Abstract 5032: Elucidating the role of CXCR4 in Ewing sarcoma.

Background: Ewing sarcoma (ES) is the second most common malignant bone tumor in pediatric patients. While intensification of chemotherapy has improved the outlook for patients with localized disease, little progress has been made for those with metastatic or relapsed disease. Prognosis for these patients remains dismal with fewer than 20% surviving. Moving forward, we need to develop molecularly targeted therapy for patients with high-risk disease. The mechanisms underlying metastatic relapse in ES remain unknown but gene encoding the chemokine receptor CXCR4 has been shown to be upregulated in metastatic ES. The CXCL12-CXCR4 axis contributes to tumor metastasis in other sarcomas but its function in ES is not yet known. Objective: In the current study we are investigating whether CXCR4 is a mediator of ES cell metastasis and may be useful as a biomarker of high-risk disease. Results: Using qRT-PCR and flow cytometry we have shown that expression of CXCR4 transcript and protein varies significantly across ES cell lines. Importantly, the frequency of CXCR4+ cells and CXCR4 expression levels were found to be highly dynamic and were altered under different experimental conditions. Specifically, both serum deprivation and hypoxia induced upregulation of CXCR4 in TC32, TC71, and A673 ES cells. CXCR4+ cell frequency was found to be consistently highest in TC32 where the frequency ranged from 35% in ambient conditions to 62% in serum deprivation. When sorted into CXCR4+ and CXCR4- populations TC32 populations returned to their baseline state of 40% CXCR4+ cells within 3 weeks indicating that CXCR4+ cells can change into CXCR4- cells and vice versa. Interestingly, CXCR4 transcript was identified to be increased in slowly proliferating (PKH-dye-retaining) TC71 and A673 cells compared to more rapidly dividing counterparts. These data implicate CXCR4 as a potential marker of putative cancer stem cells. Finally, initial immunohistochemical studies have demonstrated highly variable patterns of CXCR4 expression in primary ES tumor samples. Summary: ES cells express CXCR4 both in primary tumors and in cultured cell lines. However, the expression is both variable and highly dynamic. Specifically, we have found that ES cells can switch back and forth between CXCR4- and CXCR4+ states in response to environmental cues including serum deprivation and hypoxia. Ongoing work is now testing the hypothesis that CXCR4+ ES cells are more migratory than CXCR4- cells and may thus be a source of metastasis. To further test this hypothesis we are using correlative studies of clinically annotated tumor microarrays to determine if a high frequency of CXCR4+ cells at diagnosis is associated with an increased risk of metastatic relapse. Citation Format: Melanie A. Krook, Lauren A. Nicholls, Natashay J. Bailey, Amy Han, Chris Scannell, Dafydd Thomas, Elizabeth R. Lawlor. Elucidating the role of CXCR4 in Ewing sarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5032. doi:10.1158/1538-7445.AM2013-5032