Calcium-rich bone microenvironment promotes drug-tolerant persistence in osteosarcoma through ORAI3–Ca²⁺–PINK1–mitophagy axis

Abstract Background Drug-tolerant persister (DTP) cells contribute to local tumor recurrence, yet remain incompletely defined. Given the calcium-rich bone microenvironment and the role of mitochondrial quality control in therapy tolerance, we investigated whether calcium signaling regulates local tumor recurrence through PINK1-mediated mitophagy. Methods Osteosarcoma cells were treated with cisplatin, methotrexate, doxorubicin to establish DTP models. Calcium signaling, ORAI3, NFAT4, and PINK1 expression, mitophagy, mitochondrial homeostasis, and metabolism were assessed using molecular and cellular assays. Inducible PINK1 knockdown xenografts and lung metastasis models were used to evaluate tumor persistence and local recurrence after chemotherapy. Human osteosarcoma tissues with or without chemotherapy were analyzed by immunohistochemistry to assess the clinical relevance of ORAI3 and PINK1. Results Chemotherapy exposure increased intracellular Ca²⁺ levels in osteosarcoma DTP cells and was accompanied by elevated ORAI3 expression. Enhanced calcium signaling promoted NFAT4 nuclear translocation and was associated with increased PINK1 expression. PINK1-dependent mitophagy helped preserve mitochondrial homeostasis and supported a metabolic phenotype characterized by sustained tricarboxylic acid cycle activity and oxidative phosphorylation, which contributed to DTP cell survival under chemotherapeutic stress. In vivo, inducible PINK1 knockdown did not affect tumor growth before chemotherapy but reduced tumor burden during treatment and delayed regrowth after withdrawal. Single-cell and spatial transcriptomic analyses further supported the clinical relevance of this axis. Pharmacological targeting of calcium signaling with GSK-7975 A, particularly in combination with chloroquine, suppressed local recurrence in xenograft, orthotopic, and lung metastasis models. Conclusions These findings uncover an ORAI3-Ca²⁺-NFAT4-PINK1 signaling axis that supports osteosarcoma DTP survival by promoting mitophagy and maintaining mitochondrial metabolic homeostasis. Targeting calcium signaling and mitophagy may represent a potential therapeutic strategy to reduce persister cell survival and limit local recurrence of osteosarcoma after chemotherapy. Significance Osteosarcoma DTP cells driving local recurrence after chemotherapy are critically dependent on calcium signaling and PINK1-mediated mitophagy, revealing a therapeutic opportunity to eliminate these persister cells and enhance long-term treatment efficacy.