 DTPA 和 2-3 IRDye® 800CW 成功合成 111In-αGD2-IR800，并保留足够的量抗原结合（aGD2 的 Kd = 2.39 nM，DTPA-aGD2-IR800 的 Kd = 21.31 nM）。抗 GD2 示踪剂在具有人神经母细胞瘤异种移植物的小鼠中表现出抗原特异性摄取（抗 GD2 示踪剂在第 4 天和第 6 天的 γ 生物分布肿瘤与血液的比率分别为 3.87 和 3.88），而同种型对照示踪剂没有累积（0.414第 4 天和第 6 天为 0.514）。使用广泛使用的手术工具（Neoprobe® 和 SPY-PHI 相机）检测和可视化异种移植物中的探针积累，并有助于检测无引导切除后切除腔内的假定残留疾病。我们开发了一种基于双标记抗 GD2 抗体的示踪剂它结合了 In-111 和 IRDye® 800CW，分别用于无线电和荧光引导手术。该示踪剂与 GD2 充分结合，特异性地在表达 GD2 的异种移植肿瘤中积聚，并通过手持式近红外相机实现肿瘤可视化。这些结果鼓励开发 111In-αGD2-IR800，以供未来用于儿童神经母细胞瘤，目标是提高患者安全性、切除的完整性和患者的总体预后。© 2024。作者。

Surgical resection is integral for the treatment of neuroblastoma, the most common extracranial solid malignancy in children. Safely locating and resecting primary tumor and remote deposits of disease remains a significant challenge, resulting in high rates of complications and incomplete surgery, worsening outcomes. Intraoperative molecular imaging (IMI) uses targeted radioactive or fluorescent tracers to identify and visualize tumors intraoperatively. GD2 was selected as an IMI target, as it is highly overexpressed in neuroblastoma and minimally expressed in normal tissue.GD2 expression in neuroblastoma cell lines was measured by flow cytometry. DTPA and IRDye® 800CW were conjugated to anti-GD2 antibody to generate DTPA-αGD2-IR800. Binding affinity (Kd) of the antibody and the non-radiolabeled tracer were then measured by ELISA assay. Human neuroblastoma SK-N-BE(2) cells were surgically injected into the left adrenal gland of 3.5-5-week-old nude mice and the orthotopic xenograft tumors grew for 5 weeks. 111In-αGD2-IR800 or isotype control tracer was administered via tail vein injection. After 4 and 6 days, mice were euthanized and gamma and fluorescence biodistributions were measured using a gamma counter and ImageJ analysis of acquired SPY-PHI fluorescence images of resected organs (including tumor, contralateral adrenal, kidneys, liver, muscle, blood, and others). Organ uptake was compared by one-way ANOVA (with a separate analysis for each tracer/day combination), and if significant, Sidak's multiple comparison test was used to compare the uptake of each organ to the tumor. Handheld tools were also used to detect and visualize tumor in situ, and to assess for residual disease following non-guided resection.111In-αGD2-IR800 was successfully synthesized with 0.75-2.0 DTPA and 2-3 IRDye® 800CW per antibody and retained adequate antigen-binding (Kd = 2.39 nM for aGD2 vs. 21.31 nM for DTPA-aGD2-IR800). The anti-GD2 tracer demonstrated antigen-specific uptake in mice with human neuroblastoma xenografts (gamma biodistribution tumor-to-blood ratios of 3.87 and 3.88 on days 4 and 6 with anti-GD2 tracer), while isotype control tracer did not accumulate (0.414 and 0.514 on days 4 and 6). Probe accumulation in xenografts was detected and visualized using widely available operative tools (Neoprobe® and SPY-PHI camera) and facilitated detection ofputative residual disease in the resection cavity following unguided resection.We have developed a dual-labeled anti-GD2 antibody-based tracer that incorporates In-111 and IRDye® 800CW for radio- and fluorescence-guided surgery, respectively. The tracer adequately binds to GD2, specifically accumulates in GD2-expressing xenograft tumors, and enables tumor visualization with a hand-held NIR camera. These results encourage the development of 111In-αGD2-IR800 for future use in children with neuroblastoma, with the goal of improving patient safety, completeness of resection, and overall patient outcomes.© 2024. The Author(s).