Abstract Vascular growth factor-C (VEGF-C) is well known as an indispensable growth factor for development of the lymphatic system and its overexpression in cancers has been widely implicated in lymph node metastasis. Recently, several studies have demonstrated that VEGF-C, in addition to playing a role in lymphangiogenesis, can also contribute to tumor progression in an autocrine manner, such as to increase the proliferation and migration of tumor cells. In this work, we describe the discovery of a novel function of VEGF-C in breast cancer, in which the protein modulates the response of tumor cells to oxidative stress, thereby contributing to a tumor initiating cell phenotype. Knockdown (KD) of VEGF-C in 66cl4 mouse mammary carcinoma cells decreases ALDH activity (using an in vitro assay which measures tumor initiating cell populations) compared to scramble control KD cells and in immune competent animals in vivo, VEGF-C KD in the 66cl4 cells leads to a decrease in tumor size and tumor formation. Tumor initiating populations have been shown to have enhanced anti-oxidant activity, by which they can survive under excessive oxidative stress generated in the tumor microenvironment or by conventional therapies, thereby contributing to metastasis and recurrence. Therefore, we further measured whether VEGF-C modulates the response of tumor cells to oxidative stress. Indeed, VEGF-C KD decreases the viability of 66cl4 cells when treated with ROS (induced by H2O2), and furthermore, the knockdown decreases viability in response to several chemotherapy drugs that are known to cause DNA damage, at least in part by accumulating ROS. To identify factors downstream of VEGF-C that mediate its response to oxidative stress, we performed a qPCR array for ROS response related genes in the 66cl4 scramble and VEGF-C KD cells and identified superoxide dismutase 3 (Sod3), one of the major ROS scavenging members, as regulated by VEGF-C. Further, decreased expression of Sod3 was detected in the VEGF-C KD cells as well as in the 66cl4-VEGF-C KD tumors compared to 66cl4-control tumors. Importantly, restoration of Sod3 in the VEGF-C KD cells rescued the ability of tumor cells to survive under oxidative stress in vitro; and restored tumor growth and metastasis in the 66cl4 immune competent animal model in vivo, strongly suggesting that the VEGF-C-Sod3 axis promotes tumor progression by eliminating excessive oxidative stress generated during tumor cell growth. Finally, analyses of public breast cancer datasets demonstrate that VEGF-C is predominantly expressed in breast cancer cell lines that contain high percentages of tumor initiating cell populations and in aggressive subtypes of breast cancer. Together, our data define a novel function for VEGF-C in breast cancer, providing evidence that this growth factor can influence tumor initiating properties perhaps in part through altering anti-oxidant function. Our study also implies that blocking VEGF-C signaling will not only decrease lymphatic dissemination, but may also decrease the tumor initiating cell population and sensitize tumor cells to oxidative stress/chemotherapies, particularly in patients with aggressive subtypes of breast cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A58. Citation Format: Chu-An Wang, Ritsuko Iwanaga, Heide Ford. Targeting vascular growth factor C (VEGF-C) in cancer stem-like populations sensitizes cells to oxidative stress and inhibits tumor progression via regulation of superoxide dismutase 3 (Sod3) in a mouse model of breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A58.