MTR-01BEVACIZUMAB-INDUCED MIF DEPLETION: A NOVEL RESISTANCE MECHANISM IN GLIOBLASTOMA

Abstract

Clinical trials revealed limited response duration of glioblastomas to the anti-angiogenic therapy bevacizumab, a VEGF-neutralizing antibody. Bevacizumab-resistant glioblastomas exhibit increased invasiveness and 50% more tumor-associated macrophages (TAMs), which can be of classical M1 or pro-tumoral M2 subtypes. As macrophage migration inhibitory factor (MIF) regulates tumor invasion and macrophage recruitment, we investigated MIF's role in bevacizumab resistance in glioblastoma. Patient bevacizumab-resistant glioblastomas exhibited 2-fold less MIF protein than bevacizumab-naive glioblastomas (n = 10; p < 0.05). Bevacizumab-resistant xenografts exhibited 6-fold less MIF protein and increased invasiveness than isogeneic bevacizumab-responsive xenografts (p < 0.05). Immunoprecipitation revealed that bevacizumab bound MIF, likely reflecting its 30% homology to the VEGF protein structure. Furthermore, VEGF stimulation of U87 cells in vitro increased MIF transcription and secretion 5-fold (p < 0.05), suggesting VEGF depletion as another mechanism of MIF downregulation. Site-directed biopsies of bevacizumab-naive glioblastomas revealed enriched MIF expression at the enhancing tumor margin, which was lost in bevacizumab-resistant glioblastomas (p < 0.05). A similar regional pattern of expression was seen with VEGF, supporting its role in stimulating tumor cell MIF secretion. Continued bevacizumab treatment of bevacizumab-resistant xenografts increased M2-like CD11b+ TAMs, 3-fold, with preferential localization at the tumor margin (p < 0.05). The same pattern of recruitment and polarization was seen in U87/MIF-shRNA relative to U87/control-shRNA intracranial xenografts (p < 0.05). When co-cultured with cells from bevacizumab-resistant xenografts or U87/MIF-shRNA cells, bone marrow matured to an M2-like phenotype (p < 0.05), while an M1-like phenotype was generated with addition of recombinant MIF in a dose-dependent manner (p < 0.05). In conclusion, MIF expression is enriched at the glioblastoma margin, a pattern that may create a perimeter of inflammatory M1-like TAMs. This pattern is lost with bevacizumab resistance, likely due to bevacizumab-induced MIF depletion and diminished VEGF-induced MIF upregulation. MIF depletion leads to M2-like TAM polarization and recruitment to the tumor margin, thus softening the tumor border and promoting invasive bevacizumab resistance.