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 &#38; Sciences) to C Nielsen; NIH grant R15 CA242177-01 and RSAC award to X Chen.

All procedures reported herein were performed in accordance with Ohio University&#39;s IACUC and with the NIH.
1. Selection of nonhydrolyzable fluorescent ATP (NHF-ATP) and dextrans
<ol>
	<li>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...

<ol>
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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....

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 &#181;M 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&#39;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&#39;s instability and high turnover rates in cells, it is technically challenging to monitor ATP&#39;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&#39;s internalization and intracellular localization, following internalization in cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62768/62768fig01.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/62768/62768fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A549 cells, human lung epithelial, carcinoma</td>
			<td>National Cancer Institute</td>
			<td>n/a</td>
			<td>Less expensive source</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>S25904</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil, Reynolds</td>
			<td>Grainger</td>
			<td>6CHG6</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqueous Mounting Medium, ProLong Gold Anti-fade Reagent</td>
			<td>ThermoFisher</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP analog</td>
			<td>Jena Biosciences</td>
			<td>NK-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave, sterilizer</td>
			<td>Grainger</td>
			<td>33ZZ40</td>
			<td></td>
		</tr>
		<tr>
			<td>Blades, cryostat, high profile</td>
			<td>C. L. Sturkey, Inc.</td>
			<td>DT554550</td>
			<td></td>
		</tr>
		<tr>
			<td>Calipers, vernier</td>
			<td>Grainger</td>
			<td>4KU77</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell medium, Ham&#39;s Nutrient Mixture F12, serum-free</td>
			<td>Millipore Sigma</td>
			<td>51651C-1000ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge, refrigerated with swinging bucket rotor</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Acros Organics</td>
			<td>423555000</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube, 15 mL</td>
			<td>VWR</td>
			<td>21008-216</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube, 50 mL</td>
			<td>VWR</td>
			<td>21008-242</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips, glass, 12 mm</td>
			<td>Corning</td>
			<td>2975-245</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat, Leica CM1950</td>
			<td>Leica Biosystems</td>
			<td>CM1950</td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate task wipe, Kim Wipes</td>
			<td>Kimberly-Clark</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran, Lysine fixable, High Molecular Weight (HMW)</td>
			<td>Invitrogen</td>
			<td>D1818</td>
			<td>MW = 70,000, Tetramethylrhodamine</td>
		</tr>
		<tr>
			<td>Dextran, Neutral, High Molecular Weight (HMW)</td>
			<td>Invitrogen</td>
			<td>D1819</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM), serum-free</td>
			<td>Fisher Scientific</td>
			<td>11885076</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry ice</td>
			<td>Local delivery</td>
			<td>Custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescent imaging system, Nikon NiU and Nikon NIS Elements acquisition software</td>
			<td>Nikon</td>
			<td>Custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>ThermoFisher</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps, Dumont #7, curved</td>
			<td>Fine Science Tools</td>
			<td>11274-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps, Dumont #5, straight</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves (small, medium, large)</td>
			<td>Microflex</td>
			<td>N191, N192, N193</td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves, MAPA Temp-Ice 700 Thermal (for handling dry ice)</td>
			<td>Fisher Scientific</td>
			<td>19-046-563</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Daigger</td>
			<td>EF16034F EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator, cell culture</td>
			<td>Eppendorf</td>
			<td>Galaxy 170 S</td>
			<td></td>
		</tr>
		<tr>
			<td>Labelling tape</td>
			<td>Fisher Scientific</td>
			<td>159015R</td>
			<td></td>
		</tr>
		<tr>
			<td>Marking pen, Sharpie (ultra-fine)</td>
			<td>Staples</td>
			<td>642736</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice, immunodeficient (Nu/J)</td>
			<td>Jackson Laboratory</td>
			<td>2019</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge, accuSpin Micro17</td>
			<td>Fisher Scientific</td>
			<td>13-100-675</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifgue tubes, Eppendorf tubes (1.5 mL)</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide box</td>
			<td>Fisher Scientific</td>
			<td>50-751-4983</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle, 27 gauge</td>
			<td>Becton-Dickinson</td>
			<td>752 0071</td>
			<td></td>
		</tr>
		<tr>
			<td>Paintbrush</td>
			<td>Grainger</td>
			<td>39AL12</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td>Staples</td>
			<td>33550</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Acros Organics</td>
			<td>416785000</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Perforated spoon, 15 mm diameter, 135 mm length</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-6162</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>BP3991</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips (10 &#956;L)</td>
			<td>Fisher Scientific</td>
			<td>02-707-438</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips (200 &#956;L)</td>
			<td>Fisher Scientific</td>
			<td>02-707-411</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips (1000 &#956;L)</td>
			<td>Fisher Scientific</td>
			<td>02-707-403</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipets, serological (10 mL)</td>
			<td>VWR</td>
			<td>89130-910</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippetor, Gilson P2</td>
			<td>Daigger</td>
			<td>EF9930A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor Starter Kit, Gilson (2-10 &#956;L, 20-200 &#956;L, 200-1000 &#956;L)</td>
			<td>Daigger</td>
			<td>EF9931A</td>
			<td></td>
		</tr>
		<tr>
			<td>Platform shaker - orbital, benchtop</td>
			<td>Cole-Parmer</td>
			<td>EW-51710-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Positively-charged microscope slides, Superfrost</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel, size 10, Surgical Design, Inc.</td>
			<td>Fisher Scientific</td>
			<td>22-079-707</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors, surgical - sharp, curved</td>
			<td>Fine Science Tools</td>
			<td>14005-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for image analysis, Nikon Elements</td>
			<td>Nikon</td>
			<td>Custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for image analysis, ImageJ (FIJI)</td>
			<td>National Institutes of Health</td>
			<td>n/a</td>
			<td>Download online (free)</td>
		</tr>
		<tr>
			<td>Specimen disc 30 mm (chuck holder), cryostat accessory</td>
			<td>Leica Biosystems</td>
			<td>14047740044</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining tray, 245 mm BioAssay Dish</td>
			<td>Corning</td>
			<td>431111</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 1 cc</td>
			<td>Becton-Dickinson</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr>
			<td>Tape, laboratory, 19 mm width</td>
			<td>Fisher Scientific</td>
			<td>15-901-5R</td>
			<td></td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>Fisher Scientific</td>
			<td>14-649-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture dish, 100 x 15 mm diameter</td>
			<td>Fisher Scientific</td>
			<td>08-757-100D</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture flask, 225 cm2</td>
			<td>ThermoFisher</td>
			<td>159933</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plate, 24-well</td>
			<td>Becton-Dickinson</td>
			<td>353226</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue embedding mold, stainless steel</td>
			<td>Tissue Tek</td>
			<td>4161</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Freezing Medium, Optimal Cutting Temperature (OCT)</td>
			<td>Fisher Scientific</td>
			<td>4585</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (ethylenediaminetetraacetic acid), 0.25%</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath, Precision GP 2S</td>
			<td>ThermoFisher</td>
			<td>TSGP2S</td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescence microscopy for atp internalization mediated by macropinocytosis in human tumor cells and tumor-xenografted mice

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&#39; laboratories&#39; 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.

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 macropinosomes<sup class=...

Watch this Scientific Journal Video about Fluorescence Microscopy for ATP Internalization Mediated by Macropinocytosis in Human Tumor Cells and Tumor-xenografted Mice at JoVE.com

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

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 ...

Cancer cells have a remarkable ability to internalize nutrients, such as ATP, from the extracellular environment. Our protocol permits us to demonstrate ATP internalization and to visualize ATP localization within cells. This high resolution imaging of internalized ATP, within macropynozomes, is ideal for understanding especial localization and completing quantity of analysis of internalization.The protocol describes different experimental applications to study ATP internalization. This method can be used to study different mechanisms of cellular internalization, including macropinocytosis and exosome-mediated endocytosis. For metabolic diseases, ATP internalization in non-cancer cells can be studied with this protocol.To begin, seed the cancer cells in a 225 square centimeter flask, containing DMEM supplemented with FBS and antibiotics. Allow the cells to grow at 37 degrees Celsius and 5%carbon dioxide in the cell culture incubator. Once the 80%confluence is achieved, detach the cells and pellet down the detach cells by centrifugation, and resuspend the cells in ice cold PBS to achieve the cell density of 5 times 10 to the 6 cells per 100 microliters of PBS.Transfer the cell suspension to a 1.5 milliliter microcentrifuge tube. Clean the injection site on the flank of an immunodeficient mouse, with 75%ethanol, and wipe excess ethanol with a delicate task wipe. Draw the cells into a one milliliter latex-free syringe, equipped with a 27 gauge precision glide needle.For subcutaneous injection, hold the needle at about a 10 degree angle to the skin and insert the needle tip with the bevel facing up underneath the skin, keeping only one to two millimeters of the needle outside the skin. Dispense the cells from the syringe slowly over approximately 10 seconds. After injecting the entire volume, hold the needle in place for three to five seconds and then withdraw the needle.Apply gentle but firm pressure to the injection site for three to five seconds with fingers, to prevent leaking of the injected content. Monitor and measure the tumor growth using Vernier calipers, until the tumors reach a volume of 200 to 500 cubic millimeters. Dissolve 300 microliters of 16 milligrams per milliliter high molecular weight fluorescent dextran and serum-free DMEM, and incubate in a water bath, tempered at 37 degrees Celsius for 30 minutes, then, centrifuge the dextran solution and transfer the supernatant to a new 1.5 milliliter microcentrifuge tube.At 40 microliters, a one millimolar non hydrolyzable fluorescent ATP analog stock to 160 microliters of serum-free DMEM, to make a 0.2 millimolar nonhydrolyzable fluorescent ATP solution. Prepare the treatment solutions of DMEM or eight milligrams per milliliter's high or low molecular weight fluorescent dextran, with or without 100 micromolar nonhydrolyzable fluorescent ATP in DMEM, by mixing the stock solutions prepared previously. Use a one milliliter syringe to collect 50 microliters of one treatment solution.Inject the solution directly into the xenograft tumor and repeat for four biological replicates of each treatment. After euthanizing the mouse and isolating the tumor tissue, use a perforated spoon to scoop up the resected tumor tissue and immediately place the tissue into a freezing medium and roll the tissue, ensuring that the tissue remains submerged in the medium bathes, all the tissue surfaces. Carefully place the tissue into the embedding mold containing the freezing medium.Place the corresponding label tag vertically into the freezing medium or mold, ensuring that the written label is visible outside the medium. When the freezing medium turns opaque white, remove the tissue block from the mold, place the tissue block on dry ice and repeat freezing for each tumor section. Store the tissue blocks at minus 80 degrees Celsius for several months before the cryosectioning procedure.After making the slides of the tissue blocks, fix the tissue section slides in 95%ethanol at minus 18 degrees Celsius for five minutes. Wash the fixed section with PBS for five minutes. Add 10 to 50 microliters of an aqueous mounting medium with DAPI to the tissue sections, according to the size of the sections, and place a glass cover slip over the tissue section.After 12 to 24 hours of mounting, identify regions of interest of the fixed tumor sections and acquire the images using fluorescence microscopy as demonstrated previously. After two to 24 hours of the human tumor cell fixation and standing with DAPI, capture the images using an epifluorescence imaging system and data acquisition software. Place the slide on the stage of an upright epiflorescence microscope in binocular mode.Open the imaging program, select the 10 times objective and adjust the stage to define focus. Scan the slide from left to right in a serpentine manner to identify the regions of interest. Select the 40 times objective, and use the toggle on the microscope to switch from binocular to image capture mode.Click on the live quality icon to view and subsequently acquire the images. Use the OC panel on the acquisition toolbar to define the exposure parameters for each filter cube or fluorescent channel, then, use the multi-channel acquisition toolbar to acquire a three channel image with the defined exposure settings. Alternatively, multi-channel images can be acquired manually by toggling between the filter cubes, setting the exposure time, closing or opening the shutter between image acquisition for each channel and overlaying each image taken for individual channels.Save the image as nd2 file. Save the TIF files, including the merged channel image and individual channel images. Use the object count feature on the annotations and measurements toolbar to count the number of NHF-ATP, TMR high molecular weight fluorescent dextran and TMR low molecular weight fluorescent dextran positive cells on a saved nd2 image file and export the data to a spreadsheet through the analysis program.This protocol was developed for spatial, temporal and quantitative analysis of the cellular internalization of nonhydrolyzable ATP. The intracellular internalization of nonhydrolyzable fluorescent ATP was demonstrated by colocalization of nonhydrolyzable fluorescent ATP with high or low molecular weight fluorescent dextran in vitro, ex vivo and in vivo. To ensure that tumor cells retained internalized ATP, limited experimental time for all intratumoral injection to cryo-embedding.Also account for intratumoral variation by imaging tissue throughout the tumor. Future iterations of our method could involve real-time visualization of the E-ATP internalizing process, revealing information about macropynocytotic kinetics and trafficking in specific tissues.

fluorescence-microscopy-for-atp-internalization-mediated

Research

JoVE Journal

Cancer Research

2.6K visualizações.  Ohio University. As células cancerígenas têm uma notável capacidade de internalizar nutrientes, como o ATP, do ambiente extracelular. Nosso protocolo nos permite demonstrar a internalização da ATP e visualizar a localização de ATP dentro das células. Esta imagem de alta resolução de ATP internalizada, dentro de macropirnozomes, é ideal para entender a localização especial e completar a quantidade de análise de internalização.O protocolo descreve diferentes aplicações experimentais pa...