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.