Abstract
Glioblastoma (WHO grade IV, GBM) is a malignant, very aggressive, primary brain tumor which due to tumor cell heterogeneity, genetic alterations and therapy resistance remains incurable. GBM is heavily infiltrated with microglia and macrophages which play an important role in proliferation, diffusive growth and responses to treatment. Microglia and peripheral macrophages accumulate in malignant gliomas and constitute 30-50% of the tumor mass. Glioma cells overexpress and secrete proteins that reprogram microglia and peripheral macrophages into cells which potentiate tumor invasion and growth, at the same suppressing antitumor immunity. We have previously demonstrated that glioma-derived granulocyte macrophage colony-stimulating factor - GM-CSF (encoded by a CSF-2 gene) induces accumulation and protumorigenic activation of microglia/macrophages. We hypothesized that a humanized peptide that selectively binds to GM-CSF, would block its binding to respective receptors on myeloid cells, and inhibit activation of the receptors and downstream signaling pathways, which should result in inhibition of glioma invasiveness. First, we designed a peptide library containing 27 peptides, each 14-residues long. Next, we identified the peptide binding to GM-CSF using peptide microarrays, enzyme-linked immunosorbent assay (ELISA) and a technique based on surface plasmon resonance (SPR). Subsequently, using the Matrigel Matrix cell invasion assay we selected the peptide (G7) with most potent capacity for inhibition of human U87 MG and LN18 glioma cell invasiveness in the presence of glioma-microglia co-cultures. We further confirmed that this peptide blocks binding of GM-CSF to its receptor using SPR and its relatively good stability using mass spectrometry. Anti-tumor activity of G7 peptide in vivo was confirmed in orthotopic U87 xenograft mouse model. Intracranial delivery of this peptide inhibitor omits blood-brain barrier and minimizes nonspecific toxicity.