Altered Magnesium Environments Restrict Colorectal HT-29 Spheroid Growth by Disturbing Cellular Mg2+ Homeostasis.

Dysregulated magnesium (Mg2+) homeostasis contributes to colorectal cancer (CRC), yet its context-dependent function within the tumor microenvironment remains unresolved. This study aimed to determine how sustained low and high extracellular Mg2+ environments affect CRC spheroid (SP) growth and Mg2+ homeostasis using HT-29 SPs. We analyzed Mg2+ flux, the expression of Mg2+ transporters (e.g., Transient Receptor Potential Melastatin (TRPM) 6), viability, apoptotic and autophagic markers, and phospho-/oxidoproteomic alterations. Both Mg2+ extremes destabilized SP architecture, reduced viability, and induced apoptosis and autophagy, with SPs displaying heightened vulnerability relative to 2D cultures. Mg2+ stress impaired Mg2+ influx and eliminated adaptive transporter regulation in SPs. Loss of membrane TRPM6/7 heterodimers, driven by altered phosphorylation (e.g., TRPM6 Serine 141, Serine 1252, Threonine 1851) and elevated oxidation (e.g., Methionine 1755), suppressed channel activity. High Mg2+ caused profound metabolic failure despite increased total Mg2+, reflecting functional Mg2+ deficiency. CRC spheroids are acutely susceptible to Mg2+ imbalance due to collapsed transporter homeostasis and post-translational inhibition of Mg2+ channels. These findings reveal a targetable metabolic vulnerability and support the therapeutic potential of localized Mg2+ modulation in CRC.