Name: in vivo epr assessment of ph, po2, redox status, and concentrations of phosphate and glutathione in the tumor microenvironment
Uploaded: 2018-03-16T13:00:00
Description: Watch this Scientific Journal Video about In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment at JoVE.com

Detta arbete var delvis stöds av NIH grants CA194013, CA192064 och U54GM104942. WVCTSI är erkänt för uppstart till VVK, AB, och TDE. Författarna tackar Dr. M. Gencheva och K. Steinberger för hjälpen med de belysande experiment. Innehållet ansvarar enbart för författarna och representerar inte nödvändigtvis officiella ståndpunkter av NIH.

Alla djur arbetet utförs i enlighet med WVU IACUC godkända protokoll.
1. probe syntes och kalibrering
<ol><li>Partikelformiga pO2-känsliga LiNc-BuO sond Obs: LiNc-BuO microcrystals är syntetiserade och beredda som beskrivs i referens13. De är mycket stabila och kan förvaras vid rumstemperatur i år. Den EPR linewidth LiNc-BuO partiklar sondens är en pO2-känslig parameter. LiNc-BuO microcrystals demons...

<ol>
	<li>Siemann, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tumor Microenvironment. , (2011).</li><li>Tatum, J. 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=Hypoxia:+importance+in+tumor+biology,+noninvasive+measurement+by+imaging,+and+value+of+its+measurement+in+the+management+of+cancer+therapy.">Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy.</a> Int J Radiat Biol. 82 (10), 699-757 (2006).</li><li>Brahimi-Horn, M. C., Chiche, J., Pouyssegur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+signalling+controls+metabolic+demand.">Hypoxia signalling controls metabolic demand.</a> Curr Opin Cell Biol. 19 (2), 223-229 (2007).</li><li>Haulica, A., Ababei, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+glycolytic+activity+in+the+erythrocytes+of+animals+with+chronic+experimental+hypoxia+and+with+tumours.">Comparative study of glycolytic activity in the erythrocytes of animals with chronic experimental hypoxia and with tumours.</a> Neoplasma. 21 (1), 29-35 (1974).</li><li>Matsumoto, 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=High-resolution+mapping+of+tumor+redox+status+by+magnetic+resonance+imaging+using+nitroxides+as+redox-sensitive+contrast+agents.">High-resolution mapping of tumor redox status by magnetic resonance imaging using nitroxides as redox-sensitive contrast agents.</a> Clin Cancer Res. 12 (8), 2455-2462 (2006).</li><li>Estrela, J. M., Ortega, A., Obrador, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+in+cancer+biology+and+therapy.">Glutathione in cancer biology and therapy.</a> Crit Rev Clin Lab Sci. 43 (2), 143-181 (2006).</li><li>Voegtlin, C., Thompson, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+content+of+tumor+animals.">Glutathione content of tumor animals.</a> J. Biol. Chem. 70, 801-806 (1926).</li><li>Bobko, A. 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=Interstitial+Inorganic+Phosphate+as+a+Tumor+Microenvironment+Marker+for+Tumor+Progression.">Interstitial Inorganic Phosphate as a Tumor Microenvironment Marker for Tumor Progression.</a> Sci Rep. 7, 41233 (2017).</li><li>Gillies, R. J., Raghunand, N., Garcia-Martin, M. L., Gatenby, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH+imaging.+A+review+of+pH+measurement+methods+and+applications+in+cancers.">pH imaging. A review of pH measurement methods and applications in cancers.</a> IEEE Eng Med Biol Mag. 23 (5), 57-64 (2004).</li><li>Gade, T. 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=Imaging+intratumoral+convection:+pressure-dependent+enhancement+in+chemotherapeutic+delivery+to+solid+tumors.">Imaging intratumoral convection: pressure-dependent enhancement in chemotherapeutic delivery to solid tumors.</a> Clin Cancer Res. 15 (1), 247-255 (2009).</li><li>Bobko, A. 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=In+vivo+monitoring+of+pH,+redox+status,+and+glutathione+using+L-band+EPR+for+assessment+of+therapeutic+effectiveness+in+solid+tumors.">In vivo monitoring of pH, redox status, and glutathione using L-band EPR for assessment of therapeutic effectiveness in solid tumors.</a> Magn Reson Med. 67, 1827-1836 (2012).</li><li>Dhimitruka, I., Bobko, A. A., Eubank, T. D., Komarov, D. A., Khramtsov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphonated+Trityl+Probe+for+Concurrent+In+Vivo+Tissue+Oxygen+and+pH+Monitoring+Using+EPR-based+Techniques.">Phosphonated Trityl Probe for Concurrent In Vivo Tissue Oxygen and pH Monitoring Using EPR-based Techniques.</a> JACS. 135, 5904-5910 (2013).</li><li>Pandian, R. P., Parinandi, N. L., Ilangovan, G., Zweier, J. L., Kuppusamy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+particulate+spin+probe+for+targeted+determination+of+oxygen+in+cells+and+tissues.">Novel particulate spin probe for targeted determination of oxygen in cells and tissues.</a> Free Radic Biol Med. 35 (9), 1138-1148 (2003).</li><li>Bobko, A. A., Evans, J., Denko, N. C., Khramtsov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+Longitudinal+EPR+Monitoring+of+Tissue+Oxygenation,+Acidosis,+and+Reducing+Capacity+in+Mouse+Xenograft+Tumor+Models.">Concurrent Longitudinal EPR Monitoring of Tissue Oxygenation, Acidosis, and Reducing Capacity in Mouse Xenograft Tumor Models.</a> Cell Biochem Biophys. 75, 247-253 (2017).</li><li>Khramtsov, V. V., Yelinova, V. I., Glazachev Yu, I., Reznikov, V. A., Zimmer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+determination+and+reversible+modification+of+thiols+using+imidazolidine+biradical+disulfide+label.">Quantitative determination and reversible modification of thiols using imidazolidine biradical disulfide label.</a> J Biochem Biophys Methods. 35 (2), 115-128 (1997).</li><li>Roshchupkina, G. I., 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=In+vivo+EPR+measurement+of+glutathione+in+tumor-bearing+mice+using+improved+disulfide+biradical+probe.">In vivo EPR measurement of glutathione in tumor-bearing mice using improved disulfide biradical probe.</a> Free Rad. Biol. Med. 45, 312-320 (2008).</li><li>Khramtsov, V. V., Zweier, J. L., Hicks, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Stable Radicals: Fundamentals and Applied Aspects of Odd-Electron Compounds. , 537-566 (2010).</li><li>Bobko, A. A., Dhimitruka, I., Zweier, J. L., Khramtsov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fourier+Transform+EPR+of+Trityl+Radicals+for+Multifunctional+Assessment+of+Chemical+Microenvironment).">Fourier Transform EPR of Trityl Radicals for Multifunctional Assessment of Chemical Microenvironment).</a> Angew. Chem. Int. Edit. 53, 2735-2738 (2014).</li><li>Martin, M. L., Martin, G. J., Delpuech, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical NMR spectroscopy. , (1980).</li><li>Lin, E. 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=Progression+to+malignancy+in+the+polyoma+middle+T+oncoprotein+mouse+breast+cancer+model+provides+a+reliable+model+for+human+diseases.">Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases.</a> Am J Pathol. 163 (5), 2113-2126 (2003).</li><li>Eubank, T. D., 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=Granulocyte+macrophage+colony-stimulating+factor+inhibits+breast+cancer+growth+and+metastasis+by+invoking+an+anti-angiogenic+program+in+tumor-educated+macrophages.">Granulocyte macrophage colony-stimulating factor inhibits breast cancer growth and metastasis by invoking an anti-angiogenic program in tumor-educated macrophages.</a> Cancer Res. 69 (5), 2133-2140 (2009).</li><li>Khramtsov, V. V., 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=Quantitative+determination+of+SH+groups+in+low-+and+high-molecular-weight+compounds+by+an+electron+spin+resonance+method.">Quantitative determination of SH groups in low- and high-molecular-weight compounds by an electron spin resonance method.</a> Anal Biochem. 182 (1), 58-63 (1989).</li><li>Komarov, D. 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=Electron+paramagnetic+resonance+monitoring+of+ischemia-induced+myocardial+oxygen+depletion+and+acidosis+in+isolated+rat+hearts+using+soluble+paramagnetic+probes.">Electron paramagnetic resonance monitoring of ischemia-induced myocardial oxygen depletion and acidosis in isolated rat hearts using soluble paramagnetic probes.</a> Magnetic Resonance in Medicine. 68 (2), 649-655 (2012).</li><li>Song, Y. G., Liu, Y. P., Liu, W. B., Villamena, F. A., Zweier, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+binding+of+the+Finland+trityl+radical+with+bovine+serum+albumin.">Characterization of the binding of the Finland trityl radical with bovine serum albumin.</a> Rsc Advances. 4 (88), 47649-47656 (2014).</li><li>Khramtsov, V. V., Bobko, A. A., Tseytlin, M., Driesschaert, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exchange+Phenomena+in+the+Electron+Paramagnetic+Resonance+Spectra+of+the+Nitroxyl+and+Trityl+Radicals:+Multifunctional+Spectroscopy+and+Imaging+of+Local+Chemical+Microenvironment.">Exchange Phenomena in the Electron Paramagnetic Resonance Spectra of the Nitroxyl and Trityl Radicals: Multifunctional Spectroscopy and Imaging of Local Chemical Microenvironment.</a> Analyt. Chem. 89 (9), 4758-4771 (2017).</li><li>Samouilov, 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=In+Vivo+Proton-Electron+Double-Resonance+Imaging+of+Extracellular+Tumor+pH+Using+an+Advanced+Nitroxide+Probe.">In Vivo Proton-Electron Double-Resonance Imaging of Extracellular Tumor pH Using an Advanced Nitroxide Probe.</a> Analyt. Chem. 86 (2), 1045-1052 (2014).</li><li>Goodwin, 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=In+vivo+tumour+extracellular+pH+monitoring+using+electron+paramagnetic+resonance:+the+effect+of+X-ray+irradiation.">In vivo tumour extracellular pH monitoring using electron paramagnetic resonance: the effect of X-ray irradiation.</a> NMR Biomed. 27 (4), 453-458 (2014).</li></ol>

De presenterade metoderna möjliggör noninvasiv i vivo bedömning av kritiska parametrarna för den kemiska TME, nämligen pO2, pH, redox status och koncentrationer av interstitiell Pi och intracellulära GSH. Magnetisk resonans tekniker, såsom MRI och låg-fältet EPR, är metoderna för val av noninvasiv i vivo profilering av dessa TME-parametrar. MRT visualiserar anatomiska strukturer men saknar funktionell känslighet. I motsats till MRI ger EPR tekniker funktionella känslighe...

En nyckelroll i TME i cancer progression och terapi är alltmer uppskattad1. Bland viktiga fysiologiska parametrar av TME i vävnad hypoxi2, hög reducerande kapacitet5, acidos3,4, solida tumörer, förhöjda halter av intracellulära GSH6,7, och interstitiell Pi8 är väl dokumenterade. Noninvasiv invivo pO2, pH, Pi, GSH och redox bedömningar ger unika insikter i biologiska processer i TME och hjälpa förskott verktyg för pre-klinisk screening av läkemedel mot cancer och TME-riktade behandlingsstrategier. En rimlig radiofrekvent genomträngningsdjupet i vävnader av magnetisk resonanstomografi (MRT) och låg-fältet EPR-baserade tekniker gör dem de mest lämpliga metoderna för icke-invasiv bedömning av dessa TME-parametrar. MRI bygger till stor del på imaging vatten protoner och används ofta i kliniska inställningar för att tillhandahålla anatomisk upplösning men saknar funktionell upplösning. Fosfor-31 kärnmagnetisk resonans (31P-NMR) mätningarna extracellulära Pi koncentration och pH baserat på en signal från endogena fosfat är potentiellt attraktiva för TME karakterisering, men är vanligtvis maskerade av flera gånger högre intracellulära Pi koncentrationer9,10. I kontrast till detta, EPR mätningar lita på spektroskopi och avbildning av speciellt utformade paramagnetiska sonder att tillhandahålla funktionell upplösning. Observera att exogena EPR sonder har en fördel över exogena NMR sonder på grund av mycket högre inneboende känslighet EPR och avsaknad av endogena bakgrund EPR signaler. Den senaste utvecklingen av en dubbel funktion pH och redox nitroxyl probe11 och multifunktionella trityl sond12 ger oöverträffade möjligheter för i vivo samtidiga mätningar av flera TME parametrar och deras korrelation analyser oberoende på sonden distribution och tid för mätningen. Såvitt vi vet finns det inga andra metoder som är tillgängliga för att samtidigt bedöma i vivo fysiologiskt viktiga kemiska TME parametrar i levande ämnen, såsom pO2, pHe, Pi, redox och GSH.
Sonder för In Vivo Funktionella mått:
Figur 1 visar kemiska strukturer av de paramagnetiska sonder används för åtkomst till TME faktorer, som inbegriper partiklar och lösliga sonder. Hög funktionell känslighet, stabilitet i levande vävnad och minimal toxicitet är några fördelar som gör partiklar sonder föredrog över lösligt sonder för i vivo EPR pulsoximetri. Till exempel ökat partiklar sonder retentionstider på platsen för vävnad implantat jämfört med lösligt sonder möjliggör längsgående mätning av vävnad pO2 över flera veckor. Däremot, lösliga sonder överträffa partiklar sonder genom att ge rumsliga-löst mätningar med EPR-baserat imaging tekniker samt möjliggör samtidig analyser från flera funktioner (pO2, pH, Pi, redox, och GSH).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56624/56624fig1.jpg"> Figur 1. Kemiska strukturer i de paramagnetiska sonder som montera TME bedömning assay. Detta inkluderar partiklar pO2 sonden, LiNc-BuO (R = - O (CH2)3CH3), och lösliga sonder: dubbel funktion pH och redox probe, NR; GSH-känsliga probe, GFDFJH; och multifunktionell pO2, pH och Pi sonden av den extracellulära mikromiljö, hopp sonden. Syntesen av dessa sonder har beskrivits i den medföljande referenser 11,12. <a href="//cloudflare.jove.com/files/ftp_upload/56624/56624fig1large.jpg" target="_blank">Klicka här för att se en större version av denna siffra.</a>

<table border="0" cellpadding="0" cellspacing="0" width="703">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-band EPR spectrometer</td>
			<td style="width:201px;">Magnettech, Germany</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">L-band (1.2 GHz) electron paramagnetic resonance (EPR) spectrometer for collection in vitro and in vivo spectra of paramagnetic molecules</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;Temperature &#38; Gas Controller&#160;</td>
			<td style="width:201px;">Noxygen, Germany</td>
			<td style="width:119px;"></td>
			<td>Temperature &#38; Gas Controller designed to control and adjust the temperature and gas composition&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sonicator</td>
			<td style="width:201px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GSH (L-Glutathione reduced)</td>
			<td style="width:201px;">Sigma-Aldrich</td>
			<td style="width:119px;">G4251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MMTV-PyMT&#160; mice</td>
			<td style="width:201px;">In house</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:201px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11995065</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Met-1 murine breast cancer cells</td>
			<td style="width:201px;">In house</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C57Bl/6 wild type mice&#160;</td>
			<td style="width:201px;">Jackson Laboratory</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin</td>
			<td style="width:201px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan Blue Exclusion Dye&#160;</td>
			<td style="width:201px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">T10282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ohmeda Fluotec 3&#160;</td>
			<td style="width:201px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoflurane (IsoFlo)</td>
			<td style="width:201px;">Abbott Laboratories</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium phosphate dibasic</td>
			<td style="width:201px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9763</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium phosphate monobasic</td>
			<td style="width:201px;">sigma-Aldrich</td>
			<td style="width:119px;">S07051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Chloride</td>
			<td style="width:201px;">sigma-Aldrich</td>
			<td style="width:119px;">S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrochloric acid</td>
			<td style="width:201px;">sigma-Aldrich</td>
			<td style="width:119px;">320331</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Hydroxide</td>
			<td style="width:201px;">sigma-Aldrich</td>
			<td style="width:119px;">S8045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glucose</td>
			<td style="width:201px;">sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glucose oxydase</td>
			<td style="width:201px;">sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lauda Circulator E100</td>
			<td style="width:201px;">Lauda-Brikmann</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pH meter Orion</td>
			<td style="width:201px;">Thermo Scientific&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LiNc-BuO probe</td>
			<td style="width:201px;">In house</td>
			<td style="width:119px;"></td>
			<td>The Octa-n-Butoxy-Naphthalocyanine probe was synthesizided according to ref 13</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NR probe</td>
			<td style="width:201px;">In house</td>
			<td style="width:119px;"></td>
			<td>The Nitroxide probe was synthesizided according to ref 11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RSSR probe</td>
			<td style="width:201px;">In house</td>
			<td style="width:119px;"></td>
			<td>The di-Nitroxide probe was synthesizided according to ref 15</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HOPE probe</td>
			<td style="width:201px;">In house</td>
			<td style="width:119px;"></td>
			<td>The monophoshonated Triarylmethyl probe was synthesizided according to ref 12</td>
		</tr>
	</tbody>
</table>

in vivo epr assessment of ph, po2, redox status, and concentrations of phosphate and glutathione in the tumor microenvironment

Detta protokoll visar kapaciteten hos låg-fältet elektron paramagnetiska resonans (EPR)-baserade tekniker i kombination med funktionell paramagnetiska sonder för att ge kvantitativa uppgifter om de kemiska tumör närmiljön (TME), inklusive p O2, pH, redox status, koncentrationer av interstitiell oorganiskt fosfat (Pi) och intracellulära glutation (GSH). I synnerhet en ansökan av en nyligen utvecklad lösliga multifunktionella trityl sond ger oöverträffad möjlighet för in vivo samtidiga mätningar av pH, pO2 och Pi E xtracellular utrymme (hopp probe). Mätningar av tre parametrar med hjälp av en enda sond möjliggör deras korrelation analyser som är oberoende av sonden distribution och tiden för mätningarna.

Vävnad p O 2 Bedömning med hjälp av LiNc-BuO sonder:
Med hjälp av proceduren som beskrivs under steg 1.1, utfört vi kalibreringen av nylagade LiNc-BuO microcrystals suspension. Figur 2 visar typiska syre beroendet av linewidth av LiNc-BuO sonden, liksom dess exemplifieras EPR spectra mätt i...

Watch this Scientific Journal Video about In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment at JoVE.com

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Låg-fältet (L-band, 1,2 GHz) elektron paramagnetiska resonans med lösliga nitroxyl och trityl sonder demonstreras för bedömning av fysiologiskt viktiga parametrar i den tumör närmiljön i musmodeller av bröstcancer.

In Vivo EPR bedömning av pH, pO2, Redox Status, och koncentrationer av fosfat och glutation i den tumör mikromiljö

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

The overall goal of this in vivo electron paramagnetic resonance technology is to quantitatively and noninvasively assess the physiologically relevant parameters of local tissue microenvironment in preclinical settings in animal models of cancer. This technique is based on the combination of flourofield APR with special design functional paramagnetic probes to provide quantitative information on tissue oxygenation as CDT in dock status as well as interstitial organic phosphate and intercellular concentrations. This noninvasive technique provides unique insight into the world of tumor microenvironment and tumor regenesis and may contribute to the future rational design of tumor microenvironment targeted anticancer therapeutics.The application of this technique extend toward brain's corelation between the parameters of application of multifunctional APR probes. Therefore providing additional insight into underlying biological mechanisms. In addition to me and Andrey, demonstrating the procedure will be Marieta Gencheva and Dr.Benoit Driesschaert and a graduate student, Kayla Steinberger from IMMR Center.To prepare the particulate oxygen sensitive probe, first weigh 60 milligrams of the microcrystals. For oxygen sensitivity calibration, suspend microcrystals in three milliliters of dolbeckas modified eagle media. Sonicate for five minutes, on ice, with a probe sonicator at 20 kilohertz using seven watts of power in a five milliliter glass round bottom tube.Next, place one milliliter of the sonicated microcrystals in a glass tube in the surface coil resignator of the L-band EPR spectrometer. Acquire continuous waves EPR spectra at the physiological temperature of 37 degrees Celsius and oxygen concentrations of zero, one, two, four, eight, and 20.9 percent. To prepare the dual function pH and redox probe, dissolve 6.34 milligrams of the probe in one milliliter of saline solution.Adjust the pH to 7.2 with small allocates of hydrochloric acid or sodium hydroxide using a pH meter. To perform the pH calibration of the nitroxide probe, first add 0.1 milliliter of the probe stock solution to 0.9 milliliters of two millimolar sodium phosphate buffer, 150 millimolar sodium chloride. Then titrate the obtained one millimolar probe solution with allocates of hydrochloric acid or sodium hydroxide to the required pH using a pH meter.Record the EPR spectra of the samples in 1.5 milliliter microcentrifuge tubes using the L-band EPR spectrometer. To prepare the GSH sensitive RSSR probe, dissolve 4.05 milligrams of the probe in one milliliter of DMSO solution. Next, determine the value of the rate constant of the reaction of RSSR with GSH.To do so, first add 20 microliters of the RSSR stock solution to 0.98 milliliters of one millimolar phosphate buffer pH 7.2, 150 millimolar sodium chloride to obtain a 0.2 millimolar RSSR probe solution. Then mix equal volumes of 0.2 millimolar RSSR solution in one of the GSH solutions for a final concentration of the probe at 0.1 millimolar and of GSH at 0.5, one, or 2.5 millimolar. Immediately after RSSR and GSH solution mixing, place the sample in the EPR resignator and record the EPR spectra every 12 seconds for 10 minutes.Then calculate the kinetics of the increase of the monoradical spectral amplitude for the multifunctional HOPE probe for oxygen pH and Pi assessment. Dissolve 10.7 milligrams of the probe in one milliliter of saline solution and adjust the pH to 7.4. Then add 20 microliters of the prepared stock solution to 0.98 milliliters of the saline solution to obtain a 0.2 millimolar probe solution.For probe calibration of the pH, titrate 0.2 millimolar of the probe solution by the addition of a small volume of sodium hydroxide or hydrochloric acid so that the final dilution of sample less than 1%Acquire the EPR spectra at intermediate pH as described in the text protocol. For oxygen sensitivity probe calibration, acquire EPR spectra of the probe at various oxygen concentrations as described in the text protocol. For probe calibration of Pi, acquire EPR specra of the probe at various phosphate concentrations.Use the radical solution with a pH value near the PKA and titrate it with various phosphate concentrations. Maintain the temperature and gas composition as before. In the case of the MET1 tumor model for internalization of the particulate probe into MET1 cells, add 100 microliters of the sonicated suspension to a T75 flask with 10 milliliters of culture media containing MET1 cells.All procedures take place in a biosafety cabinet and the media contains penicillin and streptomycin to minimize potential infection. Incubate the cells at 37 degrees Celsius for 72 hours or until they reach approximately 80%confluency. Aspirate the media and wash the cells five times with 10 milliliters of PBS.Detach the cells with five milliliters of tripson EDTA. After collecting the cells and centrifuging as before, stain the sample of cells of the exclusion dye to determine cell viability and quantity. Suspend the cells at a concentration of one million cells per 100 microliters in minimal DMEM.Using an insulin syringe, slowly inject 100 microliters of the cell suspension containing the internalized microcrystals into the number four mammary fat pads of an eight week old, female, wild-type mouse as described in the text protocol. Monitor tumor initiation and growth. For EPR spectroscopic measurements, anesthetize the mice by inhalation of air isophlorane mixture using an anesthesia machine as described in the text protocol.Perform functional measurements using an L-band EPR spectrometer by placing the surface coil resignator onto a normal mammary gland or a mammary tumor. Proceed to tune the spectrometer. In the case of soluble probes, acquire the EPR spectra immediately after probe injection for five to 10 minutes.Acquire the EPR spectra from the implanted particulate probe for five to 10 minutes over several weeks after implantation. Finally, analyze the EPR spectra and the probes as described in the text protocol. The EPR spectral sensitivity of particulate LiNc-BuO probe to oxygen is shown here.The EPR spectrum was measured at 0%of pO2 and 37 degrees Celsius. Representative dependents of the line width of the EPR spectrum of the probe suspension on oxygen partial pressure is shown. The line width increases linearly with pO2.A spectrum measured in mammary tumor tissue of an anesthetized female mouse corresponds to tissue partial oxygen of 7.5 millimeters of mercury. L-band EPR spectra of the NR probe solution were acquired at 37 degrees Celsius at various pH levels. Dependents of the observed hyperfine splitting constant on pH is plotted and fitted with the standard titration curve.Shown here is the multifunctional tumor microenvironment assessment combining the oxygen sensitive LiNc-BuO probe in the dual function pH in redox NR probe. The observed triplet spectrum of the NR is superimposed with the single EPR line of LiNc-BuO probe embedded within tumor. In vivo EPR assessment of intracellular tissue glutathione concentrations is shown here.Kinetics of the monoradical spectral peek intensity change measured by L-band EPR in mammary tumor in normal mammary gland of FVBN mice immediately after intratissual injection of RSSR probe are shown. The faster kinetics reflects the higher GSH concentration in tumor tissue. The technique assists investigators in the field of cancer research with surpassed opportunity for in vivo performing of cellular physiological important parameters of tumor microenvironment and the transpotential for the translation.The sensitives of the probes according to the previous procedures requires expertise in synthetic chemistry and is critical for presentation. Our center reported the difficult of this technology and to the sites on a collaborative basis.

Research

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Cancer Research

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

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