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

Summary

We developed a reproducible method to visualize the internalization of nonhydrolyzable fluorescent adenosine triphosphate (ATP), an ATP surrogate, with high cellular resolution. We validated our method using independent in vitro and in vivo assays-human tumor cell lines and immunodeficient mice xenografted with human tumor tissue.

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.

Introduction

The opportunistic uptake of intratumoral extracellular (ie) nutrients has recently been named a key hallmark for cancer metabolism¹. One of these important nutrients is ATP, as the concentration of ieATP is 10³ and 10⁴ times higher than that found in normal tissues, in the range of several hundred µM to low mM^2,3,4,5. As a key energy and signaling molecule, ATP plays a central role in cellular metabolism in cancerous and healthy cells^6,7

Protocol

All procedures reported herein were performed in accordance with Ohio University's IACUC and with the NIH.

1. Selection of nonhydrolyzable fluorescent ATP (NHF-ATP) and dextrans

1. Select a fluorophore-conjugated NHF-ATP (Figure 1A) and endocytosis tracers, high and low molecular weight fluorescent dextrans (TMR-HMWFD and TMR-LMWFD) (Figure 1B), based on the preferred emission wavelengths (e.g., imaging system equipped wi.......

Discussion

A method was developed for spatial, temporal, and quantitative analysis of the cellular internalization of nonhydrolyzable ATP. This method is broadly applicable for use in diverse biological systems, including various tumorigenic models, for which we provide technical instruction and representative data. To acquire interpretable data during in vivo ATP internalization studies (section 4 of the protocol), it is critical to limit the experimental time elapsed from intratumoral dextran injection to cryo-embedding........

Materials

Name	Company	Catalog Number	Comments
A549 cells, human lung epithelial, carcinoma	National Cancer Institute	n/a	Less expensive source
Acetone	Fisher Scientific	S25904
Aluminum foil, Reynolds	Grainger	6CHG6
Aqueous Mounting Medium, ProLong Gold Anti-fade Reagent	ThermoFisher	P36930
ATP analog	Jena Biosciences	NK-101
Autoclave, sterilizer	Grainger	33ZZ40
Blades, cryostat, high profile	C. L. Sturkey, Inc.	DT554550
Calipers, vernier	Grainger	4KU77
Cell medium, Ham's Nutrient Mixture F12, serum-free	Millipore Sigma	51651C-1000ML
Centrifuge, refrigerated with swinging bucket rotor	Eppendorf	5810R
Chloroform	Acros Organics	423555000
Conical tube, 15 mL	VWR	21008-216
Conical tube, 50 mL	VWR	21008-242
Coverslips, glass, 12 mm	Corning	2975-245
Cryostat, Leica CM1950	Leica Biosystems	CM1950
Delicate task wipe, Kim Wipes	Kimberly-Clark	34155
Dextran, Lysine fixable, High Molecular Weight (HMW)	Invitrogen	D1818	MW = 70,000, Tetramethylrhodamine
Dextran, Neutral, High Molecular Weight (HMW)	Invitrogen	D1819
Dulbecco's Modified Eagle Medium (DMEM), serum-free	Fisher Scientific	11885076
Dry ice	Local delivery	Custom order
Epifluorescent imaging system, Nikon NiU and Nikon NIS Elements acquisition software	Nikon	Custom order
Ethanol	Fisher Scientific	BP2818-4
Fetal bovine serum (FBS)	ThermoFisher	16000044
Forceps, Dumont #7, curved	Fine Science Tools	11274-20
Forceps, Dumont #5, straight	Fine Science Tools	11254-20
Gloves (small, medium, large)	Microflex	N191, N192, N193
Gloves, MAPA Temp-Ice 700 Thermal (for handling dry ice)	Fisher Scientific	19-046-563
Hemocytometer	Daigger	EF16034F EA
Incubator, cell culture	Eppendorf	Galaxy 170 S
Labelling tape	Fisher Scientific	159015R
Marking pen, Sharpie (ultra-fine)	Staples	642736
Mice, immunodeficient (Nu/J)	Jackson Laboratory	2019
Microcentrifuge, accuSpin Micro17	Fisher Scientific	13-100-675
Microcentrifgue tubes, Eppendorf tubes (1.5 mL)	Axygen	MCT-150-C
Microscope slide box	Fisher Scientific	50-751-4983
Needle, 27 gauge	Becton-Dickinson	752 0071
Paintbrush	Grainger	39AL12
Paper towels	Staples	33550
Paraformaldehyde	Acros Organics	416785000
Penicillin/Streptomycin	Gibco	15140122
Perforated spoon, 15 mm diameter, 135 mm length	Roboz Surgical Instrument Co.	RS-6162
Phosphate buffered saline (PBS)	Fisher Scientific	BP3991
Pipet tips (10 μL)	Fisher Scientific	02-707-438
Pipet tips (200 μL)	Fisher Scientific	02-707-411
Pipet tips (1000 μL)	Fisher Scientific	02-707-403
Pipets, serological (10 mL)	VWR	89130-910
Pippetor, Gilson P2	Daigger	EF9930A
Pipettor Starter Kit, Gilson (2-10 μL, 20-200 μL, 200-1000 μL)	Daigger	EF9931A
Platform shaker - orbital, benchtop	Cole-Parmer	EW-51710-23
Positively-charged microscope slides, Superfrost	Fisher Scientific	12-550-15
Scalpel, size 10, Surgical Design, Inc.	Fisher Scientific	22-079-707
Scissors, surgical - sharp, curved	Fine Science Tools	14005-12
Software for image analysis, Nikon Elements	Nikon	Custom order
Software for image analysis, ImageJ (FIJI)	National Institutes of Health	n/a	Download online (free)
Specimen disc 30 mm (chuck holder), cryostat accessory	Leica Biosystems	14047740044
Staining tray, 245 mm BioAssay Dish	Corning	431111
Syringe, 1 cc	Becton-Dickinson	309623
Tape, laboratory, 19 mm width	Fisher Scientific	15-901-5R
Timer	Fisher Scientific	14-649-17
Tissue culture dish, 100 x 15 mm diameter	Fisher Scientific	08-757-100D
Tissue culture flask, 225 cm2	ThermoFisher	159933
Tissue culture plate, 24-well	Becton-Dickinson	353226
Tissue embedding mold, stainless steel	Tissue Tek	4161
Tissue Freezing Medium, Optimal Cutting Temperature (OCT)	Fisher Scientific	4585
Trypsin-EDTA (ethylenediaminetetraacetic acid), 0.25%	Gibco	25200072
Water bath, Precision GP 2S	ThermoFisher	TSGP2S