Name: fluorescence microscopy for atp internalization mediated by macropinocytosis in human tumor cells and tumor-xenografted mice
Uploaded: 2021-06-30T14:30:00
Description: 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

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>
	<li>Pavlova, N. N., Thompson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+hallmarks+of+cancer+metabolism.">The emerging hallmarks of cancer metabolism.</a> Cell Metabolism. 23 (1), 27-47 (2016).</li><li>Pellegatti, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+level+of+extracellular+ATP+at+tumor+sites:+in+vivo+imaging+with+plasma+membrane+luciferase.">Increased level of extracellular ATP at tumor sites: in vivo imaging with plasma membrane luciferase.</a> PLoS ONE. 3, 25992008 (2008).</li><li>Falzoni, S., Donvito, G., Di Virgilio, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+adenosine+triphosphate+in+the+pericellular+space.">Detecting adenosine triphosphate in the pericellular space.</a> Interface Focus. 3 (3), 20120101 (2013).</li><li>Michaud, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy-dependent+anticancer+immune+responses+induced+by+chemotherapeutic+agents+in+mice.">Autophagy-dependent anticancer immune responses induced by chemotherapeutic agents in mice.</a> Science. 334, 1573-1577 (2011).</li><li>Wilhelm, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graft-versus-host+disease+is+enhanced+by+extracellular+ATP+activating+P2X7R.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+Warburg+effect:+the+metabolic+requirements+of+cell+proliferation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cancer+cell+metabolism.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Warburg+effect:+evolving+interpretations+of+an+established+concept.">The Warburg effect: evolving interpretations of an established concept.</a> Free Radical Biology &amp; Medicine. 79, 253-263 (2015).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+as+a+biological+hydrotrope.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+ATP+is+internalized+by+macropinocytosis+and+induces+intracellular+ATP+increase+and+drug+resistance+in+cancer+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+ATP+a+new+player+in+cancer+metabolism:+NSCLC+cells+internalize+ATP+in+vitro+and+in+vivo+using+multiple+endocytic+mechanisms.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macropinocytosis+of+protein+is+an+amino+acid+supply+route+in+Ras-transformed+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+the+size+of+fluorescent+dextran+on+its+endocytic+pathway.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+dendritic+cell+antigen+uptake+via+alpha2+adrenoceptor-mediated+PI3K+activation+following+brief+exposure+to+noradrenaline.">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><li>Hoppe, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimalarial+quinolines+and+artemisinin+inhibit+endocytosis+in+Plasmodium+falciparum.">Antimalarial quinolines and artemisinin inhibit endocytosis in Plasmodium falciparum.</a> Antimicrobial Agents &amp; Chemotherapy. 48, 2370-2378 (2004).</li><li>Chaudry, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+ATP+cross+the+cell+plasma+membrane.">Does ATP cross the cell plasma membrane.</a> Yale Journal of Biology &amp; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+utilization+of+extracellular+[gamma-32P]ATP+for+the+phosphorylation+of+intracellular+proteins+in+the+squid+giant+axon.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+evidence+for+ATP+uptake+by+rat+tissues.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Otto+Warburg's+contributions+to+current+concepts+of+cancer+metabolism.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Links+between+metabolism+and+cancer.">Links between metabolism and cancer.</a> Genes &amp; Development. 26, 877-890 (2012).</li><li>Israelsen, W. J., Vander Heiden, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+consumption+promotes+cancer+metabolism.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+and+insulin+secretion:+the+ATP-sensitive+K++channel+(K+ATP)+connection.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+mitochondrial+ATP+synthesis+and+glucose+transport/phosphorylation+in+type+2+diabetes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+imaging+reveals+elevated+glomerular+ATP/AMP+in+diabetes/obesity+and+identifies+sphingomyelin+as+a+possible+mediator.">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>

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

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

FISH for Long Non-Coding RNA Localization in Human Osteosarcoma Cells

RNA Fluorescence In Situ Hybridization for Long Non-Coding RNA Localization in Human Osteosarcoma Cells

The important roles of long non-coding RNAs (lncRNAs) in cancer have been studied, such as regulating the proliferation, epithelial-mesenchymal transition (EMT), migration, infiltration, and autophagy of cancer cells. Localization detection of lncRNAs in cells can provide insight into their functions. By designing the lncRNA-specific antisense chain sequence followed by labeling with fluorescent dyes, RNA fluorescence in situ hybridization (FISH) can be applied to detect the cellular localization of lncRNAs. Together with the development of microscopy, the RNA FISH techniques now even allow for visualization of the poorly expressed lncRNAs. This method can not only detect the localization of lncRNAs alone, but also detect the colocalization of other RNAs, DNA, or proteins by using double-color or multicolor immunofluorescence. Here, we have included the detailed experimental operation procedure and precautions of RNA FISH by using lncRNA small nucleolar RNA host gene 6 (SNHG6) in human osteosarcoma cells (143B) as an example, to provide a reference for researchers who want to perform RNA FISH experiments, especially lncRNA FISH.

Author Spotlight: RNA FISH for Locating lncRNA-SNHG6 in Osteosarcoma Cells 

The present protocol describes a method of RNA fluorescence in situ hybridization to localize the lncRNAs in human osteosarcoma cells.

The important roles of long non-coding RNAs (lncRNAs) in cancer have been studied, such as regulating the proliferation, epithelial-mesenchymal transition ...

Preparing a Microfluidics-Based Vascularized Human Micro-Tumor Model

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.&#160;To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

Author Spotlight: Creating Human Vascularized Micro-Tumors as Models for Translational Cancer Research 

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...