Introduction Complete resection is the only possible treatment for cholangiocarcinoma in the extrahepatic biliary tree (eCCA), although current imaging modalities are limited in their ability to accurately diagnose longitudinal spread. We aimed to develop fluorescence imaging techniques for real-time identification of eCCA using an enzyme-activatable probe, which emits fluorescence immediately after activation by a cancer-specific enzyme. Methods Using lysates and small tissue fragments collected from surgically resected specimens, we selected the most specific probe for eCCA from among 800 enzyme-activatable probes. The selected probe was directly sprayed onto resected specimens and fluorescence images were acquired; these images were evaluated for diagnostic accuracy. We also comprehensively searched for enzymes that could activate the probe, then compared their expression levels in cancer and non-cancer tissues. Results Analyses of 19 samples (four cancer lysates, seven non-cancer lysates, and eight bile samples) and 54 tissue fragments (13 cancer tissues and 41 non-cancer tissues) revealed that PM-2MeSiR was the most specific fluorophore for eCCA. Fluorescence images of seven patients were obtained; these images enabled rapid identification of cancerous regions, which closely matched histopathology findings in four patients. Puromycin-sensitive aminopeptidase was identified as the enzyme that might activate the probe, and its expression was upregulated in eCCA. Discussion/Conclusion Fluorescence imaging with PM-2MeSiR, which may be activated by puromycin-sensitive aminopeptidase, yielded generally high accuracy. This technique may be useful for real-time identification of the spread of eCCA during surgery and endoscopic examinations.


中文翻译：

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使用酶激活探针进行实时荧光成像识别肝外胆管树中的胆管癌


介绍 完全切除是肝外胆管树（eCCA）胆管癌唯一可能的治疗方法，尽管目前的成像方式准确诊断纵向扩散的能力有限。我们的目标是开发荧光成像技术，使用酶激活探针实时识别 eCCA，该探针在被癌症特异性酶激活后立即发出荧光。 方法 使用从手术切除的标本中收集的裂解物和小组织碎片，我们从 800 个酶激活探针中选择了 eCCA 最具特异性的探针。将选定的探针直接喷射到切除的标本上并获取荧光图像；对这些图像的诊断准确性进行了评估。我们还全面寻找可以激活探针的酶，然后比较它们在癌症和非癌症组织中的表达水平。 结果 对 19 个样本（4 个癌症裂解物、7 个非癌症裂解物和 8 个胆汁样本）和 54 个组织碎片（13 个癌症组织和 41 个非癌症组织）的分析表明，PM-2MeSiR 是 eCCA 最具特异性的荧光团。获得7名患者的荧光图像；这些图像能够快速识别癌变区域，这与四名患者的组织病理学结果非常匹配。嘌呤霉素敏感氨肽酶被鉴定为可能激活探针的酶，并且其表达在 eCCA 中上调。 讨论/结论 PM-2MeSiR 的荧光成像可被嘌呤霉素敏感的氨肽酶激活，通常具有较高的准确度。该技术可能有助于在手术和内窥镜检查过程中实时识别 eCCA 的扩散。