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

This work was partially supported by NIH grants CA194013, CA192064 and U54GM104942. The WVCTSI is acknowledged for start-up to VVK, AB, and TDE. The authors thank Dr. M. Gencheva and K. Steinberger for the assistance with the illustrative experiments. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

All animal work was performed in accordance with WVU IACUC approved protocol.
1. Probe Synthesis and Calibration
<ol>
	<li>Particulate pO2-sensitive LiNc-BuO probe 
	Note: LiNc-BuO microcrystals are synthesized and prepared as described in reference13. They are very stable and can be kept at room temperature for years. The EPR linewidth of the LiNc-BuO particulate probe is a pO2-sensitive parameter. LiNc-Bu...

<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>

The presented methods allow for noninvasive in vivo assessment of the critical parameters of the chemical TME, namely pO2, pH, redox status, and concentrations of interstitial Pi and intracellular GSH. Magnetic resonance techniques, such as MRI and low-field EPR, are the methods of choice for noninvasive in vivo profiling of these TME parameters. MRI visualizes anatomical structures but lacks functional sensitivity. In contrast to MRI, EPR techniques provide functional sensitivity to...

A key role of the TME in cancer progression and therapy is increasingly appreciated1. Among important physiological parameters of the TME in solid tumors, tissue hypoxia2, acidosis3,4, high reducing capacity5, elevated concentrations of intracellular GSH6,7, and interstitial Pi8 are well documented. Noninvasive in vivo pO2, pH, Pi, GSH, and redox assessments provide unique insights into the biological processes in TME, and help advance tools for pre-clinical screening of anti-cancer drugs and TME-targeted therapeutic strategies. A reasonable radiofrequency penetration depth in tissues by magnetic resonance imaging (MRI) and low-field EPR-based techniques makes them the most appropriate approaches for noninvasive assessment of these TME parameters. MRI relies largely on imaging water protons and is widely used in clinical settings to provide anatomical resolution but lacks functional resolution. The phosphorus-31 nuclear magnetic resonance (31P-NMR) measurements of extracellular Pi concentration and pH based on a signal from endogenous phosphate are potentially attractive for TME characterization, but are normally masked by several times higher intracellular Pi concentrations9,10. In contrast to this, EPR measurements rely on spectroscopy and imaging of specially designed paramagnetic probes to provide functional resolution. Note that exogenous EPR probes have an advantage over exogenous NMR probes due to the much higher intrinsic sensitivity of EPR and absence of endogenous background EPR signals. The recent development of a dual function pH and redox nitroxyl probe11 and multifunctional trityl probe12&#160;provides unsurpassed opportunities for in vivo concurrent measurements of several TME parameters and their correlation analyses independent on probe distribution and time of measurement. To our knowledge, there are no other methods available to concurrently assess in vivo physiologically important chemical TME parameters in living subjects, such as pO2, pHe, Pi, redox, and GSH.
Probes for In Vivo Functional Measurements:
Figure 1 shows chemical structures of the paramagnetic probes used to access TME parameters, which include particulate and soluble probes. High functional sensitivity, stability in living tissue, and minimal toxicity are a few benefits that make particulate probes preferred over soluble probes for in vivo EPR oximetry. For example, particulate probes have increased retention times at the site of tissue implant compared to soluble probes allowing for longitudinal measurement of tissue pO2 over several weeks. On the other hand, soluble probes outperform particulate probes by providing spatial-resolved measurements using EPR-based imaging techniques as well as allowing concomitant analyses from multiple functionalities (pO2, pH, Pi, redox, and GSH).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56624/56624fig1.jpg" /> 
Figure 1. Chemical structures of the paramagnetic probes that assemble TME assessment assay. This includes the particulate pO2 probe, LiNc-BuO (R = -O(CH2)3CH3), and soluble probes: dual function pH and redox probe, NR; GSH-sensitive probe, RSSR; and multifunctional pO2, pH, and Pi probe of the extracellular&#160;microenvironment, the HOPE probe. The synthesis of these probes has been described in the provided references 11,12. <a href="//cloudflare.jove.com/files/ftp_upload/56624/56624fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</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

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

Tissue pO2 Assessment Using the LiNc-BuO Probes:
Using the procedure described under step 1.1, we performed the calibration of freshly prepared LiNc-BuO microcrystals suspension. Figure 2 shows the typical oxygen dependence of the linewidth of the LiNc-BuO probe, as well as its exemplified EPR sp...

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

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

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

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.

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Research

JoVE Journal

Cancer Research

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these drugs can be used, their target proteins must be validated as therapeutic targets in specific cancer types. This validation is often performed by knocking out the gene encoding the candidate therapeutic target in a genetically engineered mouse (GEM) model of cancer and determining what effect this has on tumor growth. Unfortunately, technical issues such as embryonic lethality in conventional knockouts and mosaicism in conditional knockouts often limit this approach. To overcome these limitations, an approach to ablating a floxed embryonic lethal gene of interest in short-term cultures of malignant peripheral nerve sheath tumors (MPNSTs) generated in a GEM model was developed.
This paper describes how to establish a mouse model with the appropriate genotype, derive short-term tumor cultures from these animals, and then ablate the floxed embryonic lethal gene using an adenoviral vector that expresses Cre recombinase and enhanced green fluorescent protein (eGFP). Purification of cells transduced with adenovirus using fluorescence-activated cell sorting (FACS) and the quantification of the effects that gene ablation exerts on cellular proliferation, viability, the transcriptome, and orthotopic allograft growth is then detailed. These methodologies provide an effective and generalizable approach to identifying and validating therapeutic targets in vitro and in vivo. These approaches also provide a renewable source of low-passage tumor-derived cells with reduced in vitro growth artifacts. This allows the biological role of the targeted gene to be studied in diverse biologic processes such as migration, invasion, metastasis, and intercellular communication mediated by the secretome.

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in vivo.

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these ...

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the tumor immune landscape. Although standard immunohistochemistry is suitable for studying tissue architecture, it is limited to the analysis of a small number of markers. Conversely, techniques such as flow cytometry can evaluate multiple markers simultaneously, although information about tissue morphology is lost. In recent years, multiplexed strategies that integrate phenotypic and spatial analysis have emerged as comprehensive approaches to the characterization of the tumor immune landscape. Herein, we discuss an innovative technology combining metal-labeled antibodies and secondary ion mass spectrometry focusing on the technical steps in assay development and optimization, tissue preparation, and image acquisition and processing. Before staining, a metal-labeled antibody panel must be developed and optimized. This hi-plex image system supports up to 40 metal-tagged antibodies in a single tissue section. Of note, the risk of signal interference increases with the number of markers included in the panel. After panel design, particular attention should be given to the metal isotope assignment to the antibody to minimize this interference. Preliminary panel testing is performed using a small subset of antibodies and subsequent testing of the entire panel in control tissues. Formalin-fixed, paraffin-embedded tissue sections are obtained and mounted on gold-coated slides and further stained. The staining takes 2 days and closely resembles standard immunohistochemical staining. Once samples are stained, they are placed in the image acquisition instrument. Fields of view are selected, and images are acquired, uploaded, and stored. The final stage is image preparation for the filtering and removal of interference using the system&#39;s image processing software. A disadvantage of this platform is the lack of analytical software. However, the images generated are supported by different computational pathology software.

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the ...

Industrialized, Artificial Intelligence-guided Laser Microdissection for Microscaled Proteomic Analysis of the Tumor Microenvironment

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell populations. To characterize proteome-level variation and tumor heterogeneity at scale, high-throughput methods are needed to selectively isolate discrete cellular populations in solid tumor malignancies. This protocol describes a high-throughput workflow, enabled by artificial intelligence (AI), that segments images of hematoxylin and eosin (H&#38;E)-stained, thin tissue sections into pathology-confirmed regions of interest for selective harvest of histology-resolved cell populations using laser microdissection (LMD). This strategy includes a novel algorithm enabling the transfer of regions denoting cell populations of interest, annotated using digital image software, directly to laser microscopes, thus enabling more facile collections. Successful implementation of this workflow was performed, demonstrating the utility of this harmonized method to selectively harvest tumor cell populations from the TME for quantitative, multiplexed proteomic analysis by high-resolution mass spectrometry. This strategy fully integrates with routine histopathology review, leveraging digital image analysis to support enrichment of cellular populations of interest and is fully generalizable, enabling harmonized harvests of cell populations from the TME for multiomic analyses.

This protocol describes a high-throughput workflow for artificial intelligence-driven segmentation of pathology-confirmed regions of interest from stained, thin tissue section images for enrichment of histology-resolved cell populations using laser microdissection. This strategy includes a novel algorithm enabling the transfer of demarcations denoting cell populations of interest directly to laser microscopes.

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell ...