Interleukin-6 Producing Tumor Associated Macrophages (Tams) Increase Myc Expression And Promote Tumor Growth In A Novel Murine Neuroblastoma Model

The concept of tumor-promoting inflammation is a recognized enabling characteristic of cancers. Recent studies have demonstrated the prognostic significance of tumor-associated macrophages (TAM) in several cancers. Our group has also demonstrated the significance of TAMs and interleukin-6 receptor in predicting poor outcome in children diagnosed with metastatic MYCN non-amplified neuroblastoma (NBL-NA). Previous in vitro work has shown that co-culture of a human neuroblastoma MYCN non-amplified cell line with monocytes enhanced proliferation, an effect mediated in part by interleukin-6 (IL6). To address the mechanism by which inflammation contributes to tumorigenesis in neuroblastoma, we have established a novel, 100% penetrant, murine model of metastatic NBL-NA. The tumors of these transgenic mice (NBL-Tag) are detected by MRI only after 12 weeks of age, which coincide with IL6 levels becoming detectable and increasing in blood. DNA profiling of tumors reveal no amplification of the MYCN locus, while RNA profiling and FACS analyses of tumors show high expression of IL6, and infiltration by IL6-producing TAMs. Thus, a pro-inflammatory microenvironment mimicking that observed in human metastatic NBL-NA characterizes the growth of NBL-Tag murine tumors. Immune-related gene expression analysis of NBL-Tag adrenal glands in mice at pre-tumor development age (4, 8 and 12 weeks old) show age-dependent increase in expression of CCL2 (aka monocyte chemotactic protein 1) and fibronectin (FN1) compared to wildtype adrenal glands demonstrating early monocyte chemotaxis and establishment of a pro-inflammatory niche. In vitro co-culture of spleen or peritoneal macrophages from wildtype mice increases proliferation of a NBL-Tag cell line (NBT2) by 30 - 50% over its basal rate, as measured by Brdu incorporation. This effect coincides with two-fold increase in IL6 protein level in the co-cultured media. However, these proliferative changes do not require cell-cell contact and are only partially reversed by blocking IL6, suggesting other soluble factors also contribute to the observed effects. Co-cultured NBT2 cells also increase their expression of MYC by two-fold, providing insight into the tumor proliferative mechanism facilitated by TAMs. In summary, this study demonstrates the recruitment and growth-promoting effects of IL6 producing TAMs in early pathogenesis of neuroblastoma in a novel MYCN non-amplified murine model. The NBL-Tag mice provide a robust transgenic model to study neuroblastoma pathogenesis and provide a platform to assess therapies targeting tumor cells and tumor-associated inflammatory cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 383. doi:1538-7445.AM2012-383