在肺癌手术过程中，明确肿瘤边界、确定手术切缘距离非常重要。在以往的研究中，荧光探针的系统应用可以帮助医疗专业人员确定肿瘤的边界，发现小肿瘤和转移灶，从而提高手术切除的准确性。然而，目前能够应用于临床试验的安全有效的探针还很少，这限制了荧光成像的临床应用。这里我们开发了一种新技术，可以在手术过程中快速识别切除的肺组织中的肿瘤区域，并区分肿瘤边界和转移淋巴结。

对于动物研究，建立了肺癌 PDX 模型。通过手术切除荷瘤小鼠的肿瘤、肺部和瘤周肌肉组织，并与靶向表皮生长因子受体（EGFR）的探针一起孵育20分钟，然后通过闭场近红外二区（NIR）成像-II) 荧光成像系统。对于临床样本，10例接受根治性切除的肺癌患者的10例手术切除的肺组织和60个淋巴结在术中切除后立即与靶向探针一起孵育并成像以识别肿瘤区域并区分肿瘤边界和转移淋巴结。通过HE染色和免疫组化证实荧光成像的准确性。

离体动物成像实验显示肿瘤组织荧光增强。对于临床样本，我们的结果表明，这项新技术在识别切除肺组织中的肿瘤区域方面具有超过 85.7% 的敏感性和 100% 的特异性。平均荧光肿瘤与背景比为 2.5 ± 1.3。此外，我们还在术中使用该技术对转移淋巴结进行成像，结果显示转移淋巴结比正常淋巴结具有更亮的荧光，平均荧光肿瘤与背景信号比为2.7±1.1。对与转移淋巴结数量相关的荧光信号结果的计算得出灵敏度为 77.8%，特异性为 92.1%。我们期望这项新技术成为快速术中病理检测和切缘确定的有用诊断工具。

通过使用荧光标记的抗人EGFR重组抗体scFv片段在术中孵育新鲜分离的组织，探针可以在肺癌组织中快速积累，可用于快速识别切除肺组织中的肿瘤区域，区分肿瘤边界并发现肿瘤。淋巴结转移。该技术有望用于术中快速病理检测和切缘确定。

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Purpose

During lung cancer surgery, it is very important to define tumor boundaries and determine the surgical margin distance. In previous research, systemically application of fluorescent probes can help medical professionals determine the boundaries of tumors and find small tumors and metastases, thereby improving the accuracy of surgical resection. However, there are very few safe and effective probes that can be applied to clinical trials up to now, which limits the clinical application of fluorescence imaging. Here we developed a new technology that can quickly identify the tumor area in the resected lung tissue during the operation and distinguish the tumor boundary and metastatic lymph nodes.

For animal studies, a PDX model of lung cancer was established. The tumors, lungs, and peritumoral muscle tissues of tumor-bearing mice were surgically removed and incubated with a probe targeting epidermal growth factor receptor (EGFR) for 20 min, and then imaged by a closed-field near-infrared two-zone (NIR-II) fluorescence imaging system. For clinical samples, ten surgically removed lung tissues and 60 lymph nodes from 10 lung cancer patients undergoing radical resection were incubated with the targeting probe immediately after intraoperative resection and imaged to identify the tumor area and distinguish the tumor boundary and metastatic lymph nodes. The accuracy of fluorescence imaging was confirmed by HE staining and immunohistochemistry.

The ex vivo animal imaging experiments showed a fluorescence enhancement of tumor tissue. For clinical samples, our results showed that this new technology yielded more than 85.7% sensitivity and 100% specificity in identifying the tumor area in the resected lung tissue. The average fluorescence tumor-to-background ratio was 2.5 ± 1.3. Furthermore, we also used this technique to image metastatic lymph nodes intraoperatively and showed that metastatic lymph nodes have brighter fluorescence than normal lymph nodes, as the average fluorescence tumor-to-background signal ratio was 2.7 ± 1.1. Calculations on the results of the fluorescence signal in relation to the number of metastatic lymph nodes yielded values of 77.8% for sensitivity and 92.1% for specificity. We expect this new technology to be a useful diagnostic tool for rapid intraoperative pathological detection and margin determination.

By using fluorescently labeled anti-human EGFR recombinant antibody scFv fragment to incubate freshly isolated tissues during surgery, the probes can quickly accumulate in lung cancer tissues, which can be used to quickly identify tumor areas in the resected lung tissues and distinguish tumor boundaries and find metastases in lymph nodes. This technology is expected to be used for rapid intraoperative pathological detection and margin determination.