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

Abstract

To study vascular anatomy on oral cancer-draining lymph nodes before metastasis in mice. 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. 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-β, Lysyl Oxidase-like 2, Slit homolog 3 and Protease Serine 22. The upregulation of these genes was confirmed in xenografts. 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.