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

Abstract

Background: 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. Methods: 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. Results: 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. Conclusion: 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.

Analysis of miR-9-5p, miR-124-3p, miR-21-5p, miR-138-5p, and miR-1-3p in Glioblastoma Cell Lines and Extracellular Vesicles

“…FREM2 and SPRY1 have been proposed as potential new markers of GBM, and have been demonstrated to be significantly upregulated in GBM at the mRNA and/or protein level in GBM cell lines and GBM tissues compared to references (astrocytes or reference non-malignant brain tissue) [6,19]. Furthermore, VIM, NCL, and NAP1L1 were significantly overexpressed in GBM tissues and/or cells compared to the reference [20,21]. Some of the proposed genes have also been implicated in other cancers, for example gastric cancer (vimentin) [37], large B-cell lymphoma (nucleolin) [38], and pancreatic cancer (NAP1L1) [39].…”

“…Finally, we investigated whether the target genes of the selected miRNAs-NAP1L1, FREM2, SPRY1, VIM, and NCL-that have been associated with GBM in our previous studies [6, [19][20][21] were also present in the sEVs of different cell types and showed any particular expression pattern in GBM-isolated sEVs compared to normal astrocyte cells. qPCR was used for analysis.…”

“…We then compared the gene expression in sEVs with the expression in cells types, as previously published [19,21]. The expression profile of VIM gene in sEVs followed the expression in cells, while the expression profile of the NAP1L1, NCL, SPRY1, and FREM2 genes in sEVs did not follow the expression profile of the same genes in different cell types [19,21].…”

“…1. Selection of a Set of miRNAs (miR-9-5p, miR-21-5p, miR-124-3p, miR-138-5p, and miR-1-3p) That Target VIM, NCL, NAP1L1, FREM2, and SPRY1 Genes Candidate miRNAs were selected based on their target genes, vimentin (VIM), nucleolin (NCL), nucleosome assembly protein-1 like-1 (NAP1L1), FRAS1-related extracellular matrix protein 2 (FREM2), and sprouty RTK signaling antagonist 1 (SPRY1) [6, [19][20][21], which were previously associated with GBM in our studies. The selection was based on the prediction of the high probability and consistency of miRNA candidates among TargetScan [22], miRDB [23,24], and microT-CDS datasets [25,26].…”

TUFM‑knockdown inhibits the migration and proliferation of gastrointestinal stromal tumor cells

“…Therefore, the results of the present study, which demonstrated that late apoptotic cells were increased in the TUFM-silenced GIST-T1 cell line, are consistent with the aforementioned research revealing inhibited mitochondrial autophagy and damaged mitochondrial scavenging in TUFM stable knockout mice and TUFM-silenced 293T cells. These changes lead to considerable mitochondrial disintegration factor release and increased apoptosis (40). TUFM silencing directly induces mitochondrial protein synthesis inhibition, membrane dysfunction and mitochondrial structural dysfunction, which eventually leads to mitochondrial damage or disintegration, which is an important contributor to tumor cell apoptosis (41).…”