Glioblastoma multiforme (GBM) is a glioma, a tumor found in central nervous system. It is the most common form of brain tumors, which affects 3-5 patients per 100,000 people. The average survival period of patients with GBM is 12 to 18 months, which includes resection and combination of postoperative therapy with temozolomide. Glioblastoma stem cells (GMC) are responsible for high genetic heterogeneity of GBM, and due to their resistance to chemotherapy and radiotherapy they successfully invade healthy tissue. The treatment is further aggravated, due to difficult transition of chemotherapeutics through blood-brain barrier (BBB). To improve GBM treatment and the outcome of patients, it is necessary to continuously deliver drugs to the glioma cells while reducing the effect of drugs on adjacent, healthy neurons and glial cells. New GBM treatment approaches are therefore much needed. For the development of targeted GBM treatment, we still need the discovery of more specific GMC biomarkers and the corresponding targeting drugs that would pass BBB. This can be achieved by a proteomic approach based on nanobodies, single-domain antigen-binding fragments, derived from camelid heavy chain antibodies that can, due to their small size, pass BBB.
In the doctoral thesis, we constructed a nanobody library and biopannings were made on the whole GMCs. We obtained a nanobody specific for a GMC antigen. Mass spectrometry determined that the new biomarker of GMC is the mitochondrial translational-elongation factor TUFM. Differential expression of TUFM was studied at the protein and mRNA levels in the GBM cell lines (U87MG, and U251MG), GMC and GBM tissue, compared to its expression in neural stem cells (NSC) and normal brain tissue. Western blot and qPCR confirmed the TUFM overexpression in GMC. With immunohistochemistry, on paraffin-embedded GBM tissue, we confirmed the TUFM overexpression, whereas the normal brain tissue was negative for TUFM. Immunocytochemistry confirmed the entry of anti-TUFM nanobodies to the U87MG, U251MG and GMC cells and its binding to mitochondria. The cytotoxic effect of anti-TUFM nanobodies on GBM-related cell lines (U87MG, U251MG and GMC), and on control cell lines (astrocytes, NSC and human immortal keratinocytes) was measured through metabolic assays. Anti-TUFM nanobody had cytotoxic effect on all GBM cell lines, while on the other hand no toxicity of anti-TUFM on control cell lines was observed.
Anti-TUFM nanobodies were encapsulated to archeosomes and used to verify their delivery in cells. Aerheosomes are not cytotoxic in vivo and have unique structural properties for the development of new drug delivery systems. These properties are stability at high temperatures, extreme pH values, resistance to phospholipases and bile salts and lower membrane permeability. Successfully, anti-TUFM nanobodies were encapsulated in the archeosomes and were found that they could enter the U251MG cells. Therefore, an encapsulated anti-TUFM nanobody exhibited lower cytotoxic effect on GBM cells than the anti-TUFM nanobody itself.
In the doctoral thesis, we showed the specificity and pronounced inhibitory effect of anti-TUFM nanobody on GMC growth, which could in the future contribute to the development of a specific approach for the treatment of glioblastoma.