Tolerance to extracellular acidic pH facilitates tumor plasticity

Cancer cells maintain glycolytic metabolism despite oxygen availability, resulting in an acidic microenvironment mediated by enhanced proton and lactate secretion. We have previously reported that this acidic tumor microenvironment induces the activation of the cholesterol biosynthesis pathway and polyamine pathway. Although these metabolic adaptations have been well documented, the mechanisms governing cell survival under acidic conditions are poorly understood. We first demonstrated that severe acidification (pH 5.6) triggered necroptotic cell death, whereas moderate acidification (pH 6.8) prevented necroptosis and allowed anchorage-independent survival and tumor initiation. At pH 5.6, PANC1 and MIA-PaCa2 pancreatic cancer cells exhibited phosphorylation of RIP1 and MLKL, and Calcein-AM live imaging confirmed membrane-rupturing cell death; these features were not observed at pH 6.8. In addition, when PANC1 floating cells (pH 7.4 or pH 6.8) were subcutaneously injected into SCID/SCID mice, tumor initiation was observed only in the mice implanted with pH 6.8 floating cells by day 7. RNA sequencing comparing adherent and floating cells at pH 6.8 and pH 7.4 revealed activation of the complement pathway specifically in pH 6.8 floating cells, suggesting that complement inhibition may selectively eliminate acid-tolerant cancer cells. A genome-wide CRISPR-Cas9 knockout screen in chronically acid-exposed PANC1 cells identified FAM129C as a critical regulator of acid tolerance and cell survival. In xenograft models, FAM129C overexpression led to downregulation of PIGR. Conversely, in murine pancreatic Pan02 cells overexpressing PIGR, immune profiling showed enhanced immune cell infiltration, particularly increased macrophage infiltration. Finally, we evaluated the therapeutic efficacy of combining an anti-PD-L1 antibody (alphaPD-L1) with PMX53, a complement inhibitor. This synergistic treatment markedly suppressed tumor growth in PIGR-overexpressing Pan02 tumors. Immunohistochemistry revealed that the combination therapy increased IFN-gamma⁺ CD8⁺ T cells and reduced macrophage infiltration within the tumor microenvironment. Our findings reveal a previously uncharacterized pathway that evades necroptotic cell death and enhances cellular plasticity under acidic stress, offering new therapeutic approaches for cancer treatment through pH-dependent cell death modulation.