Abstract
Malignant gliomas are among the rarest brain tumours, and they have the worst prognosis. Grade IV astrocytoma, known as glioblastoma multiforme (GBM), is a highly lethal disease where the standard therapies of surgery, followed by radiation and chemotherapy, cannot significantly prolong the life expectancy of the patients. Tumour recurrence shows more aggressive form compared to the primary tumour, and results in patient survival from 12 to 15 months only. Although still controversial, the cancer stem cell hypothesis postulates that cancer stem cells are responsible for early relapse of the disease after surgical intervention due to their high resistance to therapy. Alternative strategies for GBM therapy are thus urgently needed. Nanobodies are single-domain antigen-binding fragments of heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small and stable, and they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments. We first immunised an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. Next, a nanobody library was constructed in a phage-display vector. Using nanobody phage-display technology, we selected specific GBM stem-like cell binders through a number of affinity selections, using whole cell protein extracts and membrane protein-enriched extracts from eight different GBM patients, and membrane protein-enriched extracts from two established GBM stem-like cell lines (NCH644 and NCH421K cells). After the enrichment, periplasmic extract ELISA was used to screen for specific clones. These nanobody clones were recloned into the pHEN6 vector, expressed in Escherichia coli WK6, and purified using immobilised metal affinity chromatography and size-exclusion chromatography. Specific nanobody:antigen pairs were obtained and mass spectrometry analysis revealed two proteins, TRIM28 and β-actin, that were up-regulated in the GBM stem-like cells compared to the controls.
Highlights
Several hallmarks of cancer have been described recently, which include resistance to current treatment modalities of a fraction of tumour cells that show stem-like properties, and are known as cancer stem cells [1, 2].Gliomas, classified as astrocytomas, account for about half of all primary brain tumours [3]
The American Brain Tumour Association has estimated that brain tumours are the second leading cause of cancer-related deaths in children under the age of 20 years and in males aged below 40 years [6] with the incidence of Glioblastoma multiforme (GBM) increasing with age from 30 years onwards [7]
Despite all the research performed in this field, patients suffering from GBM currently have survival prognoses from 12 to 15 months [5], whereas those suffering from recurrent GBM have survival of about 6 months [8]
Summary
Several hallmarks of cancer have been described recently, which include resistance to current treatment modalities of a fraction of tumour cells that show stem-like properties, and are known as cancer stem cells [1, 2].Gliomas, classified as astrocytomas, account for about half of all primary brain tumours [3]. The blood–brain barrier represents a specific problem in the treatment of brain tumours, as it generally prevents the passage of molecules greater than 500 Da into the brain [9]. This puts serious restrictions on the use of chemotherapy, a number of clinical trials employing more targeted treatments have been carried out with the hope of improving GBM patients’ outcomes [10,11,12,13]
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