Abstract 466: Systemic early immune priming via tumor-secreted cytokines facilitates breast cancer metastasis in syngeneic mouse model
Authors: Korkaya Hasan , Lee Eunmi , Ouzounova Maria , El Andaloussi Abdeljabar , Novakovic Ena , Piranlioglu Raziye , Demirci Mehmet F. , Clouthier Shawn G. , Wicha Max S. 
Published: August 2015

Abstract
Metastatic breast cancer is the second leading cause of cancer-related death among women in the United States. Although the genetic and epigenetic differences between the metastatic versus non-metastatic breast tumors have been well studied, early events between tumor and immune system in metastatic process remain poorly understood. In order to determine early events, we utilized murine mammary tumors (4T1 as metastatic, EMT6 or 67NR as non-metastatic) in syngeneic mouse model. The 4T1 tumor contained a higher proportion of cancer stem cell (CSC) population compared to the non-metastatic EMT6 or 67NR clones. Although, both murine tumor cell lines (50K each) grow to same size tumors within 8 weeks, 4T1 tumors develop spontaneous metastasis in 100% of animals most of which do not survive more than 8 weeks due to extensive wide spread metastasis to lung, liver and bone. We observed immune infiltrates in the lungs of 4T1 tumor bearing mice as early as 1 week. We next assessed the cytokine profile of metastatic 4T1 tumor compared to non-metastatic counterparts (EMT6 or 67NR) secretes significantly higher levels of inflammatory cytokines, including the IL6, IL8, RANTES, GCSF, GM-CSF, IL12, CXCL16 and CXCL5.
MDSCs are potent suppressor of anti-tumor immunity and a significant impediment to cancer therapy. We therefore hypothesized that the tumor secreted inflammatory cytokines promotes the systemic expansion of MDSCs that down regulate immune surveillance and anti-tumor immunity, thus facilitating tumor progression. We sought to determine whether 4T1 tumors could induce MDSCs in mice. Murine 4T1 or EMT6 tumor cells (at 50K cells each) were implanted into the fat pads of BALB/c mice, then sacrificed (4 mice from each group) at weeks 1, 2, 3 and 4 for subsequent evaluation of the MDSC expansion in bone marrow, spleen, lung and tumors. The MDSC induction and infiltration in bone marrow, spleen, lung and tumors were observed as early as one-week post-implantation of 4T1 tumor compared to the EMT6. Furthermore, the MDSCs isolated from 4T1 tumor bearing animals were more suppressive than that of the EMT6 tumor bearing mice.
We determined that non-metastatic EMT6-Luciferase tumor growth and metastasis is robustly enhanced in pre-primed animals (in which metastatic 4T1 cells were pre-implanted in the fat pads and resected after 10 days when tumors were 2mm in size) or by IP injection of inflammatory cytokine rich 4T1 conditioned medium when compared to injection of EMT6-Luci cells into naïve animals. Our preliminary findings suggested that 4T1 tumors within 10 days of implantation created a systemic tumor-promoting microenvironment and thus promoted the metastatic spread of EMT6-Luci. Together these studies strongly suggest that metastatic 4T1 tumor with high CSC phenotype generate a permissive systemic microenvironment for successful metastasis via secretion of inflammatory cytokines in syngeneic BALB/c mice.
Citation Format: Hasan Korkaya, Eunmi Lee, Maria Ouzounova, Abdeljabar El Andaloussi, Ena Novakovic, Raziye Piranlioglu, Mehmet F. Demirci, Shawn G. Clouthier, Max S. Wicha. Systemic early immune priming via tumor-secreted cytokines facilitates breast cancer metastasis in syngeneic mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 466. doi:10.1158/1538-7445.AM2015-466