Abstract
Glioblastoma multiforme (GBM) is the most common form of brain cancer, with an average life expectancy of fewer than two years post-diagnosis. We have previously reported that cancer cell originated exosomes, including GBM, have NANOG and NANOGP8 DNA associated with them. The exosomal NANOG DNA has certain differences as compared to its normal counterpart that are of immense importance as a potential cancer biomarker. NANOG has been demonstrated to play an essential role in the maintenance of embryonic stem cells, and its pseudogene, NANOGP8, is suggested to promote the cancer stem cell phenotype. Similarly, SOX2 is another stemness gene highly expressed in cancer stem cells with an intimate involvement in GBM progression and metastasis as well as promotion of tumorigenicity in Neuroblastoma (NB). Since exosomes are critical in intercellular communication with a role in dissipating hallmark biomolecules responsible for cancer, we conducted a detailed analysis of the association of the SOX2 gene with exosomes whose sequence modulations with further research and appropriate sample size can help to identify diagnostic markers for cancer. We have detected SOX2 DNA associated with exosomes and have identified some of the SNPs and nucleotide variations in the sequences from a GBM and SH-SY5Y sample. Although a further systematic investigation of exosomal DNA from GBM and NB patient’s blood is needed, finding of SOX2 DNA in exosomes in the current study may have value in clinical research. SOX2 is known to be misregulated in cancer cells by changes in miRNA function, such as SNPs in the binding sites. Our finding of cancer-specific SNPs in exosomal SOX2 DNA sequence may reflect those changes in the cancer stem cells as well as cancer cells. A series of our study on embryonic stem cell gene analysis in exosomal DNA may lead to a minimally invasive exosome-based diagnosis, and give us a key in understanding the mechanisms of cancer formation, progression, and metastasis.
Highlights
In our previously published study, we have reported that differential sequences found in pseudo/retro-oncogene NANOGP8 can be used as potential diagnostic biomarker for Glioblastoma multiforme (GBM)
To detect SOX2 DNA, exosomes precipitated from the spent media of neural stem cells (NSC), GBM cancer cells, CD133+ GBM and neuroblastoma cancer cells SH-SY5Y were used
We have reported that GBM tissue contains GBM cancer cells (CD133-), GBM cancer stem cells (CSCs) (CD133+), NSCs and normal neural cells
Summary
Methods and materialsCell cultureHuman glioblastoma tumor masses were removed from a patient undergoing craniotomy for primary resection of newly diagnosed tumor identified by magnetic resonance imaging. The patient provided Institutional Review Board-approved informed consent for the study prior to the surgery. Primary GBM cells were collected by dissociation of one human brain tumor patient specimen in accordance with a human subject protection protocol approved by Florida Hospital Institutional Review Board (Florida Hospital renamed as “AdventHealth”). Following the manufacturer’s protocol, the CSCs ( mentioned as CD133+ GBM) were separated from the GBM primary cells using CD133 antibody conjugated with magnetic beads (Miltenyi Biotec, CD133 microbeads, human, Mat. No 120-000-312). The cells were cultured in NSC media containing Heparin (0.5 U/ mL), EGF 20 ng/mL, bFGF 20 ng /mL and 2% B27 in DMEM/F12. Human neuroblastoma cell line SH-SY5Y was procured from ATCC (#CRL-2266) and the cells were cultured in tissue culture treated adherent flasks using NT2 (NTERA-2 human embryonal carcinoma cell line) media containing DMEM-F12 supplemented with 10% exosome-depleted FBS
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