Cell density drives glutamine synthesis in fibroblast-like cells in the tumor microenvironment

Dense fibrous tissue, a hallmark of increased breast cancer risk, is composed of abundant extracellular matrix (ECM) and fibroblast-like cells that shape the tumor microenvironment (TME). These fibroblast-like cells can create regions of variable cell density, which in turn influences local nutrient availability. In solid tumors, poor vascularization generates a nutrient-deprived environment where amino acids, particularly glutamine, become limiting. Glutamine is essential for many cell processes, including replenishment of the TCA cycle, antioxidant defense, protein synthesis, and nucleotide production. Aggressive triple-negative breast cancer cells are often unable to synthesize their own glutamine de novo using glutamine synthetase (GS) and rely on exogenous sources for growth and survival. Fibroblast-like cells within the TME may compensate by producing and supplying glutamine through GS activity. Although this metabolic dependency has been demonstrated in ovarian cancer, how GS is regulated in fibroblast-like cells within the breast TME, and whether it is influenced by cell density, remains unknown. We hypothesize that high cell density acts as a non-cell-autonomous metabolic cue, upregulating GS in fibroblast-like cells under glutamine-deprived conditions. To test this, fibroblasts from mammary, skin, and lung tissue, as well as progenitor fibroblast-like cell lines (10T1/2 and MC3T3), were cultured at varying densities in the presence or absence of exogenous glutamine. Preliminary data show that near-confluent fibroblast-like cells upregulate GS and maintain active protein translation despite glutamine deprivation, suggesting that dense cultures adopt a metabolic state that gives them an added advantage compared to less-confluent cells. These findings point to a potential mechanism by which fibroblast-like cells in the breast TME promote glutamine availability and, consequently, tumor progression. Because direct GS inhibition is neurotoxic, identifying upstream, density-dependent regulators of GS represents a promising therapeutic avenue for selectively disrupting glutamine supply to aggressive breast cancers.