Chemopreventive Targeting of Oncogenic Stemness: EGCG-Mediated Suppression of the HIF-1α-PPARγ-Cancer Stem Cells Transcriptional Signature in 3D Glioblastoma Spheroids

<b>Background:</b> Preventing the emergence and persistence of cancer stemness represents a promising strategy to reduce tumor aggressiveness and therapeutic failure. Cancer stem cells (CSCs), which contribute significantly to therapy resistance, recurrence, and metastasis, are sustained in part by metabolic reprogramming that enhances survival and self-renewal under stress conditions. <b>Methods:</b> To model the hypoxic core of solid tumors, three-dimensional (3D) glioblastoma (GBM) spheroid cultures were generated using human U87, U118, U138, and U251 cell lines and compared to their respective two-dimensional (2D) monolayer cultures. Total RNA was extracted, and gene expression was analyzed via RT-qPCR and targeted gene arrays. Transient gene silencing was performed using specific siRNAs, while pharmacological intervention involved treatment with (-)-Epigallocatechin-3-gallate (EGCG), a bioactive phytochemical derived from green tea. Adipogenesis was evaluated using Oil Red O staining. <b>Results:</b> Compared to conventional 2D cultures, 3D spheroids exhibited elevated expression of hypoxia-inducible factor-1 alpha (HIF-1α) and upregulation of peroxisome proliferator-activated receptor gamma (PPARγ), identified through adipogenesis array screening. Adipogenic activity persisted in the 3D spheroid model, and EGCG treatment effectively suppressed the upregulation of <i>HIF-1α</i> and <i>PPARG</i> transcripts. This led to a significant downregulation of adipogenic genes (<i>CEBPD</i>, <i>FOXO1</i>, <i>BMP2</i>, <i>BMP7</i>) and CSC-associated markers (<i>CD44</i>, <i>PROM1</i>, <i>ABCB5</i>, <i>ABCG2</i>), accompanied by reduced spheroid growth. <b>Conclusions:</b> These findings underscore EGCG's chemopreventive potential in disrupting early HIF-1α-mediated molecular pathways that reinforce GBM stemness. By targeting hypoxia-driven metabolic reprogramming, EGCG offers a dietary-based approach to modulate the CSC niche and potentially delay or prevent GBM progression. Moreover, the use of 3D spheroid models highlights their relevance in preclinical chemoprevention research, bridging the gap between simplistic 2D cultures and the complex biology of solid tumors.