Detection of epidermal growth factor receptor expressing lung squamous cell carcinoma by activatable fluorescence imaging

Background: Detection of mediastinal lymph node metastases in patients with lung cancer is important in reducing risks of cancer recurrence and poor prognosis. We employed the activatable fluorescence imaging (FLI) technique to detect mediastinal epidermal growth factor receptor (EGFR)-positive lymph nodes in this preclinical study. Activatable FLI enables only metastatic lymph nodes to be visualized as bright lesions, while quenched fluorescence dye shows no signals. Indocyanine green (ICG) that emits fluorescence at NIR excitation wavelengths was chosen for the activatable probe in this study. Due to its long history of clinical use, ICG is a preferable fluorescence dye for future clinical applications. Video-assisted thoracic surgery (VATS) is a widely available procedure in thoracic surgery that utilizes a charge coupled device (CCD) camera that can visualize images with visible light. Near infrared (NIR) light has an advantage in better penetration of tissues. FLI using NIR enables lesions in deep areas, such as mediastinal lymph node metastases, to be detected by FLI. Potentially VATS operation can eliminate the need for rapid pathological diagnosis during the operation.

Objectives: We investigated the feasibility of the application of activatable FLI to detect and visualize mediastinal lymph node metastases of EGFR-positive lung squamous cell carcinoma (LSCC).

Methods: ICG was conjugated to the monoclonal antibody panitumumab (Pan) with the ratio of 4:1. The quenching ability of the conjugate was evaluated by 1% SDS treatment. LSCC EGFR-expressing (H226) and non-expressing (H520) cell lines were used. Fluorescence microscopy examination and flow cytometry were performed to certificate the conjugate’s specificity in binding to EGFR and its activatable ability. To make mediastinal lymph node metastases, H226 and H520 cell lines were separately injected to five mice each, with direct injection of the cells into the lung parenchymal tissue at 5 mm depth from the skin. The size of lung tumors was examined by CT scan every week. When lung tumors were visually recognized and their sizes became greater than 8 mm, Pan-ICG (50 μg/100 μl) was injected through the tail vein. Forty-eight hours after the injection, the mice were sacrificed and their chests were opened for fluorescent imaging acquisition. Lymph node metastases with five highest fluorescent signal intensities in each mouse were chosen for the statistical analyses of the average fluorescence signal (AFS) ratios of the lymph node metastasis to the liver.

Results: With regard to the quenching capacity of Pan-ICG, the conjugate had almost no fluorescence in PBS, but after 1% SDS treatment, a 20-fold increase in fluorescent signals was detected in vitro. Both fluorescent microscopy study and flow cytometry showed the specific binding between the conjugate and H226, but almost no specific binding with H520. The EGFR-positive mediastinal lymph node metastases (n = 25) showed significantly higher AFS ratios than the EGFR-negative ones (n = 25) 48 hours after the conjugate administration (70.1 ± 4.5% v.s. 13.3 ± 1.8%; p< 0.05).

Conclusions: The activatable FLI using Pan-ICG was able to detect EGFR-positive mediastinal lymph node metastases with high specificity.