Extracellular ATP Acts as Messenger in Immunity and Cancer [Apr. 1, 2025] DOJINDO LABORATORIES

Previous Science Note

Extracellular ATP, released from dying or stressed cells, acts as a key damage-associated molecular pattern (DAMP) that signals immune cells and neighboring cells via purinergic receptors such as P2X7 and P2Y2. This Science Note introduces recent advances in our understanding of how extracellular ATP influences the immune system and the tumor microenvironment.

Colon tumour cell death causes mTOR dependence by paracrine P2X4 stimulation (Nature, 2022)

Colon tumor cells undergoing chemotherapy-induced cell death release extracellular ATP, which acts on neighboring tumor cells to activate mTOR signaling via P2X4 and promote their survival.

Highlighted technique: This study used Lgr5-EGFP-DTR(+) mice, which allow selective induction of apoptosis in Lgr5-positive (stem) cells by diphtheria toxin. Colon tumor organoids derived from these mice enabled targeted ablation of Lgr5-positive tumor cells, allowing analysis of the impact of their death on neighboring Lgr5-negative cells.

Related technique Extracellular ATP Assay, Annexin V Apoptosis Plate Assay

Cancer cells dying from ferroptosis impede dendritic cell-mediated anti-tumor immunity (Nature Communications, 2022)

To study the immune effects of ferroptosis cells, the researchers divided ferroptosis into initial, intermediate, and terminal phases, defined by lipid peroxidation, ATP release, and HMGB1 release with membrane rupture. Unlike apoptotic cells, ferroptosis cells fail to activate immunity through extracellular ATP and other signals, particularly in the initial phase, where they suppress dendritic cell maturation and phagocytosis.

Highlighted technique: This study established a model to precisely control cell death by inducing GPX4 knockdown, which triggers ferroptosis, and temporarily blocking ferroptosis by adding ferrostatin-1. Removal of ferrostatin-1 triggers synchronised ferroptosis, allowing the process of ferroptosis to be precisely controlled.

Related technique Intracellular Iron detection, Lipid Peroxide Detection

P2RX7 Enhances Tumor Control by CD8⁺ T Cells in Adoptive Cell Therapy (Cancer Immunology Research, 2022)

In melanoma, extracellular ATP enhances the antitumor activity of CD8⁺ T cells by maintaining their mitochondrial function through the purinergic receptor P2RX7.

Highlighted technique: Adoptive cell therapy (ACT) is a cancer treatment in which ex vivo activated immune cells are reinfused into the patient. As mitochondrial function is key to ACT efficacy, this study comprehensively evaluates T cell function using multiple parameters, including mitochondrial mass, mitochondrial ROS, oxygen consumption rate and extracellular acidification rate.

Application Note I
> Apoptosis Inhibition Enhances ATP Release

Jurkat cells were treated with or without Z-VAD, an apoptosis inhibitor, and then treated with staurosporine. After treatment, changes in extracellular ATP release, cell viability and extracellular LDH release were assessed over time. The results showed that cell death was inhibited by Z-VAD, but extracellular ATP released during the initial phase of apoptosis increased over time.

＜Product in use＞
Extracelluar ATP
Extracellular ATP Assay Kit-Luminescence

LDH release
Cytotoxicity LDH Assay Kit-WST

Cell viability
Cell Counting Kit-8

Application Note II
> Ferroptosis Induction and ATP Release Profile

Changes in extracellular ATP release, cell viability, extracellular LDH release, phosphatidylserine, and intracellular Fe²⁺ were evaluated in HeLa cells treated with various concentrations of Erastin, a ferroptosis inducer, for 24 hours. The results showed that cell viability decreased and extracellular ATP release and extracellular LDH increased in cells treated with Erastin concentration of 25 μmol/l, indicating that cell death was induced under high concentration conditions. Interestingly, the increase in extracellular ATP in the early phase of stimulation, which was observed with the apoptosis inducer Staurosporine, was not observed with Erastin (See Experimental Example: Evaluation using Staurosporine-treated Cells.). Although the apoptosis-related marker phosphatidylserine was not significantly altered by Erastin treatment at any concentration. The amount of intracellular Fe²⁺, a ferroptosis-related marker, was significantly increased under the low-concentration treatment condition, indicating that it tends to increase before actual cell death occurs.

＜Product in use＞
Extracellular ATP:
Extracellular ATP Assay Kit-Luminescence

Extracellular LDH:
Cytotoxicity LDH Assay Kit-WST

Cell viability: Cell Counting Kit-8

Phosphatidylserine:
Annexin V Apoptosis Plate Assay Kit

Intracellular Fe²⁺: FerroOrange

Related Techniques

Target	Kit & Probes
Apoptosis detection in multiple samples	Annexin V Apoptosis Plate Assay Kit
Intracellular / mitochondrial ferrous ion (Fe²⁺) detection	FerroOrange, Mito-FerroGreen
Intracellular / mitochondrial lipid peroxidation detection	Liperfluo, MitoPeDPP
Mitochondrial membrane potential detection	JC-1 MitoMP Detection Kit, MT-1 MitoMP Detection Kit
Mitochondrial superoxide detection	MitoBright ROS Deep Red - Mitochondrial Superoxide Detection
Total ROS detection	Highly sensitive DCFH-DA or Photo-oxidation Resistant DCFH-DA
Cell proliferation/ cytotoxicity assay	Cell Counting Kit-8 and Cytotoxicity LDH Assay Kit-WST