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.

<ol><li>Siemann, D. W. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSiemann%2C+DW+%22Tumor+Microenvironment%22">Tumor Microenvironment</a>. , John Wiley & Sons Ltd.,. Chichester, UK; Hoboken, NJ, USA. (2011).</li>
<li>Tatum, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypoxia%3A+importance+in+tumor+biology%2C+noninvasive+measurement+by+imaging%2C+and+value+of+its+measurement+in+the+management+of+cancer+therapy%5BTitle%5D)+AND+%22Int+J+Radiat+Biol%22%5BJournal%5D)">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/pubmed?term=((Hypoxia+signalling+controls+metabolic+demand%5BTitle%5D)+AND+%22Curr+Opin+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((Comparative+study+of+glycolytic+activity+in+the+erythrocytes+of+animals+with+chronic+experimental+hypoxia+and+with+tumours%5BTitle%5D)+AND+%22Neoplasma%22%5BJournal%5D)">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/pubmed?term=((High-resolution+mapping+of+tumor+redox+status+by+magnetic+resonance+imaging+using+nitroxides+as+redox-sensitive+contrast+agents%5BTitle%5D)+AND+%22Clin+Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((Glutathione+in+cancer+biology+and+therapy%5BTitle%5D)+AND+%22Crit+Rev+Clin+Lab+Sci%22%5BJournal%5D)">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.jbc.org/content/70/3/801.full.pdf">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/pubmed?term=((Interstitial+Inorganic+Phosphate+as+a+Tumor+Microenvironment+Marker+for+Tumor+Progression%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">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/pubmed?term=((pH+imaging.+A+review+of+pH+measurement+methods+and+applications+in+cancers%5BTitle%5D)+AND+%22IEEE+Eng+Med+Biol+Mag%22%5BJournal%5D)">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/pubmed?term=((Imaging+intratumoral+convection%3A+pressure-dependent+enhancement+in+chemotherapeutic+delivery+to+solid+tumors%5BTitle%5D)+AND+%22Clin+Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((In+vivo+monitoring+of+pH%2C+redox+status%2C+and+glutathione+using+L-band+EPR+for+assessment+of+therapeutic+effectiveness+in+solid+tumors%5BTitle%5D)+AND+%22Magn+Reson+Med%22%5BJournal%5D)">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://pubs.acs.org/doi/abs/10.1021/ja401572r">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. Novel particulate spin probe for targeted determination of oxygen in cells and tissues. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPandian%2C+RP+%22Free+Radic+Biol+Med%22">Free Radic Biol Med</a>. 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/pubmed?term=((Concurrent+Longitudinal+EPR+Monitoring+of+Tissue+Oxygenation%2C+Acidosis%2C+and+Reducing+Capacity+in+Mouse+Xenograft+Tumor+Models%5BTitle%5D)+AND+%22Cell+Biochem+Biophys%22%5BJournal%5D)">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/pubmed?term=((Quantitative+determination+and+reversible+modification+of+thiols+using+imidazolidine+biradical+disulfide+label%5BTitle%5D)+AND+%22J+Biochem+Biophys+Methods%22%5BJournal%5D)">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/pubmed?term=((In+vivo+EPR+measurement+of+glutathione+in+tumor-bearing+mice+using+improved+disulfide+biradical+probe%5BTitle%5D)+AND+%22Free+Rad.+Biol.+Med%22%5BJournal%5D)">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>Hicks, R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKhramtsov%2C+VV+%22Stable+Radicals%3A+Fundamentals+and+Applied+Aspects+of+Odd-Electron+Compounds%22">Stable Radicals: Fundamentals and Applied Aspects of Odd-Electron Compounds</a>. , John Wiley & Sons, Ltd. 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/pubmed?term=((Fourier+Transform+EPR+of+Trityl+Radicals+for+Multifunctional+Assessment+of+Chemical+Microenvironment%29%5BTitle%5D)+AND+%22Angew.+Chem.+Int.+Edit%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMartin%2C+ML+%22Practical+NMR+spectroscopy%22">Practical NMR spectroscopy</a>. , Heyden. (1980).</li>
<li>Lin, E. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progression+to+malignancy+in+the+polyoma+middle+T+oncoprotein+mouse+breast+cancer+model+provides+a+reliable+model+for+human+diseases%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">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/pubmed?term=((Granulocyte+macrophage+colony-stimulating+factor+inhibits+breast+cancer+growth+and+metastasis+by+invoking+an+anti-angiogenic+program+in+tumor-educated+macrophages%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((Quantitative+determination+of+SH+groups+in+low-+and+high-molecular-weight+compounds+by+an+electron+spin+resonance+method%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">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/pubmed?term=((Electron+paramagnetic+resonance+monitoring+of+ischemia-induced+myocardial+oxygen+depletion+and+acidosis+in+isolated+rat+hearts+using+soluble+paramagnetic+probes%5BTitle%5D)+AND+%22Magnetic+Resonance+in+Medicine%22%5BJournal%5D)">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/pubmed?term=((Characterization+of+the+binding+of+the+Finland+trityl+radical+with+bovine+serum+albumin%5BTitle%5D)+AND+%22Rsc+Advances%22%5BJournal%5D)">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/pubmed?term=((Exchange+Phenomena+in+the+Electron+Paramagnetic+Resonance+Spectra+of+the+Nitroxyl+and+Trityl+Radicals%3A+Multifunctional+Spectroscopy+and+Imaging+of+Local+Chemical+Microenvironment%5BTitle%5D)+AND+%22Analyt.+Chem%22%5BJournal%5D)">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/pubmed?term=((In+Vivo+Proton-Electron+Double-Resonance+Imaging+of+Extracellular+Tumor+pH+Using+an+Advanced+Nitroxide+Probe%5BTitle%5D)+AND+%22Analyt.+Chem%22%5BJournal%5D)">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/pubmed?term=((In+vivo+tumour+extracellular+pH+monitoring+using+electron+paramagnetic+resonance%3A+the+effect+of+X-ray+irradiation%5BTitle%5D)+AND+%22NMR+Biomed%22%5BJournal%5D)">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 authors have nothing to disclose.

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

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Cancer Research

8.3K Views.  West Virginia University. 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. 

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

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Medicine

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

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 ...

In vitro Monitoring of Extracellular pH in Real-Time

Early accumulation of neutrophils (PMN) is a hallmark of acute intestinal inflammation. This acute inflammation is either resolved or progresses to chronic inflammation. Without efficient PMN clearance at sites of infiltration, PMN can accumulate and contribute to chronic inflammatory conditions, including the intestinal diseases ulcerative colitis (UC) and Crohn's Disease (CD). The pH in the distal colon in individuals with active UC can range between a pH of 5 and 6, whereas healthy individuals maintain colonic pH in the range of 6.8-7.4. Extracellular pH has been shown to influence both intestinal epithelial cells and the infiltrating immune cells. More specifically, extracellular acidosis significantly impacts PMN. At pH below 6.5, there are increases in the production of H2O2, inhibition of apoptosis, and increases in the functional lifespan of PMN. Given the significant presence of PMN and extracellular acidification at sites of inflammation, we developed a novel model that allows for the monitoring of extracellular pH during PMN transepithelial migration in real time. Here, we describe this model and how it can be utilized to measure both the apical and basal pH during PMN trafficking. This model can be utilized to monitor extracellular pH under a wide range of conditions; including, hypoxia, PMN transepithelial migration, and for extended periods of time.

In vitro Monitoring of Extracellular pH in Real-Time

This article represents a useful in vitro assay to measure changes in extracellular pH during neutrophil (PMN) transepithelial migration (TEM)

Early accumulation of neutrophils (PMN) is a hallmark of acute intestinal inflammation. This acute inflammation is either resolved or progresses to ...

Biochemistry

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of mucin-secreting tumor cells in the peritoneal cavity. PMP can arise from various types of cancers, including appendiceal, ovarian, and colorectal, though appendiceal neoplasms are by far the most common etiology. PMP is challenging to study due to its (1) rarity, (2) limited murine models, and (3) mucinous, acellular histology. The method presented here allows real-time visualization and interrogation of these tumor types using patient-derived ex vivo organotypic slices in a preparation where the tumor microenvironment (TME) remains intact. In this protocol, we first describe the preparation of tumor slices using a vibratome and subsequent long-term culture. Second, we describe confocal imaging of tumor slices and how to monitor functional readouts of viability, calcium imaging, and local proliferation. In short, slices are loaded with imaging dyes and are placed in an imaging chamber that can be mounted onto a confocal microscope. Time-lapse videos and confocal images are used to assess the initial viability and cellular functionality. This procedure also explores translational cellular movement, and paracrine signaling interactions in the TME. Lastly, we describe a dissociation protocol for tumor slices to be used for flow cytometry analysis. Quantitative flow cytometry analysis can be used for bench-to-bedside therapeutic testing to determine changes occurring within the immune landscape and epithelial cell content.

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

We describe a protocol for the production, culture, and visualization of human cancers, which have metastasized to the peritoneal surfaces. Resected tumor specimens are cut using a vibratome and cultured on permeable inserts for increased oxygenation and viability, followed by imaging and downstream analyses using confocal microscopy and flow cytometry.

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

Bioengineering

Guided Tumor Inoculation and Multimodal Imaging in Mice

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor microenvironment and its response to therapy. These imaging modalities have micron-scale resolution but have limited use in the clinic due to their shallow penetration depth into tissue. More clinically applicable imaging modalities such as CT, MRI, and PET have much greater penetration depth but have comparatively lower spatial resolution (mm scale). 
To translate preclinical intravital imaging findings into the clinic, new methods must be developed to bridge this micro-to-macro resolution gap. Here we describe a dorsal skinfold window chamber tumor mouse model designed to enable preclinical intravital and clinically applicable (CT and MR) imaging in the same animal, and the image analysis platform that links these two disparate visualization methods. Importantly, the described window chamber approach enables the different imaging modalities to be co-registered in 3D using fiducial markers on the window chamber for direct spatial concordance. This model can be used for validation of existing clinical imaging methods, as well as for the development of new ones through direct correlation with "ground truth" high-resolution intravital findings. 
Finally, the tumor response to various treatments-chemotherapy, radiotherapy, photodynamic therapy-can be monitored longitudinally with this methodology using preclinical and clinically applicable imaging modalities. The dorsal skinfold window chamber tumor mouse model and imaging platforms described here can thus be used in a variety of cancer research studies, for example, in translating preclinical intravital microscopy findings to more clinically applicable imaging modalities such as CT or MRI.

Author Spotlight: Integrating High-Resolution Intravital Imaging and MRI to Enhance Stereotactic Body Radiation Therapy Planning

Translation of Intravital microscopy findings is challenged by its shallow depth penetration into tissue. Here we describe a dorsal window chamber mouse model that enables co-registration of intravital microscopy and clinically applicable imaging modalities (e.g., CT, MRI) for direct spatial correlation, potentially streamlining clinical translation of intravital microscopy findings.

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Preclinical Imaging of Tumor Oxygen Levels Using EPRI and Ultrasound

Tumor Hypoxia Assessment: In Vivo 3D Oxygen Imaging Through Electron Paramagnetic Resonance

The precise and real-time measurement of oxygen partial pressure (pO2) brings valuable information in many pathologies, including cancer. Low tumor pO2 (i.e., hypoxia) is connected to tumor aggressiveness and poor response to therapy. The quantification of tumor pO2 allows the evaluation of treatment effectiveness. Electron Paramagnetic Resonance Imaging (EPRI), particularly Pulse EPRI, has emerged as an advanced three-dimensional (3D) method of assessing tissue oxygenation in vivo. This innovation was enabled by the technological developments in EPR (Electron Paramagnetic Resonance) and the application of the water-soluble oximetric spin probes from the triaryl family, offering fast and sensitive oxygenation data. The relaxation time of the spin probe (T1 and/or T2) provides accurate information about pO2 in selected voxels.
Human glioblastoma LN229 tumors were grown in the interscapular fad pad of BALB/c nude mice. Ultrasound (US) imaging was used as a reference for tumor anatomical information. To image tissue pO2, the animals were placed in a fixed position in the animal bed with fiducials, enabling registration between the imaging modalities. After the OX071 contrast agent was administered, EPRI was performed, followed by US B-mode. Because of the low spin probe toxicity, the procedure can be repeated during tumor growth or treatment. Following imaging, the registration process was carried out using software written in MATLAB. Ultimately, the hypoxic fraction can be calculated for a specific tumor, and the histogram of pO2 tissue distribution can be compared over time. EPRI combined with ultrasound is an excellent tool for oxygen mapping of tumors in the preclinical setting.

Quantitative 3D oxygen maps of murine tumors were imaged noninvasively using Pulse Electron Paramagnetic Resonance. Ultrasound B-mode and Power Doppler were used for anatomy and vascular structure. Images from both modalities were superimposed enabling multi-parametric tumor analysis.

The precise and real-time measurement of oxygen partial pressure (pO2) brings valuable information in many pathologies, including cancer. Low tumor pO2 ...

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

Summary

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A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

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Tumor Hypoxia Assessment: In Vivo 3D Oxygen Imaging Through Electron Paramagnetic Resonance