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.
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.
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 mM2,3,4,5. As a key energy and signaling molecule, ATP plays a central role in cellular metabolism in cancerous and healthy cells6,7,8. Extracellular ATP is not only involved in cancer cell growth, but it also promotes drug resistance9. Previously unrecognized functions of ATP, such as hydrotropic activity, have recently been identified, thus implicating ATP involvement in diseases such as Alzheimer's10. Indeed, it seems our understanding of ATP and its functions in cancer cells, healthy cells, and other diseased cells is far from complete. However, due to ATP's instability and high turnover rates in cells, it is technically challenging to monitor ATP's movement across the cell membrane and into the cell.
To address this problem and fill the need of this research area, a method was developed in which nonhydrolyzable fluorescent ATP (NHF-ATP) (Figure 1) was used as a surrogate to visualize the internalization of ATP and observe the intracellular spatial localization of internalized ATP, both in vitro and in vivo11,12. NHF-ATP has been demonstrated to substitute for endogenous ATP to investigate ATP movement across animal cell membranes, both in cancer cell lines and in human tumor tissue xenografted on immunodeficient mice11,12. Moreover, administering macropinocytosis inhibitors to cells blocked eATP internalization, suggesting that intracellular uptake of eATP involves a macropinocytotic mechanism9,11,12. This protocol permits immunobased colabeling against cell-specific proteins and thus identification of which cell type internalizes NHF-ATP. Using in vivo tumor xenografts and high-resolution microscopy, NHF-ATP can be visualized spatially across the tissue sample and even within a single cell. These methods also permit quantitative analysis, such as the percentage of cellular uptake, number of macropinocytotic vesicles, and internalization kinetics. This paper describes in detail how NHF-ATP, working alone or together with endocytosis-tracer fluorescent dextrans13,14,15,16, can be used in different experimental settings to study ATP's internalization and intracellular localization, following internalization in cells.
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. Please click here to view a larger version of this figure.
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
2. ATP localization studies, in vitro (Figure 2)
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. Please click here to view a larger version of this figure.
3. ATP internalization in tumors, ex vivo (Figure 3)
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. Please click here to view a larger version of this figure.
4. ATP internalization in tumors, in vivo
In vitro study
Intracellular internalization of NHF-ATP was demonstrated by co-localization of NHF-ATP with HMWFD or LMWFD (Figure 4). The success of this procedure primarily relies on the use of appropriate concentrations of NHF-ATP and dextrans and on determining the appropriate type(s) of dextrans (poly-lysine vs. neutral). For example, to investigate macropinocytosis, HMWFD was chosen as it is internalized only by macropinosomes13,14,15,16. Alternatively, if clathrin- and/or caveolae-mediated endocytoses are studied, then LMWFD is to be selected because the smaller sizes of these endocytosis-associated endosomes only allow them to engulf LMWFD14,15,16. Fluorescent dextrans are also available in two different forms: poly-lysine dextran and neutral dextran. These dextrans generate different fluorescence intensity and background staining; thus, their recommended applications vary. For example, poly-lysine dextran produces higher fluorescence intensity but also higher background. Neutral dextrans generate sufficient fluorescence intensity with relatively low background signal and are preferred for experiments using cancer cell lines. Using this cost-efficient assay, we generated high-intensity and high signal-to-noise NHF-ATP fluorescent labeling, which matched the fluorescence intensity of dextrans without confounding background fluorescence.
As the selection of the fixation agent and the fixation procedure significantly influenced the outcome of the assay, fixation conditions must be experimentally determined and selected for each specific experiment (i.e., specific cell line or tissue type). Extracellular ATP is not needed in the assay; in fact, high concentrations of eATP in the assay solution may lead to increased background. Importantly, after cell seeding, the optimal time frame for serum starvation is ~15-18 h. If serum starvation is too long, the lack of nutrients will affect cell attachment and lead to the loss of cells in the following steps. If serum starvation is too short, the cell cycle will not be properly arrested, and nuclear staining will not be uniform across the slide. Rinsing the coverslip-bound tissue in warm PBS five times is adequate to remove background staining. It is important to be very gentle with tissue washes. Avoid excessive rinsing, cold rinsing, or forceful washing, as these may damage the morphology of the cells.
While any cancer cell lines can be used in this assay, different cell lines may show different degrees of internalization. We have shown that cancer cell lines with KRAS proto-oncogene mutations are advantageous for studying NHF-ATP internalization. While KRAS mutations are not required for the internalization of eATP, KRAS mutations are associated with increased macropinocytosis in vitro13. For these experiments, we selected A549 lung cancer cells, which harbor a KRAS mutation. Indeed, macropinocytosis of eATP in A549 cells has been previously shown using an in vitro ATP internalization assay13.
Ex vivo study
Figure 5 shows fluorescence microscopic images of NHF-ATP internalization in tumor tissue sections. The success of the ex vivo study relies heavily on the proper incubation of NHF-ATP with the tumor tissue and thorough post-collection washing of the tumor sections. A shorter or longer incubation time may disrupt internalization. Determine incubation times, experimentally and in advance, for different tumors. Inadequate rinsing of tumor tissue permits high background staining and thus a low signal-to-noise ratio.
Selection of the fixation agent is also critical. Fixation in methanol, formaldehyde, acetone, and ethanol can be tested individually and compared to identify the best fixation agent for the specific study system. It is noteworthy that the mounted slides need to be photographed within 12-24 h, but not longer, to prevent the internalized fluorescent molecules from being released from cells and contributing to high background. Finally, to avoid the edge-effect phenomenon-intense staining at the edges of tumor tissue sections-it is critical to collect uniform tissue sections before ex vivo incubation.
Given the heterogeneity of tumor tissue, it is important to obtain tissue sections throughout the tumor. The described method of collecting serial tumor sections, taken every 100-200 µm apart, ensures that representative data from different regions of the tumor can be acquired and analyzed. For example, just as different tumor types might generate different ATP internalization data, select intratumoral regions may also vary in ATP internalization kinetics and mechanism.
In vivo study
Figure 6 shows NHF-ATP internalization in injected (xenografted) tumors. An important parameter for a successful and positive in vivo study was the NHF-ATP injection and the time between injection and animal euthanasia. During the injection procedure, position the injection needle to reach as much tumor area as possible, and keep the needle intact within the tumor for as long as 1 min to ensure that NHF-ATP remains inside the tumor and does not leak out. The short time interval between the injection and euthanasia ensures that injected NHF-ATP is transported into the tumor cells, but that transport is not too long, so that recipient cells will not significantly metabolize and degrade NHF-ATP, resulting in intracellular smears. After euthanasia and tumor removal, document the injection procedure for each tumor, including the injection site and injection direction. In this way, tumors can be sectioned in specific orientations and along specific anatomical planes so that tumor sections run parallel to the injection direction, leading to the identification of more NHF-ATP-positive cells.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Fixing tissue slides-post-tumor sectioning-is a necessary step before fluorescence microscopic imaging. Together, these two critical steps ensure that tumor cells retain the internalized ATP during the imaging process. Another important consideration during the analysis of ATP internalization is to account for the heterogeneity of xenograft tumors. As tumorigenesis proceeds differently among cancer cell types, it may be necessary to troubleshoot aspects of this method to determine ideal experimental conditions for different cancer cells. To ensure a comprehensive assessment of macropinocytosis of ATP in tumors, it is critical to image tissue sections throughout the tumor as there may be regional variation in terms of resident cells and their ability to internalize eATP. Indeed, this method may uncover different mechanisms of eATP internalization among cancer/tumor cells in future studies.
It is important to note that NHF-ATP can only replace unconjugated ATP or radioactive ATP for the internalization study. It cannot be used for other studies, such as metabolic studies, as NHF-ATP will behave and be metabolized differently once it is released from macropinosomes and/or endosomes inside cells. Traditionally, ATP internalization was investigated using radioactive ATP17,18,19. However, given the instability of radioactive ATPs, measurable radioactivity inside target cells is not necessarily intact ATP. Because NHF-ATP is nonhydrolyzable and can be visualized microscopically, its use is recommended over radioactive ATP.
The procedure described herein is simple to perform, quick, and cost-effective. If an imaging system is equipped with video capability in future applications, then the dynamic process of ATP internalization can be visualized in real time, revealing information about macropinocytotic kinetics and trafficking in specific tissues. This procedure has been successfully used in other cancer cell lines such as H1299 cells12, noncancerous cells such as NL-20 cells12, and neuronal cells, with no or minor modifications (data not shown). As ATP is intimately involved in the Warburg effect in cancer metabolism6,7,8,20,21,22, in diabetes23,24,25, and other diseases involving energy metabolism, and as eATP may play important roles in those diseases, the described procedure is likely to have broad applications.
The authors have nothing to disclose.
Cryosectioning was performed on-site at the Ohio University Histopathology Core. This work was supported partly by start-up funds (Ohio University College of Arts & Sciences) to C Nielsen; NIH grant R15 CA242177-01 and RSAC award to X Chen.
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 |