Article
Impact of the CRISPR/Cas9-mediated editing of the Prom1/CD133 gene on viability, stemness and therapy-sensitivity of stem-like glioma cells
Auswirkungen der CRISPR/Cas9-vermittelten Editierung des Prom-1/CD133-Gens auf Viabilität, Stammzellcharakter und Therapiesensitivität von stammzellähnlichen Gliomzellen
Search Medline for
Authors
-
Till Jasper Kahl
- Universitätsklinikum Schleswig-Holstein, Experimentelle Neuroonkologie, Lübeck, Deutschland
-
Jan Kruse
- Universitätsklinikum Schleswig-Holstein, Experimentelle Neuroonkologie, Lübeck, Deutschland
-
Enrico Riebel
- Universitätsklinikum Schleswig-Holstein, Experimentelle Neuroonkologie, Lübeck, Deutschland
-
Edith Pawlak
- Universitätsklinikum Schleswig-Holstein, Experimentelle Neuroonkologie, Lübeck, Deutschland
-
Volker M. Tronnier
- Universitätsklinikum Schleswig-Holstein, Experimentelle Neuroonkologie, Lübeck, Deutschland
-
Christina Zechel
- Universitätsklinikum Schleswig-Holstein, Experimentelle Neuroonkologie, Lübeck, Deutschland
| Published: | June 26, 2020 |
|---|
Outline
Text
Objective: The glioblastoma multiforme (GBM) and the gliosarcoma (GSarc) are the most aggressive and lethal primary brain tumors in adults. Therapy options which include chemotherapy with temozolomide (TMZ), radiotherapy and surgery are basically palliative. Even under optimized chemotherapy complete tumor regression is not achievable. To a large extent therapy resistance is caused by so-called stem-like glioma cells (SLGCs). SLGCs display characteristics of neural stem cell cells like self-renewal, indefinite proliferation and the expression of neural markers. Moreover, SLGCs tolerate irradiation and TMZ treatment much better than non-SLGCs. Controversy exists about the significance and function of the cell surface protein Prominin-1 (Prom1)/CD133.
Methods: We generated functional Prom1/CD133 knock-outs in genetically distinct GBM- and GSarc-derived human SLGCs in order to investigate the impact of this protein on stemness, viability and therapy resistance. We infected nine GBM-derived and two GSarc-derived SLGC lines with a replication-deficient lentiviral vector containing the genetic information for targeting exon 4 of the Prom1/CD133 gene with the prokaryotic endonuclease Cas9. In this, the CRISPR-Cas9 system targeted the sequence which essentially encodes the transmembrane domain 1 of the protein.
Results: Infected cells, which survived a 7-day Puromycin selection were subjected to limited dilution assays followed by further expansion. The proof of successful editing was obtained by Sanger sequencing, as well as analyses of protein expression by Western blotting, immunocytochemistry and flow-cytometry (FACS). SLGCs harboring large deletions and/or truncations of the Prom1/CD133 protein showed self-renewal and expressed high levels of the pluripotency factor Sox2 (sex determining region Y (SRY)-box 2). Clones carrying functional Prom1/CD133 inactivation displayed a similar differentiation capacity as their wildtype counterparts. Ongoing experiments compare the TMZ and radiation sensitivity of the Prom1/CD133-edited clones with that of their wildtype counterparts.
Conclusion: Overall it appeared that the functional biallelic knock-out of the Prom-1/CD133 gene does not impair viability and stemness of stem-like glioma cells.
