Jonathan W. Wojtkowiak, Jennifer M. Rothberg, Virendra Kumar, Karla J. Schramm, Edward Haller, Joshua B. Proemsey, Mark C. Lloyd, Bonnie F. Sloane, Robert J. Gillies

Tumor cell survival relies upon adaptation to the acidic conditions of the tumor microenvironment. To investigate potential acidosis survival mechanisms, we examined the effect of low pH (6.7) on human breast carcinoma cells. Acute low pH exposure reduced proliferation rate, induced a G1 cell cycle arrest, and increased cytoplasmic vacuolization. Gene expression analysis revealed elevated levels of ATG5 and BNIP3 in acid-conditioned cells, suggesting cells exposed to low pH may utilize autophagy as a survival mechanism. In support of this hypothesis, we found that acute low pH stimulated autophagy as defined by an increase in LC3-positive punctate vesicles, double-membrane vacuoles, and decreased phosphorylation of AKT and ribosomal protein S6. Notably, cells exposed to low pH for approximately 3 months restored their proliferative capacity while maintaining the cytoplasmic vacuolated phenotype. Although autophagy is typically transient, elevated autophagy markers were maintained chronically in low pH conditioned cells as visualized by increased protein expression of LC3-II and double-membrane vacuoles. Furthermore, these cells exhibited elevated sensitivity to PI3K-class III inhibition by 3-methyladenine. In mouse tumors, LC3 expression was reduced by systemic treatment with sodium bicarbonate, which raises intratumoral pH. Taken together, these results argue that acidic conditions in the tumor microenvironment promote autophagy, and that chronic autophagy occurs as a survival adaptation in this setting.