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.

<ol><li>Pavlova, N. N., Thompson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+emerging+hallmarks+of+cancer+metabolism%5BTitle%5D)+AND+%22Cell+Metabolism%22%5BJournal%5D)">The emerging hallmarks of cancer metabolism.</a> Cell Metabolism. 23 (1), 27-47 (2016).</li>
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<li>Wilhelm, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Graft-versus-host+disease+is+enhanced+by+extracellular+ATP+activating+P2X7R%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Graft-versus-host disease is enhanced by extracellular ATP activating P2X7R.</a> Nature Medicine. 16, 1434-1438 (2010).</li>
<li>Vander Heiden, M. G., Cantley, L. C., Thompson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Understanding+the+Warburg+effect%3A+the+metabolic+requirements+of+cell+proliferation%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Understanding the Warburg effect: the metabolic requirements of cell proliferation.</a> Science. 324, 1029-1033 (2009).</li>
<li>Cairns, R. A., Harris, I. S., Mak, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+cancer+cell+metabolism%5BTitle%5D)+AND+%22Nature+Reviews+Cancer%22%5BJournal%5D)">Regulation of cancer cell metabolism.</a> Nature Reviews Cancer. 11, 85-95 (2011).</li>
<li>Chen, X., Qian, Y., Wu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Warburg+effect%3A+evolving+interpretations+of+an+established+concept%5BTitle%5D)+AND+%22Free+Radical+Biology+%26+Medicine%22%5BJournal%5D)">The Warburg effect: evolving interpretations of an established concept.</a> Free Radical Biology & Medicine. 79, 253-263 (2015).</li>
<li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+ATP%2C+as+an+energy+and+phosphorylating+molecule%2C+induces+different+types+of+drug+resistances+in+cancer+cells+through+ATP+internalization+and+intracellular+ATP+level+increase%5BTitle%5D)+AND+%22Oncotarget%22%5BJournal%5D)">Extracellular ATP, as an energy and phosphorylating molecule, induces different types of drug resistances in cancer cells through ATP internalization and intracellular ATP level increase.</a> Oncotarget. 8 (5), 87860-87877 (2017).</li>
<li>Patel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ATP+as+a+biological+hydrotrope%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">ATP as a biological hydrotrope.</a> Science. 356, 753-756 (2017).</li>
<li>Qian, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+ATP+is+internalized+by+macropinocytosis+and+induces+intracellular+ATP+increase+and+drug+resistance+in+cancer+cells%5BTitle%5D)+AND+%22Cancer+Letters%22%5BJournal%5D)">Extracellular ATP is internalized by macropinocytosis and induces intracellular ATP increase and drug resistance in cancer cells.</a> Cancer Letters. 351, 242-251 (2014).</li>
<li>Qian, Y., Wang, X., Li, Y., Cao, Y., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+ATP+a+new+player+in+cancer+metabolism%3A+NSCLC+cells+internalize+ATP+in+vitro+and+in+vivo+using+multiple+endocytic+mechanisms%5BTitle%5D)+AND+%22Molecular+Cancer+Research%22%5BJournal%5D)">Extracellular ATP a new player in cancer metabolism: NSCLC cells internalize ATP in vitro and in vivo using multiple endocytic mechanisms.</a> Molecular Cancer Research. 14, 1087-1096 (2016).</li>
<li>Commisso, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macropinocytosis+of+protein+is+an+amino+acid+supply+route+in+Ras-transformed+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells.</a> Nature. 497, 633-637 (2013).</li>
<li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+the+size+of+fluorescent+dextran+on+its+endocytic+pathway%5BTitle%5D)+AND+%22Cell+Biology+International%22%5BJournal%5D)">The effect of the size of fluorescent dextran on its endocytic pathway.</a> Cell Biology International. 39, 531-539 (2015).</li>
<li>Yanagawa, Y., Matsumoto, M., Togashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+dendritic+cell+antigen+uptake+via+alpha2+adrenoceptor-mediated+PI3K+activation+following+brief+exposure+to+noradrenaline%5BTitle%5D)+AND+%22Journal+of+Immunology%22%5BJournal%5D)">Enhanced dendritic cell antigen uptake via alpha2 adrenoceptor-mediated PI3K activation following brief exposure to noradrenaline.</a> Journal of Immunology. 185, 5762-5768 (2010).</li>
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<li>Chaudry, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Does+ATP+cross+the+cell+plasma+membrane%5BTitle%5D)+AND+%22Yale+Journal+of+Biology+%26+Medicine%22%5BJournal%5D)">Does ATP cross the cell plasma membrane.</a> Yale Journal of Biology & Medicine. 55, 1-10 (1982).</li>
<li>Pant, H. C., Terakawa, S., Yoshioka, T., Tasaki, I., Gainer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+for+the+utilization+of+extracellular+%5Bgamma-32P%5DATP+for+the+phosphorylation+of+intracellular+proteins+in+the+squid+giant+axon%5BTitle%5D)+AND+%22Biochimica+et+Biophysica+Acta%22%5BJournal%5D)">Evidence for the utilization of extracellular [gamma-32P]ATP for the phosphorylation of intracellular proteins in the squid giant axon.</a> Biochimica et Biophysica Acta. 582, 107-114 (1979).</li>
<li>Chaudry, I. H., Baue, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Further+evidence+for+ATP+uptake+by+rat+tissues%5BTitle%5D)+AND+%22Biochimica+et+Biophysica+Acta%22%5BJournal%5D)">Further evidence for ATP uptake by rat tissues.</a> Biochimica et Biophysica Acta. 628, 336-342 (1980).</li>
<li>Koppenol, W. H., Bounds, P. L., Dang, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Otto+Warburg%27s+contributions+to+current+concepts+of+cancer+metabolism%5BTitle%5D)+AND+%22Nature+Reviews+Cancer%22%5BJournal%5D)">Otto Warburg's contributions to current concepts of cancer metabolism.</a> Nature Reviews Cancer. 11, 325-337 (2011).</li>
<li>Dang, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Links+between+metabolism+and+cancer%5BTitle%5D)+AND+%22Genes+%26+Development%22%5BJournal%5D)">Links between metabolism and cancer.</a> Genes & Development. 26, 877-890 (2012).</li>
<li>Israelsen, W. J., Vander Heiden, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ATP+consumption+promotes+cancer+metabolism%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">ATP consumption promotes cancer metabolism.</a> Cell. 143, 669-671 (2010).</li>
<li>Koster, J. C., Permutt, M. A., Nichols, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diabetes+and+insulin+secretion%3A+the+ATP-sensitive+K%2B+channel+%28K+ATP%29+connection%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Diabetes and insulin secretion: the ATP-sensitive K+ channel (K ATP) connection.</a> Diabetes. 54, 3065-3072 (2005).</li>
<li>Szendroedi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Muscle+mitochondrial+ATP+synthesis+and+glucose+transport%2Fphosphorylation+in+type+2+diabetes%5BTitle%5D)+AND+%22PLoS+Medicine%22%5BJournal%5D)">Muscle mitochondrial ATP synthesis and glucose transport/phosphorylation in type 2 diabetes.</a> PLoS Medicine. 4, 154(2007).</li>
<li>Miyamoto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mass+spectrometry+imaging+reveals+elevated+glomerular+ATP%2FAMP+in+diabetes%2Fobesity+and+identifies+sphingomyelin+as+a+possible+mediator%5BTitle%5D)+AND+%22EBioMedicine%22%5BJournal%5D)">Mass spectrometry imaging reveals elevated glomerular ATP/AMP in diabetes/obesity and identifies sphingomyelin as a possible mediator.</a> EBioMedicine. 7, 121-134 (2016).</li>
</ol>

The authors declare no competing interests.

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><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&#039;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&#039;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 &amp;mu;L)</td><td>Fisher Scientific</td><td>02-707-438</td><td></td></tr><tr><td>Pipet tips (200 &amp;mu;L)</td><td>Fisher Scientific</td><td>02-707-411</td><td></td></tr><tr><td>Pipet tips (1000 &amp;mu;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 &amp;mu;L, 20-200 &amp;mu;L, 200-1000 &amp;mu;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 cm&lt;sup&gt;2&lt;/sup&gt;</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 ...

fluorescence-microscopy-for-atp-internalization-mediated

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Cancer Research

2.7K Views.  Ohio University. Kankercellen hebben een opmerkelijk vermogen om voedingsstoffen, zoals ATP, uit de extracellulaire omgeving te internaliseren. Ons protocol stelt ons in staat om ATP-internalisatie aan te tonen en ATP-lokalisatie in cellen te visualiseren. Deze hoge resolutie beeldvorming van geïnternaliseerde ATP, binnen macropynozomen, is ideaal voor het begrijpen van speciale lokalisatie en het voltooien van de hoeveelheid analyse van internalisatie.Het protocol beschrijft verschillende experimente...

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

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Immunology and Infection

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Medicine

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Quantitative Assessment of Macropinocytosis in mTORC1-Hyperactive Cells using Flow Cytometry

Macropinocytosis is a highly conserved, actin-dependent endocytic process that allows the uptake of extracellular material, including proteins and lipids. In proliferating cells, macropinocytosis can deliver extracellular nutrients to the lysosome, processed into critical macromolecule building blocks. Recent studies have highlighted the dependence of multiple cancers on macropinocytosis, including breast, colorectal and pancreatic cancer. Ras mutations are thought to be the driver events behind macropinocytosis initiation, leading to the activation of cellular anabolic processes via the mTORC1 signaling pathway. Interestingly, mTORC1 can also be activated by macropinocytosis independently of Ras. Therefore, macropinocytosis represents a metabolic vulnerability that can be leveraged to target macropinocytic tumors by limiting their access to nutrients therapeutically.
In Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM), mTORC1-hyperactivation leads to enhanced macropinocytosis and metabolic reprogramming. Here, we describe a flow cytometry-based protocol to assess macropinocytosis in mammalian cells quantitatively. TSC2-deficient MEFs are employed, which exhibit aberrant activation of mTORC1 and have been shown to have increased macropinocytosis compared to TSC2-expressing cells. Cells treated with pharmacologic inhibitors of macropinocytosis are incubated with fluorescently labeled, lysine-fixable, 70 kDa dextran, or fluorescently labeled bovine serum albumin (BSA) assayed by flow cytometry. To date, robust image-based techniques have been developed to quantitatively assess macropinocytosis in tumor cells in vitro and in vivo. This analysis provides a quantitative assessment of macropinocytosis in multiple experimental conditions and complements existing image-based techniques.

This protocol provides experimental tools to evaluate macropinocytic uptake of nutrients (carbohydrate and protein) by mTORC1-hyperactive cells. Detailed steps to quantify the uptake of fluorescently labeled dextran and bovine serum albumin (BSA) are described.

Macropinocytosis is a highly conserved, actin-dependent endocytic process that allows the uptake of extracellular material, including proteins and lipids. ...

Biology

Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and cell debris, through bulk endocytosis. This pathway plays an essential role in a variety of cellular processes, including the regulation of immune responses and cancer cell metabolism. Given this importance in biological function, examining cell culture conditions can provide valuable information by identifying regulators of this pathway and optimizing conditions to be employed in the discovery of novel therapeutic approaches. The study describes an automated imaging and analysis technique using standard laboratory equipment and a cell imaging multi-mode plate reader for the rapid quantification of the macropinocytic index in adherent cells. The automated method is based on the uptake of high molecular weight fluorescent dextran and can be applied to 96-well microplates to facilitate assessments of multiple conditions in one experiment or fixed samples mounted onto glass coverslips. This approach is aimed at maximizing reproducibility and reducing experimental variation while being both time-saving and cost-effective.

Automated assays using multi-well microplates are advantageous approaches for identifying pathway regulators by allowing the assessment of a multitude of conditions in a single experiment. Here, we have adapted the well-established macropinosome imaging and quantification protocol to a 96-well microplate format and provide a comprehensive outline for automation using a multi-mode plate reader.

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and ...

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

This protocol demonstrates dextran imaging in live cells using continuous uptake and inverse images to optimize visualization of ruffling, macropinosome maturation, and analysis of dextran and other cell labelings.

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase ...

In Vivo Fluorescence Imaging to Localize Antibodies in a Mouse Tumor Xenograft Model

Source: Shivange, G. et al., <a href="https://app.jove.com/v/60727">Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody.</a>&#160;J. Vis. Exp. (2020)
The video demonstrates a fluorescence imaging method for in vivo antibody localization in a mouse tumor xenograft model. It involves the injection of tumor cells in a mouse to generate solid xenografts beneath the skin. Subsequent injection of fluorescent antibodies confirms specific localization through enhanced fluorescence signals within the tumor xenograft.

1. Expression and purification of antibodies

	Maintenance of CHO cells
	
		Grow CHO cells in FreeStyle CHO Media supplemented with commercially available ...

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

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