Research Article: Activatable fluorescence detection of epidermal growth factor receptor positive mediastinal lymph nodes in murine lung cancer model

Date Published: June 1, 2018

Publisher: Public Library of Science

Author(s): Xieyi Zhang, Takahito Nakajima, Mai Kim, Aiko Yamaguchi, Oyunbold Lamid-Ochir, Huong Nguyen-Thu, Anu Bhattarai, Hirofumi Hanaoka, Yoshito Tsushima, Aamir Ahmad.


It is important to detect mediastinal lymph node metastases in patients with lung cancer to improve outcomes, and it is possible that activatable fluorescence imaging with indocyanine green (ICG) can help visualize metastatic lymph nodes. Therefore, we investigated the feasibility of applying this method to mediastinal lymph node metastases in an epidermal growth factor receptor (EGFR)-positive squamous cell carcinoma of the lung. Tumors were formed by injecting H226 (EGFR-positive) and H520 (EGFR-negative) cell lines directly in the lung parenchyma of five mice each. When computed tomography revealed tumors exceeding 8 mm at their longest or atelectasis that occupied more than half of lateral lung fields, a panitumumab (Pan)–ICG conjugate was injected in the tail vein (50 μg/100 μL). The mice were then sacrificed 48 hours after injection and their chests were opened for fluorescent imaging acquisition. Lymph node metastases with the five highest fluorescent signal intensities per mouse were chosen for statistical analysis of the average signal ratios against the liver. Regarding the quenching capacity, the Pan–ICG conjugate had almost no fluorescence in phosphate-buffered saline, but there was an approximate 61.8-fold increase in vitro after treatment with 1% sodium dodecyl sulfate. Both the fluorescent microscopy and the flow cytometry showed specific binding between the conjugate and H226, but almost no specific binding with H520. The EGFR-positive mediastinal lymph node metastases showed significantly higher average fluorescence signal ratios than the EGFR-negative ones (n = 25 per group) 48 hours after conjugate administration (70.1% ± 4.5% vs. 13.3% ± 1.8%; p < 0.05). Thus, activatable fluorescence imaging using the Pan–ICG conjugate detected EGFR-positive mediastinal lymph node metastases with high specificity.

Partial Text

Lung cancer is one of the most frequently diagnosed cancers and leading causes of cancer-related death worldwide [1]. Moreover, it remains the primary cause of death from malignancy in the United States of America [2]. In addition to standard therapeutic strategies of lung cancers including surgery, chemotherapy, and radiotherapy, the molecular therapy targeting the epidermal growth factor receptor (EGFR) transmembrane glycoprotein has been developed in recent decades [3–6]. The EGFR status has mainly been evaluated by fluorescence in situ hybridization or immunohistochemistry [7], both of which require invasive procedures. The imaging modalities may also offer a more comprehensive means of evaluating EGFR status [8, 9]. Since FDG-PET can visualize the glucose metabolism, primary cancers, metastatic lesions and lymph node metastases can be detected [10, 11]. However, the residual lesions or lymph node metastases can be difficult to detect during surgery for lung cancer, which increases the risk of untreated cancer and leads to a poorer prognosis [12]. Intra-operative fluorescence-guided methods is emerging as a viable technique for the complete resection of cancer [8].

ICG itself is widely applied in clinical settings such as ophthalmic or intraoperative angiography and lymphangiography for sentinel lymph node detection where ICG binds to serum albumin, resulting in an enhancement of its fluorescence ability. Since the half-life of ICG in human body is about 3 to 4 minutes and ICG is metabolized by the liver, ICG is also clinically used as an index for liver function.

In conclusion, we have synthesized an activatable Pan-ICG conjugate and we have shown that this activatable conjugate can detect mediastinal EGFR-positive lymph node metastases of squamous cell carcinoma with such high contrast that this activatable Pan-ICG has a potential in assisting surgeons during endoscopic lung surgery.




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