Abstract
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Introduction: Lymph node metastasis (LNM) is one of the most important prognostic factors in esophageal squamous cell carcinoma (ESCC) [1]. The esophageal lymphatic system is connected transversally and longitudinally; therefore, the pattern of LNM is pretty complex. Early-stage ESCC frequently exhibits lymph nodes skipped metastasis [2], using sentinel node navigation for surgery cannot be performed. How extensional for lymph node dissection depending on how far away the metastatic lymph nodes can be detected. Preclinical models of LNM in mice are often used for the study of certain tumors with LNM. CXCR4 is a biomarker highly expressed in ESCC, and numerous studies have demonstrated that CXCR4 is involved in the LNM of ESCC [3]. PET molecular imaging is a non-invasive visualization method based on molecular recognition at the in vivo level, with high sensitivity and specificity, which can tracer metastatic lymph nodes. Therefore, in this study, a PET molecular probe [64Cu]NOTA-CP01, which specifically targets CXCR4, was used to visualize LNM in mice bearing squamous esophageal carcinoma and to investigate how far away the metastatic lymph node can be detected.
Methods: EC109 human esophageal squamous cancer cells (EC109/Luc), stably expressing the firefly luciferase reporter gene, were used. The LNM model was established by injecting EC109/Luc cells at a concentration of 2 × 105 cells/25 μl into the left paw pads of mice. LNM were monitored using an in vivo bioluminescence imaging system (IVIS) after intraperitoneal injection of D-luciferin. Synthesis and radiolabeling of [64Cu]NOTA-CP01 has already been described in our group study previously [4]. The PET/CT imaging in mice for [64Cu]NOTA-CP01 was performed analogously to the PET/CT protocol previously [4]. Further quantitative analysis was performed. Primary tumor tissues and metastatic lymph nodes were collected using HE staining to verify metastatic tumor cells. The expression levels of CXCR4 in metastatic lymph nodes were determined using immunohistochemistry.
Results: Approximately 50 days after implantation of EC109/Luc cells in the left paw pad, we palpated an enlarged lymph node measuring about 3×3 mm in the left inguinal region and monitored a fluorescent signal in the left inguinal region after intraperitoneal injection of D-luciferin, tentatively indicating metastatic lymph nodes in the groin. PET imaging detected radioactive high uptake signals in inguinal MLN 6 h after tail intravenous of [64Cu]NOTA-CP01. Further quantitative analysis showed that the SUV of the inguinal lymph node was 1.2 ± 0.05 (VS muscle, P<0.05). To further verify lymph node metastases, an autopsy search revealed enlarged lymph nodes in the groin. Further pathological tissue staining confirmed metastatic tumor cells and CXCR4 expression levels in the lymph nodes. Preliminary results show that inguinal metastatic lymph nodes can be detected using the PET molecular probe [64Cu]NOTA-CP01. More sites of metastatic lymph nodes of ESCC need to be verified in our further studies.
Conclusions: In this study, in vivo biological experiments showed that inguinal metastatic lymph nodes of ESCC in mice can be detected by the probe [64Cu]NOTA-CP01. In all, a noninvasive method for visualization of metastatic lymph nodes in ESCC was explored.