Detection of Extracellular ATP in the Tumor Microenvironment, Using the pmeLUC Biosensor
ATP is one of the main components of the tumor microenvironment, where it affects cell growth, tumor progression and antitumor immune response. The development of the pmeLUC probe, a luciferase engineered to be expressed on the outer facet of the plasma membrane, allowed real-time measurement of extracellular ATP in vitro and in vivo systems, among which the tumor microenvironment. Here we describe the experimental procedures to measure extracellular ATP levels in the tumor microenvironment of three different cancer models generated by the implant of pmeLUC-expressing tumor cells into the appropriate mice strain: ACN human neuroblastoma (nude/nude mice host), WEHI-3B murine leukemia (BALB/c host), and B16F10 murine melanoma (C57Bl/6 host). The procedure to obtain stable expression of pmeLUC in different cell types and methods for the measurement of extracellular ATP with pmeLUC in vitro are also described.
Key wordsExtracellular ATP Plasma membrane luciferase Luciferin Luminescence Luminometry
F.D.V. is supported by grants from the Italian Association for Cancer Research (n. IG 13025 and IG 18581), the Ministry of Health of Italy (n. RF-2011-02348435), the Ministry of Education of Italy (n. 20178YTNWC). E.A. is supported by grant from the Italian Association for Cancer Research (n. IG 16812). Both F.D.V. and E.A. are supported by institutional funds from the University of Ferrara. This work was funded with support from the European H2020 office through the COST Action BM1406 “Ion channels and Immune response.”