The Identification and Validation of Novel Aptamers to Glioma

Abstract

Glioma is a cancer derived from transformed glial cells; these tumours are often invasive and display a heterogeneous cell population, with various phenotypes. Current treatment modalities for glioma are ineffective, with minimal improvement to overall patient survival over the last decade. Targeted approaches that take advantage of the various biomarkers displayed within glioma are required to deliver therapeutics more specifically. Aptamers are short oligonucleotides that conform to unique three dimensional structures based on sequence and are able to target molecules selectively, in order to deliver therapeutic agents directly to the tumour. Aptamers can be selected against whole cells via cell-SELEX, for production of a targeting ligand, which may also have the secondary advantage of revealing novel biomarkers if the target of the aptamer is elucidated. Previously, the glioblastoma cell line U87MG has been utilised in cell-SELEX for aptamer identification, however cell lines may not ubiquitously express all biomarkers indicative of glioblastoma. Consequently, cell-SELEX using short term cultures from human tissue may produce aptamers that target novel biomarkers. As primary cells are only available in small numbers, a cell-SELEX procedure was developed that was able to identify aptamers using 100 cells. Utilising this procedure, seven novel DNA aptamers were selected to short term cultures. Validation studies using oligohistochemistry showed the aptamers may interact with a protein on the cell surface. DNA aptamers were also identified towards two known biomarkers important for gliomagenesis; EGFRvIII and PDE1C. Ten DNA aptamers were isolated towards a peptide within the mutated region for EGFRvIII and four aptamers were targeted towards the PDE1C protein. Whilst validation studies via Western blot showed no aptamer-protein interaction when using the EGFRvIII aptamers, denaturing and native Western blots showed positive results displaying 3 bands indicative of PDE1C isoforms. PDE1C aptamer targets were pulled out of native cell lysate via an aptoprecipitation assay, which showed similar banding patterns to the Western blots. The results presented suggest the potential of aptamers to target short term cultures and also protein biomarkers that are over-expressed or truncated within glioma. Whilst further studies are required to elucidate binding characteristics, these aptamers have the potential to assist therapeutic agents become more specific.