Premetastatic vasculogenesis in oral squamous cell carcinoma xenograft-draining lymph nodes

Objective: To study vascular anatomy on oral cancer-draining lymph nodes before metastasis in mice. Material and methods: Cell lines: highly lymph metastatic oral squamous cell carcinoma SASL1m and non-metastatic human adenoid cystic carcinoma ACC2. Bone marrow transplants and xenografts: Nude mice were lethally irradiated and transplanted with bone marrow cells from EGFP(+) mice. SASL1m or ACC2 cells were implanted in the tongue. Non-xenografted mice were used as controls. In addition, we injected conditioned medium from SASL1m or ACC2 in transplanted mice. Immunohistochemistry: Primary tumors and neck lymph nodes were resected and stained with anti-mouse Podoplanin and CD31. Images were visualized in a confocal microscope. Image analysis: Areas covered by EGFP, CD31 and Podoplanin were measured and compared statistically. Expression microarrays: Transcriptomic microarray analysis compared SASL1 to ACC2 cells. Interactomes were generated to reveal altered pathways. Results: SASL1m cells induced the assemblage of blood vessels in cancer-free, tumor-draining lymph nodes. These blood vessels incorporated bone marrow-derived EGFP(+)CD31(+) cells. Notably, SASL1m-conditioned medium induced a similar reaction. Non-metastatic cells failed to produce any change. Microarray and pathway analyses revealed the upregulation of Transforming Growth Factor-beta, Lysyl Oxidase-like 2, Slit homolog 3 and Protease Serine 22. The upregulation of these genes was confirmed in xenografts. Conclusions: This study suggests that a blood supply for new tumors is established in lymph nodes before metastasis. It also suggests that premetastatic vasculogenesis and primary tumor angiogenesis may be mediated by different mechanisms. (c) 2012 Elsevier Ltd. All rights reserved.