An orthotopic floor-of-mouth cancer model allows quantification of tumor invasion

Objectives: To establish an orthotopic murine floor-of-mouth cancer model of the analysis of the role of proteases such as urokinase-type plasminogen activator (u-PA) and the matrix metalloprotease MMP-9 (MMP-9) in in vivo invasion. Study Design: Randomized, prospective animal study. Methods: Two human squamous cell carcinoma cell lines, UM-SCC-1 and 022, were assayed via zymography for their in vitro secretion levels of u-PA and MMP-9. Both cell lines (5 x 10(6) cells) were injected into the cervical subcutaneous tissues of female athymic nude (nu/nu) mice superficial to the mylohyoid muscle. Mice were sacrificed after 30 days, and tumor invasion characteristics were histologically compared. Additional mice were then inoculated with invasive UM-SCC-1 cells and sacrificed 10, 30, and 40 days after inoculation to identify distinct stages of invasion. Results: In vitro secretion levels of MMP-9 and activity of u-PA were higher in UM-SCC-1 cells than in 022 cells. In the in vivo studies, tumors formed from 022 cells were found to be noninvasive, whereas tumors derived from UM-SCC-1 cells progressed through distinct and readily identifiable histologic stages of invasion. These stages included invasion of adjacent muscle layers (mylohyoid, geniohyoid, and genioglossus muscles) and of associated structures (blood vessels, bone, nerve, and regional lymph nodes). A staging system was devised accordingly. Conclusion: We developed an in vivo quantitative cancer invasion model that allows determination of the effect of the expression and activity levels of the proteases MMP-9 and u-PA. Tumor invasion occurred in an orderly and stepwise fashion involving muscles and related vascular, nervous, and bony structures of the floor of the mouth and tongue. This orderly invasion allowed the development of a staging system. We anticipate that this model will have wide applicability in the study of in vivo tumor response to a variety of novel therapeutic approaches.