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Purpose: Medulloblastoma in children can be categorized into at least four molecular subgroups, offering the potential for targeted therapeutic approaches to reduce treatment-related morbidities. Little is known about the role of tumor microenvironment in medulloblastoma or its contribution to these molecular subgroups. Tumor microenvironment has been shown to be an important source for therapeutic targets in both adult and pediatric neoplasms. In this study, we investigated the hypothesis that expression of genes related to tumor-associated macrophages (TAM) correlates with the medulloblastoma molecular subgroups and contributes to a diagnostic signature. Methods: Gene-expression profiling using human exon array (n = 168) was analyzed to identify medulloblastoma molecular subgroups and expression of inflammation-related genes. Expression of 45 tumor-related and inflammation-related genes was analyzed in 83 medulloblastoma samples to build a gene signature predictive of molecular subgroups. TAMs in medulloblastomas (n = 54) comprising the four molecular subgroups were assessed by immunohistochemistry (IHC). Results: A 31-gene medulloblastoma subgroup classification score inclusive of TAM-related genes (CD163 and CSF1R) was developed with a misclassification rate of 2%. Tumors in the Sonic Hedgehog (SHH) subgroup had increased expression of inflammation-related genes and significantly higher infiltration of TAMs than tumors in the Group 3 or Group 4 subgroups (P < 0.0001 and P < 0.0001, respectively). IHC data revealed a strong association between location of TAMs and proliferating tumor cells. Conclusions: These data show that SHH tumors have a unique tumor microenvironment among medulloblastoma subgroups. The interactions of TAMs and SHH medulloblastoma cells may contribute to tumor growth revealing TAMs as a potential therapeutic target. Clin Cancer Res; 21(6); 1457–65. ©2014 AACR.

Background: The concept of tumor-promoting inflammation is a recognized enabling characteristic of cancers. Our group has demonstrated the significance of inflammation-related genes in poor outcome of children diagnosed with metastatic MYCN non-amplified neuroblastoma (NBL-NA). We investigated the effect of TAMs on neuroblastoma growth using a novel murine model. Methods: We have established a novel 100% penetrant NBL-NA murine model driven by SV40's Large T-antigen (NBL-Tag) and characterized expression of immune-related genes in tumors at various developmental stages. Single cell suspensions of murine tumors were analyzed using flow cytometry for determination of immune cells frequency and IL6 levels. NBL-Tag mice were crossed with IL6 knockout mice (NBL-Tag/IL6-/-) to investigate the role of IL6 in tumor pathogenesis. Results: NBL-Tag tumor growth coincided with IL6 levels becoming detectable and increasing in blood. Immune-related gene expression analysis of NBL-Tag adrenal glands in mice at pre-tumor development age (4, 8 and 12 week old) showed age-dependent increase in expression of IL6 and CCL2 (MCP1) compared to wild-type adrenal glands demonstrating early monocyte chemotaxis and establishment of a pro-inflammatory niche. FACS of tumors showed high expression of IL6, and infiltration by IL6-producing TAMs. In vitro co-culture of peritoneal macrophages from wild type mice increased proliferation of a NBL-Tag mice driven cell line (NBT2) by average of 50% over its basal rate, as measured by Brdu incorporation. This effect coincided with two-fold increase in IL6 protein level in the co-cultured media. However, the proliferative changes were only partially reversed by blocking IL6, suggesting other soluble factors also contribute to the observed effects. IL6 genetic ablation studies were conducted to generate NBL-Tag/IL6-/- mice, which convincingly demonstrated IL6 was not required for tumor growth and development. Macrophages from IL6-/- mice increased in vitro proliferation of tumor to similar levels as those from wild-type mice, while IHC staining of NBL-Tag/IL6-/- tumors revealed similar level of TAM infiltration and positivity for phospo-STAT3, indicating an IL6-independent pathway for STAT3 activation. Lastly, gene expression analyses revealed upregulation of MYC in tumor cells during co-culture and was confirmed by western analysis. Conclusions: This study demonstrates the recruitment and growth-promoting effects of TAMs in early pathogenesis of neuroblastoma in a novel MYCN non-amplified murine model. The macrophage and neuroblastoma cell cross-talk lead to tumor cell proliferation with phosphorylation of STAT3 and MYC upregulation in tumor cells. These effects were found to be independent of IL6 activity. Citation Format: Michael D. Hadjidaniel, Soheila Shirinbak, Sakunthala Muthugounder, Long Hung, Michael Sheard, Janahan Gnanachandran, Jin Kim, Richard Sposto, Hiroyuki Shimada, Shahab Asgharzadeh. Tumor-associated macrophages (TAMs) promote murine neuroblastoma tumor growth through upregulation of MYC and independent of IL6 expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3668. doi:10.1158/1538-7445.AM2014-3668

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