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Anti-vimentin, anti-TUFM, anti-NAP1L1 and anti-DPYSL2 nanobodies display cytotoxic effect and reduce glioblastoma cell migration.

Therapeutic advances in medical oncology (2020-05-20)
Alja Zottel, Ivana Jovčevska, Neja Šamec, Jernej Mlakar, Jernej Šribar, Igor Križaj, Marija Skoblar Vidmar, Radovan Komel
ABSTRACT

Glioblastoma is a particularly common and very aggressive primary brain tumour. One of the main causes of therapy failure is the presence of glioblastoma stem cells that are resistant to chemotherapy and radiotherapy, and that have the potential to form new tumours. This study focuses on validation of eight novel antigens, TRIM28, nucleolin, vimentin, nucleosome assembly protein 1-like 1 (NAP1L1), mitochondrial translation elongation factor (EF-TU) (TUFM), dihydropyrimidinase-related protein 2 (DPYSL2), collapsin response mediator protein 1 (CRMP1) and Aly/REF export factor (ALYREF), as putative glioblastoma targets, using nanobodies. Expression of these eight antigens was analysed at the cellular level by qPCR, ELISA and immunocytochemistry, and in tissues by immunohistochemistry. The cytotoxic effects of the nanobodies were determined using AlamarBlue and water-soluble tetrazolium tests. Annexin V/propidium iodide tests were used to determine apoptotsis/necrosis of the cells in the presence of the nanobodies. Cell migration assays were performed to determine the effects of the nanobodies on cell migration. NAP1L1 and CRMP1 were significantly overexpressed in glioblastoma stem cells in comparison with astrocytes and glioblastoma cell lines at the mRNA and protein levels. Vimentin, DPYSL2 and ALYREF were overexpressed in glioblastoma cell lines only at the protein level. The functional part of the study examined the cytotoxic effects of the nanobodies on glioblastoma cell lines. Four of the nanobodies were selected in terms of their specificity towards glioblastoma cells and protein overexpression: anti-vimentin (Nb79), anti-NAP1L1 (Nb179), anti-TUFM (Nb225) and anti-DPYSL2 (Nb314). In further experiments to optimise the nanobody treatment schemes, to increase their effects, and to determine their impact on migration of glioblastoma cells, the anti-TUFM nanobody showed large cytotoxic effects on glioblastoma stem cells, while the anti-vimentin, anti-NAP1L1 and anti-DPYSL2 nanobodies were indicated as agents to target mature glioblastoma cells. The anti-vimentin nanobody also had significant effects on migration of mature glioblastoma cells. Nb79 (anti-vimentin), Nb179 (anti-NAP1L1), Nb225 (anti-TUFM) and Nb314 (anti-DPYSL2) nanobodies are indicated for further examination for cell targeting. The anti-TUFM nanobody, Nb225, is particularly potent for inhibition of cell growth after long-term exposure of glioblastoma stem cells, with minor effects seen for astrocytes. The anti-vimentin nanobody represents an agent for inhibition of cell migration.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-Vimentin antibody, Mouse monoclonal, clone V9, purified from hybridoma cell culture
Sigma-Aldrich
Anti-Mouse IgG (whole molecule)−Alkaline Phosphatase antibody produced in goat, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Anti-CRMP2 antibody produced in rabbit, ~1.0 mg/mL, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Monoclonal Anti-NAP1L1 antibody produced in mouse, clone 2A9, purified immunoglobulin, buffered aqueous solution
Sigma-Aldrich
Anti-DPYSL2 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Anti-Aly antibody, Mouse monoclonal, clone 11G5, purified from hybridoma cell culture
Sigma-Aldrich
Monoclonal Anti-TUFM antibody produced in mouse, Prestige Antibodies® Powered by Atlas Antibodies, clone CL2242, purified immunoglobulin, buffered aqueous glycerol solution
Sigma-Aldrich
Anti-Rabbit IgG (H+L), F(ab′)2 fragment, CF640R antibody produced in goat, ~2 mg/mL, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Anti-Rabbit IgG (whole molecule)–Alkaline Phosphatase antibody produced in goat, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Anti-CRMP1 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Anti-Mouse IgG (H+L), F(ab′)2 fragment, CF640R antibody produced in goat, ~2 mg/mL, affinity isolated antibody, buffered aqueous solution