In this study, we visualized the origin of Lewis lung carcinoma metastasis after transducing tumor cells with green fluorescent protein (GFP) and transplanting them orthotopically in the middle lobe of the right lung of nude mice. Metastasis was visualized in live tissue at single cell resolution by GFP-expression as early as 18 h post-tumor transplant. At this time, single-file streaming lung carcinoma cells already had invaded inferiorly via a tubular lymphatic structure crossing the lower lobes of the lung to the ipsilateral diaphragmatic surface. By post-implantation day 2, the ipsilateral lower lobes of the lung were involved with metastatic cells. By post-implantation day 3, the ipsilateral lower lobes of the lung and the ipsilateral diaphragmatic surface were highly involved with streaming metastatic cells trafficking in single file. By day 4 post-implantation, cancer cells invaded across the diaphragm to the contralateral diaphragmatic surface. Metastatic cells then invaded superiorly through a lymphatic vessel to involve the contralateral mediastinal lymph nodes. In this model of lung cancer, the origin of metastasis was an inferior invasion from the implanted tumor via a lymphatic duct to the ipsilateral diaphragmatic surface. The cancer cells from this site invaded on the surface of the diaphragm to the contralateral diaphragmatic surface and proceeded superiorly through a lymphatic duct to contralateral lymph nodes. Other organs such as the kidneys and the adrenal glands later became involved with metastasis with the contralateral mediastinal lymph nodes as the source. The use of GFP and the highly metastatic orthotopic lung cancer model allowed the visualization of the origin of metastasis at the single-cell level and demonstrated the critical role of lymphatic ducts and the diaphragmatic surface as the path to the contralateral side.
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