Osteopontin is a secreted glycosylated phosphoprotein known to be involved in numerous physiologic functions and associated with the late stages of various cancers. We used preneoplastic and neoplastic mouse models of prostate cancer to determine the onset of elevated expression of osteopontin in the development of this disease. Osteopontin alterations occurred early in the disease with dysregulated expression observed in lesions of low-grade prostatic intraepithelial neoplasia (PIN). Over time, osteopontin expressing dysplastic cells seemed to increase in number in high-grade PIN and increased further in adenocarcinoma, and in metastasis, almost all of the cancer cells immunohistochemically stained positive for osteopontin overexpression. We examined the biological properties of human prostate cancer cell lines LNCaP and PC-3, in which osteopontin overexpression was achieved via lentiviral gene transduction. Evidence was obtained that osteopontin could contribute to a proliferative advantage in both cell types, although more significantly in LNCaP than PC-3. Osteopontin also influenced their in vitro invasive ability, and again, most strikingly in the weakly oncogenic LNCaP. Furthermore, excess osteopontin induced the LNCaP cells to acquire a strong intravasation potential in vivo in the chicken embryo chorioallantoic membrane assay for blood vessel penetration. These results establish a correlation between an increased gradient of osteopontin expression throughout the stages of murine prostate cancer, beginning from the preneoplastic lesions to distant metastases that suggests a proliferative and invasive advantages to those prostate tumor cells overexpressing osteopontin. Together, these findings support a strategy designed to target osteopontin in the context of prostate cancer therapy. (Cancer Res 2006; 66(2): 883-8)