Fluorescence Microscopy for ATP Internalization Mediated by Macropinocytosis in Human Tumor Cells and Tumor-xenografted Mice

Abstract

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as drug resistance, epithelial-mesenchymal transition (EMT), and metastasis. Intratumoral eATP is 10³ to 10⁴ times higher in concentration than in normal tissues. While eATP functions as a messenger to activate purinergic signaling for EMT induction, it is also internalized by cancer cells through upregulated macropinocytosis, a specific type of endocytosis, to perform a wide variety of biological functions. These functions include providing energy to ATP-requiring biochemical reactions, donating phosphate groups during signal transduction, and facilitating or accelerating gene expression as a transcriptional cofactor. ATP is readily available, and its study in cancer and other fields will undoubtedly increase. However, eATP study remains at an early stage, and unresolved questions remain unanswered before the important and versatile activities played by eATP and internalized intracellular ATP can be fully unraveled. These authors' laboratories' contributions to these early eATP studies include microscopic imaging of non-hydrolysable fluorescent ATP, coupled with high- and low-molecular weight fluorescent dextrans, which serve as macropinocytosis and endocytosis tracers, as well as various endocytosis inhibitors, to monitor and characterize the eATP internalization process. This imaging modality was applied to tumor cell lines and to immunodeficient mice, xenografted with human cancer tumors, to study eATP internalization in vitro and in vivo. This paper describes these in vitro and in vivo protocols, with an emphasis on modifying and finetuning assay conditions so that the macropinocytosis-/endocytosis-mediated eATP internalization assays can be successfully performed in different systems.

Figure 1:. Structures of nonhydrolyzable fluorescent ATP and tetramethylrhodamine labeled high molecular weight fluorescent dextran.

(A) Structure of NHF-ATP. (B) Schematic representation of HMWFD. Abbreviations: ATP = adenosine triphosphate; NHF-ATP = nonhydrolyzable fluorescent ATP; TMR = tetramethylrhodamine; HMWFD = high molecular weight fluorescent dextran.

Figure 2:. In vitro procedure to examine ATP internalization.

Schematic representation of the protocol to visualize the internalization of extracellular ATP in cultured cancer cells using fluorescence microscopy.

Figure 3:. In vivo procedure to examine ATP internalization.

Schematic representation of the protocol to visualize the internalization of extracellular ATP in tumor xenografts using cryosectioning and fluorescence microscopy.

Figure 4:. A549 cells internalize NHF-ATP, which co-localizes with HMWFD in vitro.

Fluorescence microscopy of A549 cells incubated with both 1 mg/mL HMWFD (left panel, red) and 10 μM NHF-ATP (middle panel, green) for 30 min. HMWFD and NHF-ATP co-localize in macropinosomes (right panel, merged yellow). Insets show high magnification of boxed regions. Scale bars = 20 μm. Abbreviations: NHF-ATP = nonhydrolyzable fluorescent ATP; HMWFD = high molecular weight fluorescent dextran.

Figure 5:. A549 cells internalize NHF-ATP along with HMWFD ex vivo.

Fluorescence microscopy of tumorigenic A549 cells xenografted into immunodeficient (Nu/J) mice; NHF-ATP internalization was performed ex vivo. Surgically removed tumors were incubated (ex vivo) with 8 mg/mL HMWFD (left panel, red) and 100 μM NHF-ATP (middle panel, green). Co-localization of cellular HMWFD and NHF-ATP is shown as a merged image (right panel, yellow). Scale bars = 20 μm. Abbreviations: NHF-ATP = nonhydrolyzable fluorescent ATP; HMWFD = high molecular weight fluorescent dextran.

Figure 6:. A549 cells internalize NHF-ATP along with HMWFD in vivo.

Fluorescence microscopy of tumorigenic A549 cells, xenografted into immunodeficient (Nu/J) mice; NHF-ATP internalization was performed in vivo with 8 mg/mL HMWFD and 100 μM NHF-ATP being directly injected into tumors of living mice. Co-localization of cellular HMWFD and NHF-ATP is shown as merged images in A and B (right panels, yellow). (A) High-magnification images highlight cellular internalization of NHF-ATP and HMWFD in tumors in vivo. Scale bars = 20 μm. (B) Low-magnification images depict regional internalization within a tumor tissue section. Scale bars = 100 μm.