In Vitro Functional Validation of an Anti-FREM2 Nanobody for Glioblastoma Cell Targeting.

Abstract

Background/Objectives: Glioblastomas are the most common brain malignancies. Despite the implementation of multimodal therapy, patient life expectancy after diagnosis is barely 12 to 18 months. Glioblastomas are highly heterogeneous at the genetic and epigenetic level and comprise multiple different cell subpopulations. Therefore, small molecules such as nanobodies, able to target membrane proteins specific to glioblastoma cells or specific cell types within the tumor are being investigated as novel tools to treat glioblastomas. Methods: Here, we describe the identification of such a nanobody and its in silico and in vitro validation. NB3F18, as we named it, is directed against the membrane-associated protein FREM2, overexpressed in glioblastoma stem cells. Results: Three dimensional in silico modeling indicated that NB3F18 and FREM2 form a stable complex. Surface plasmon resonance confirmed their interaction with moderate affinity. As we demonstrated by flow cytometry, NB3F18 binds to glioblastoma stem cells to a greater extent than to differentiated glioblastoma cells and astrocytes. Immunocytochemistry revealed surface localization of NB3F18 on glioblastoma stem cells, whereas cytoplasmic localization of NB3F18 was observed in other cell lines. NB3F18 was detected by transmission electron microscopy on the plasma membrane and in various compartments of the endocytic pathway, from endocytic vesicles to multivesicular bodies (endosomes) and lysosomes. Interestingly, NB3F18 was cytotoxic to glioblastoma stem cells. Conclusions: Collectively, NB3F18 has been qualified as an interesting tool to target glioblastoma cells and as a potential vehicle to deliver biological or pharmaceutical agents to these cells.