Early urinary protein changes in the tumor-forming process of the NuTu-19 lung metastasis

The early detection of cancer is essential for effective intervention. Urine, which lacks homeostatic control, has been used to reflect early systemic changes in subcutaneous cancer models. However, urine has not been used to predict whether tumors will be formed in animal models. In this study, a cancer model was established by the tail-vein injection of 2 million NuTu-19 tumor cells. Approximately half of the rats formed lung metastatic carcinoma tumors. Urine samples were randomly selected from 4 tumor-forming rats and 4 non-tumor-forming rats on day 0/12/27/39/52 and analyzed by label-free proteomic quantitative analysis. Compared to day 0, a total of 180 and 118 urinary proteins in tumor-forming and non-tumor-forming rats, respectively, showed significant changes. Functional enrichment analysis of the differential proteins in tumor-forming rats revealed similar events during cancer metastasis cascades and tumor progression, such as the migration of tumor cell lines, coagulation system, TGF-{beta} signaling, the STAT3 pathway and the adhesion of myeloid cells and alveolar macrophages. The differential proteins in non-tumor-forming rats were associated with agranulocyte/granulocyte adhesion and diapedesis, glutathione biosynthesis, IL-12 signaling and vitamin metabolism. After validation by parallel reaction monitoring (PRM) targeted proteomics quantitative analysis, a total of 22 urinary proteins showed significant changes in the early phase of the lung tumor formation process, 5 of which have been associated with the mechanisms of lung cancer, while 3 have been suggested as lung cancer biomarkers. Another 14 urinary proteins showed significant changes in the early phase in non-tumor-forming rats. Our results indicate that urine proteins could differentiate early tumor-forming and non-tumor-forming status.