Phenotypic Plasticity upon GSC Differentiation

At the cellular level, the malignant characteristics of GBM are largely attributed to GBM stem cells (GSCs). These cells, endowed with stem-like properties, can self-renew, generate diverse cancerous cell populations, and initiate tumors in vivo. The GSC lines, NCH644 and NCH421k, when cultured as neurospheres (NS) — a condition that preserves their stem-like state, as evidenced by the expression of stem cell markers like SOX2, CD133, and Nestin — were found to align with the PN subclass and CL-B/C respectively. For the NCH644 and NCH421k lines in both stem-like and differentiated states, total RNA was isolated, and biotinylated single-strand cDNA targets were prepared using the Ambion WT Expression Kit (Cat #4411974) and the Affymetrix GeneChip® WT Terminal Labeling Kit (Cat #900671), following manufacturer guidelines. The cDNAs were hybridized onto GeneChip® Human Gene 2.0 ST arrays (Affymetrix), targeting over 40,000 RefSeq transcripts and approximately 11,000 LncRNAs. Chips were processed and scanned using Affymetrix equipment and software. Raw data was then analyzed and processed using Affymetrix Expression Console software version 1.4.1 with the default RMA settings.