Omega-3 polyunsaturated fatty acids and epigallocatechin-3-gallate attenuate obesity-associated CD44-high stemness in colorectal cancer through modulation of Notch1, Wnt/β-catenin and PI3K/mTOR signaling

Obesity is a major risk factor for colorectal cancer (CRC), promoting tumor initiation through chronic inflammation and metabolic dysregulation. Cancer stem cells (CSCs) drive CRC progression through Notch1, Wnt/β-catenin (Wnt), and PI3K/mTOR (mTOR) signaling. Omega-3 polyunsaturated fatty acids (EPA and DHA) and epigallocatechin-3-gallate (EGCG) may modulate these pathways. This study investigated the effects of obesogenic-like stimuli on CSC-related CRC signaling and the preventive potential of EPA:DHA-EGCG in patient-derived organoids (PDOs) from normal mucosa (NM) of non-obese (non-OB) patients. CD133 and CD44 expression was evaluated in tumor tissues and matched NM from obese (OB) and non-OB CRC patients. PDOs derived from NM of non-OB patients were exposed to an adipocyte-conditioned medium reproducing an obesogenic adipokine profile, with or without EPA:DHA (1:1) plus EGCG (EDE). Viability, morphology, and molecular markers of stemness, differentiation, and CRC-related pathways were assessed. OB CRC tissues exhibited a CSC change from CD133-high/CD44-low to CD44-high/CD133-low compared with non-OB CRC. In PDOs, OB-EDE recapitulated this CD44-high/CD133-low phenotype, increasing organoid viability and size while maintaining KRT20 comparable to control (CTRL). HES1 was significantly upregulated, consistent with modulation of Notch1 signaling. OB-EDE downregulated Wnt target genes and increased the mTOR downstream effector p-S6R. OB + EDE exerted marker- and pathway-specific effects, maintaining PROM1 at CTRL levels, reducing CD44 and HES1, and increasing KRT20, while LGR5 remained suppressed. Compared with OB-EDE, OB + EDE further reduced the expression of Wnt target genes and reduced p-S6R to CTRL, whereas p-mTOR/mTOR was not significantly affected. Overall, obesogenic-like stimuli were associated with a CD44-high stem-like phenotype accompanied by coordinated changes in Notch1, Wnt and mTOR signaling. EDE exerted differential effects on these molecular alterations, supporting its potential as a complementary bioactive strategy in obesity-associated CRC. Pharmacological inhibition using DAPT provided preliminary functional support for a contribution of Notch1 signaling to the maintenance of this obesogenic phenotype.