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Updated: Oct 27, 2025
Fluorescence Microscopy for ATP Internalization Mediated by Macropinocytosis in Human Tumor Cells and Tumor-xenografted Mice
Published on: June 30, 2021
Fluorescence Microscopy for ATP Internalization Mediated by Macropinocytosis in Human Tumor Cells and
Corinne M Nielsen1, Yanrong Qian2, Subhodip Adhicary3
1Department of Biological Sciences, Ohio University; Molecular and Cellular Biology Program, Ohio University; Neuroscience Program, Ohio University.
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.
Insights
Extracellular ATP (eATP) is internalized by cancer cells via macropinocytosis, influencing tumorigenesis. This study details methods to track eATP internalization in vitro and in vivo for further research.
Area of Science:
- Biochemistry
- Cell Biology
- Cancer Research
Background:
- Extracellular ATP (eATP) plays a role in cancer progression, including drug resistance, epithelial-mesenchymal transition (EMT), and metastasis.
- Intratumoral eATP concentrations are significantly elevated compared to normal tissues.
- Cancer cells internalize eATP via macropinocytosis for various intracellular functions, including energy supply and gene regulation.
Purpose of the Study:
- To describe and validate protocols for monitoring and characterizing eATP internalization in cancer cells.
- To provide methods for studying eATP internalization both in vitro and in vivo.
Main Methods:
- Utilized microscopic imaging with fluorescently labeled non-hydrolyzable ATP analogs.
- Employed fluorescent dextrans as tracers for macropinocytosis and general endocytosis.
- Incorporated endocytosis inhibitors to characterize the internalization pathways.
- Applied the imaging modality to cancer cell lines and human cancer xenografts in immunodeficient mice.
Main Results:
- Successfully monitored and characterized eATP internalization in cancer cell lines and in vivo tumor models.
- Established and refined protocols for in vitro and in vivo eATP internalization assays.
- Demonstrated the utility of fluorescent tracers and inhibitors in studying eATP uptake.
Conclusions:
- The described imaging protocols enable detailed study of eATP internalization mechanisms in cancer.
- Further research into eATP's roles in tumorigenesis can be facilitated by these validated methods.
- Understanding eATP internalization is crucial for unraveling its versatile functions in cancer biology.
