Metformin inhibits NF-κB p65/RelA-NLRP3 inflammasome-IL-1β axis, attenuates lipid droplet accumulation, and reprograms CD14/CD16 expression in monocytes exposed to colorectal tumor-conditioned medium.

Background: Colorectal cancer (CRC) generates a complex tumor microenvironment (TME) known to profoundly alter the function and phenotype of immune cells, including monocytes. Key aspects of this environment can be mimicked by tumor-conditioned medium (TCM). Metformin has emerged as a promising candidate to counteract TCM-induced immune and inflammatory dysregulation. Therefore, this study aimed to evaluate the effects of metformin on NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome expression, monocyte subset distribution, and lipid droplet (LD) accumulation upon exposure to colorectal TCM.

Methods: Assays were performed on primary human monocytes exposed to colorectal TCM in the presence or absence of metformin.

Results: TCM significantly increased nitric oxide (NO, p < 0.001) and hydrogen peroxide (H₂O₂, p < 0.001) production, intracellular free calcium ions (_ifCa²⁺) (p < 0.001) levels, expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB p65/RelA, p < 0.001), NLRP3 (p < 0.01), and interleukin-1 beta (IL-1β, p < 0.0001), as well as intracellular triacylglycerols (_iTAGs, p < 0.05), total cellular cholesterol content (_tccCHOL, p < 0.001), and lipid droplet accumulation (p < 0.05). It also impaired phagocytic activity (p < 0.05) and altered monocyte phenotype, along with a shift toward a CD14^low, CD16^high phenotype (p < 0.0001 for both markers). Notably, metformin treatment exerted broad and significant reversing effects, specifically on respiratory burst (NO, p < 0.05; H₂O₂, p < 0.01), _ifCa²⁺ levels (p < 0.01), NF-κB p65/RelA and NLRP3 expression, and IL-1β production (p < 0.0001 for all), and the lipid droplet accumulation (p < 0.0001), _iTAGs (p < 0.001), and _tccCHOL (p < 0.01). Metformin also significantly restored CD14 expression (p < 0.05) and increased CD14⁺ monocyte frequency (p < 0.01), while reducing CD16 expression (p < 0.05) and CD16⁺ monocyte frequency (p < 0.01). However, it had no significant effect on phagocytosis in TCM-exposed monocytes (p > 0.05).

Conclusions: Our findings highlight metformin's selective immunometabolic reprogramming capacity in monocytes exposed to colorectal tumor-derived signals, supporting its potential as a context-specific immunomodulator. This study lays the groundwork for future translational research on metformin as an adjunctive agent in inflammation-driven tumor settings.