Intra-operative Localization of Lymph Node Metastases Using a Replication-competent Herpes Simplex Virus
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Objective: Lymph node (LN) status is the most important prognostic factor determining the recurrence and survival in patients with mesothelioma and thoracic malignancies. Accurate localization of LN metastases in mediastinum and other LN stations is therefore necessary to improve selection of resectable and curable patients for surgery. This study investigates the potential to identify LN metastases intraoperatively using herpes-guided cancer cell-specific expression of green fluorescent protein (GFP).
Methods: Following infection with NV1066, a herpes simplex virus carrying GFP transgene; eighteen human lung, mesothelioma and esophageal cancer cell lines were assessed for cancer cell specific infection, GFP expression and viral replication. We then established murine models of lymphatic metastasis by surgical implantation of A549 / H1299 (lung) or MSTO-211H / VAMT (mesothelioma) or BE3 / SKG-T4 (esophageal) cancer cells. Cancer cells were injected into the preauricular (drainage to cervical LN), pleural (mediastinal and retroperitoneal LN) or the paraesophageal space (celiac LN) of athymic mice. Fluorescent thoracoscopy, laparoscopy and stereomicroscopy were used to localize LN metastases.
Results: In Vitro, NV1066 replicated (5 to 3000 -fold) and expressed GFP in all cancer cells even when infected at a low ratio of one viral plaque forming unit per 100 tumor cells. In vivo, NV1066 injected into primary tumors was able to transit and infect LN metastases producing GFP that was visualized by fluorescent imaging (Figure A). Histology confirmed lymphatic metastases (B) and immunohistochemistry confirmed viral presence (C) in GFP expressing regions (D). LN metastases that were not obvious on bright-field examination (E) were easily visualized by fluorescent imaging (F).
Conclusions: We introduce a novel method of LN mapping that utilizes cancer cell-specific viral production of GFP to facilitate accurate localization of metastases. Fluorescent filters that detect GFP can be incorporated into operative scopes to precisely localize and biopsy LN metastases. 
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