IMMUNOTHERAPY AGAINST THE RADIAL GLIA MARKER GLAST EFFECTIVELY TRIGGERS SPECIFIC ANTITUMOR EFFECTORS WITHOUT AUTOIMMUNITY

Glioblastoma (GB) is the most aggressive and fatal tumor of the central nervous system (CNS). Despite promising results of different therapeutic approaches, conventional treatments such as surgery, radiation and chemotherapy with Temozolomide (TMZ) are largely palliative. The identification of antigens preferentially expressed in GB and involved in its malignant phenotype is critical for developing therapeutic strategies. Murine and human GB contain a fraction of cancer cells with stem-like features (CSC) and it has been proposed that only this population may be responsible for glioma recurrence. We previously found that GLAST, a membrane protein with relevant action in glutamate trafficking, is highly expressed in murine GL261 glioma stem-like cells (GSC). In this study, we first investigated the functional consequences of GLAST enrichment using an immune-magnetic sorting approach to isolate GLAST+ cells from murine GSC. GLAST+ cells were significantly more tumorigenic than GLAST- or unsorted cells (p< 0.0006) when injected intracranially, showing the highly invasive nature of this subpopulation. GLAST expression and its impact on aggressiveness in vivo constituted a background of translational relevance. To determine whether GLAST peptide-based immunotherapy protects against GL261-gliomas, we treated glioma-bearing mice with three subcutaneous injections of four GLAST-derived peptides emulsified with Montanide™ ISA 51 VG in association with granulocyte macrophage colony-stimulating factor (GM-CSF) injections. We found that immunization with GLAST peptides efficiently induces specific anti-tumor response in the murine GL261-glioma preventing the tumor progression in 40% of immunized mice. The immunization induced relevant effects on tumor microenvironment by up-regulating IFNγ and TNFα as well as downregulating TGFβ1 and β2. GLAST expression significantly decreased in III gliomas from immunized mice, as evaluated by histological analysis and real-time PCR. Beyond these changes, trafficking of NK cells, CD8+ T cells and CD4+ T cells in the spleens and lymph nodes and their homing ability into the brain significantly increased in immunized mice when compared with controls. A local chemotactic gradient characterized by expression of CXCL10 (which may be responsible for the recruitment of CTL), CCL3, CCL4 and CCL5 (which are involved in NK cell migration), and NKG2D ligand in immunized mice may play a role in the accumulation of immune cells (particularly of NK cells) at the sites of tumor formation. Autoimmune reactions were not observed in immunized mice supporting evidence that GLAST may constitute a glioma antigen against which immune responses can be efficiently induced without toxicity. In order to emphasize the translational relevance of GLAST as marker and glioma-associated antigen we will extend our studies to human GB. GLAST expression was investigated by immunohistochemistry in 39 human primary GB. High percentage of GLAST+ cells, and a moderate or strong reactivity, was associated with a decreased overall survival (p= 0.02). The main result of this study is the definition of GLAST as an attractive glioma-associated antigen for targeting in peptide-based immunotherapy in GB patients and a potential clinical marker in association with the patient prognosis.