Abstract:
Glioblastoma multiforme is the most frequent primary malignancy of the central nervous system. Despite the clinical management, patients succumb to the disease in 12 to 18 months after initial diagnosis, while for recurrent glioblastoma life expectancy is reduced to 6 months. Glioblastoma lethality is attributed to late diagnosis, difficult surgical removal due to intracranial location, tumor heterogeneity, aggressiveness, infiltration into surrounding brain tissue and resistance to available therapy. Targeting glioblastoma is difficult due to lack of specific biomarkers. Despite the constant efforts of researchers to identify glioblastoma biomarkers, there are no major improvements in their discovery. Our work presents an unconventional nanobody-based approach for selection of glioblastoma specific proteins (PMID: 25419715, PMID: 28498803 and PMID: 29707108). Nanobodies are llama heavy-chain only antibodies which are small in size (14 kDa), hydrophilic, resistant to non-physiological pH and elevated temperatures, and with high sequence similarity to the VH part of human immunoglobulins which makes them non-immunogenic. With nanobodies we selected a panel of proteins (ACTB/NUCL complex, VIM, NAP1L1, TUFM, DPYSL2, CRMP1, ALYREF and TRIM28) which showed differential expression when compared to reference samples. Highest change in protein expression levels was observed for ALYREF, CRMP1 and VIM. Then, OncoFinder software was used for creating an interaction network among these proteins. Results were also analyzed using TCGA dataset, which suggested NAP1L1, NUCL, CRMP1, ACTB and VIM genes to differentiate between glioblastoma and lower grade gliomas, and proposed the use of DPYSL2 as a “glioma versus normal” biomarker. In silico findings were confirmed using a small scale study that showed significant changes at mRNA expression levels for VIM, DPYSL2, ACTB and TRIM28. Afterwards, we examined cytotoxic effect of all nanobodies on different glioblastoma cell lines and astrocytes. Most promising results were observed for NB179 and NB314 targeted against NAP1L1 and DPYSL2 respectively, which reduced NCH421k cell growth down to 74% and 68%. Moreover, migration assay was performed to test possible use of our nanobodies in inhibiting glioblastoma cell migration. The strongest effect was observed for NB79 targeted against the intracellular protein vimentin, which completely inhibited U87MG cell migration. At last, vimentin was found overexpressed in FFPE glioblastoma tissue samples. We report here the development of a unique method for selection of deregulated glioblastoma proteins. Due to selectivity of NB179 and NB314 towards NCH421k glioblastoma stem cell line we suggest their possible use for development of targeting strategies. As indicated by the migration assay, we anticipate that vimentin plays an important role in the migratory and invasive nature of glioblastoma. Although widely expressed, we recommend further exploration of vimentin as a biomarker for glioblastoma malignancy
