Glioblastoma is the most common primary brain tumor, occurring with a frequency of 3.2 cases per 100 000 population. Despite established treatment, which includes surgical removal of the tumor, chemotherapy with temozolomide, and radiotherapy, most patients do not survive more than 18 months after diagnosis. One of the modern possible ways of treating GBM is the use of nanobodies, antigen-recognizing parts of heavy chain-only antibodies produced by only some animals, e.g. llamas. Namely, nanobodies have many advantages over classical antibodies, such as high stability, possibility of production with E. coli and faster transition and penetration to the tumor.
In the doctoral thesis, we studied the influence of nanobodies against eight possible biomarkers of glioblastoma on cell survival, migration and colony formation. First, using immunohistochemistry, we found that, based on differences in expression or presence, the biomarker vimentin could distinguish between glioblastoma, lower-grade gliomas, and normal brain, while biomarkers TUFM, DPYSL1, and CRMP1 distinguished between glioblastoma and normal brain. The results of the study of the cytotoxic action of nanobodies showed that nanobodies Nb79 (anti-vimentin), Nb179 (anti-NAP1L1), Nb225 (anti-TUFM) and Nb314 (anti-DPYSL2) have a cytotoxic effect on glioblastoma cells. The anti-TUFM nanobody (Nb225) has a particularly large effect on the cytotoxicity of glioblastoma stem cells. The migration of glioblastoma cells is mostly affected by the anti-vimentin nanobody (Nb79), which completely inhibited the migration of cells of the glioblastoma cell line U87MG.
In the second part of the study, we developed a delivery system based on extracellular vesicles, exosomes, into which we packaged nanobodies to improve their delivery and increase efficiency. Exosomes are the smallest extracellular vesicles secreted by cells and are thought to pass more rapidly into target cells than comparable delivery systems, while also having a longer half-life. Exosomes were isolated from the U251MG glioblastoma cell line cells and characterized by detection of exosomal markers using Western blot, a nanoparticle tracking analysis method to determine their number and size, and by electron microscopy to determine their shape. Nanobodies were successfully packaged into exosomes by 0.4% saponin incubation and sonication methods, which were approximately equally effective, while indirect packaging by incubating cells with nanobodies was not successful.
In addition to being carriers of a potential delivery system, exosomes are also a possible source of biomarkers. In our study, we analyzed the expression of selected mRNAs, miRNAs, and proteins in the exosomes of glioblastoma cell lines using the qPCR method. We found that miR-9-5p, miR-124-3p, TUFM mRNA, and CRMP1 mRNA are possible markers of glioblastoma stem cell exosomes, and VIM mRNA is a suitable marker of differentiated glioblastoma cell exosomes. In contrast to the mentioned RNA molecules, proteins were less represented in the exosomes of the studied cells, in which only the protein biomarkers ALYREF and DPYSL2 could be detected.
In our doctoral dissertation, we showed that nanobodies are a suitable means of achieving a cytotoxic effect and reducing the migration of glioblastoma cells. We have successfully developed a delivery vehicle, exosomes that contain nanobodies, and these could be used in the future to increase the efficiency of the nanobodies themselves. Our results suggest that cytotoxic nanobodies and exosomes, as a delivery system, are a promising form of efficient and specific therapy for the treatment of GBM. In parallel, in this study we also identified possible exosomal markers of glioblastoma cells, for which we propose evaluation in the further study of exosomes isolated from patients’ body fluids. These could in the future serve as potential biomarkers of GBM from the blood or cerebrospinal fluid of patients with GBM.
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