Abstract LB-98: Reprogramming towards glioma stem-like cells by cytokines

Gliomas are the most common primary brain tumor in adults and are very aggressive, invasive and destructive malignancies. Recent studies suggest that glioma stem cells (GSCs) are responsible for the initiation, recurrence of gliomas, and resistance to radiological and chemotherapies, indicating that targeting GSCs is a more effective therapeutic strategy against gliomas. However, it is still unclear about the molecular mechanisms that govern GSC biology. TGF-beta and IL-1beta are both highly active in high grade gliomas and their elevated activities have been associated with poor prognosis in glioma patients. The link between TGF-beta and IL-1beta and malignant phenotype of gliomas suggests that TGF-beta and IL-1beta may contribute to glioma stem cell development. To test the hypothesis, we explored human glioma cell line LN229 and the serum-free condition. The serum-free condition supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) has been commonly used to identify GSCs, which readily form spheres, called "glioma spheres". However, LN229 cells cannot form spheres in serum-free medium, suggesting that these cells contain very few stem cells. We found that addition of IL-1beta and TGF-beta, but not either cytokine alone, to the serum-free medium resulted in sphere formation of LN229 cells even at clonal density, indicating that the two cytokines act in a synergistic fashion to induce self-renewal. Immunocytochemical analysis demonstrated that most IL-1beta/TGF-beta-induced sphere cells were nestin-positive cells. In addition, quantative PCR analyses showed that the induced spheres had significantly increased expression of other stemness markers including LIF, Notch-2 and Bmi-1. To further validate the self-renewal of the induced spheres, sphere cells were dissociated and plated in the absence of the cytokines in soft agar. The sphere cells formed more and larger colonies than the control cells by about two fold in number and size. Furthermore, the induced sphere cells demonstrated significantly increased invasion and drug resistance. Meanwhile, the gene expression of invasion-related genes including SIP1, beta-integrin and N-cadherin were also highly increased in the induced spheres compared to the control. More importantly, the induced sphere cells demonstrated their oncogenic potential by forming larger tumor with less number of cells in the brain of immunocompromised mice than the control cells. Overall, these results indicate that the spheres induced by IL-1beta and TGF-beta are cancer stem-like cells with the properties of self-renewal, drug resistance, invasion and oncogenic potential. Our finding suggests that GSCs can be a dynamic stage and microenvironmental conditions can promote the acquisition of a stem cell-like state from differentiated cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-98. doi:10.1158/1538-7445.AM2011-LB-98