ARTICLE ABSTRACTIntratumoral extracellular ATP concentrations are 1000 times higher than those in normal tissues of the same cell origin. However, whether or not cancer cells use the abundant extracellular ATP was unknown until we recently reported that cancer cells internalize ATP. The internalized ATP was found to substantially increase intracellular ATP concentration and promote cell proliferation and drug resistance in cancer cells. Here, using a nonhydrolyzable fluorescent ATP (NHF-ATP), radioactive and regular ATP, coupled with high and low molecular weight dextrans as endocytosis tracers and fluorescence microscopy and ATP assays, cultured human NSCLC A549 and H1299 cells as well as A549 tumor xenografts were found to internalize extracellular ATP at concentrations within the reported intratumoral extracellular ATP concentration range. In addition to macropinocytosis, both clathrin- and caveolae-mediated endocytosis significantly contribute to the ATP internalization, which led to an approximately 30% (within 45 minutes) or more than 50% (within 4 hours) increase in intracellular ATP levels after ATP incubation. This increase could not be accounted for by either purinergic receptor signaling or increased intracellular ATP synthesis rates in the ATP-treated cancer cells. These new findings significantly deepen our understanding of the Warburg effect by shedding light on how cancer cells in tumors, which are heterogeneous for oxygen and nutrition supplies, take up extracellular ATP and use the internalized ATP to perform multiple previously unrecognized functions of biological importance. They strongly suggest the existence of ATP sharing among cancer and stromal cells in tumors and simultaneously identify multiple new anticancer targets.Implications: Extracellular ATP is taken up by human lung cancer cells and tumors via macropinocytosis and other endocytic processes to supplement their extra energy needs for cancer growth, survival, and drug resistance, thus providing novel targets for future cancer therapy. Mol Cancer Res; 14(11); 1087–96. ©2016 AACR.