Name: in vivo epr assessment of ph, po2, redox status, and concentrations of phosphate and glutathione in the tumor microenvironment
Uploaded: 2018-03-16T13:00:00.000+00:00
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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.

<ol>
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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 the local parameters of the microenvironment when used in combination with functional spin probes. 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.
The described protocols are based on using low-field L-band EPR and specially designed paramagnetic probes. A set of four probes presented in Figure 1 can be considered an effective in vivo TME assessment assay. Availability of the probes is an important prerequisite for the described in vivo EPR measurements. The synthesis of the probes according to the published procedures11,12,13,15 requires expertise in synthetic organic chemistry and may become a critical step for the method implementation.
Application of the particulate probe enables repeated measurements of tissue pO2 for up to weeks after implantation14,21. Soluble probes demonstrate sensitivity to an extended number of the parameters beyond oxygen concentration25 and provide an opportunity for spatial-resolved measurements26. In particular, an application of the multifunctional HOPE probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2, and Pi in extracellular space. The measurements of the three parameters using a single probe allows for their correlation analyses independent of probe distribution and time of the measurements8.
The probes cannot be used simultaneously, except for the implanted LiNc-BuO particulate probe, which can be used in combination with any of the soluble probes as demonstrated in Figure 6. Therefore, the experimental design should be based on separate measurements of individual injections of specific functional soluble probes. The hydrophilic extracellular pH and redox NR probe can be delivered both via i.t. injection11,14,26 and systemic delivery27, while two other soluble probes, RSSR and HOPE, allow for i.t. delivery only8,11,12,16. Note that the future structural modifications of RSSR and HOPE probes may overcome the latter limitation of probe delivery by the development of more hydrophilic and less toxic probe analogs for systemic delivery25.
Note that spectroscopic EPR modality provides the average values of the parameters measured in the TME, which is often characterized by its high heterogeneity. This partially masks the potential differences between the parameters or decreases significance in correlation analysis. The use of the described probes in imaging modalities allows for obtaining spatially-resolved functional information26. We believe that the future of multi-functional EPR imaging relies on the further development of relatively new methodologies such as rapid scan EPR imaging and Overhauser-enhanced MRI (OMRI, also termed proton-electron double-resonance imaging, PEDRI). The new directions in the development of functional EPR probes and advanced imaging techniques have been recently discussed in the corresponding feature article25.

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.bdigitaluss.remotexs.co/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 spectra measured in buffer suspension and in breast tumor tissue in female C57Bl/6 mice grown after tumor initiation by injection of cells with internalized particles (step 3.1). Using the linewidth calibration allows for the calculation of the tissue pO2 value (equal to 7.5 mmHg for the spectrum (b) shown in the Figure 2).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56624/56624fig2.jpg" /> 
Figure 2. EPR spectral sensitivity of the particulate LiNc-BuO probe to oxygen. Representative dependence of the linewidth of the EPR spectrum of the LiNc-BuO probe suspension in DMEM on oxygen partial pressure is shown. The EPR spectrum (insert a) was measured at 0% of pO2, 37 &#176;C, and represents a pure Lorentzian line with the linewidth &#916;Hpp = 113 mG. The linewidth increases linearly with pO2 with a slope of 11.7 mG/mmHg. The spectrum shown in (insert b) was measured in mammary tumor tissue of anesthetized female C57Bl/6 mice showing &#916;Hpp = 204 mG, which corresponds to tissue pO2 of 7.5 mmHg. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56624/56624fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Assessment of Tissue Extracellular pH and Redox Using the NR Probes:
Using the procedure described under step 1.2, we performed the calibration of the pH and redox probe, NR. Figure 3 shows the observed hyperfine splitting constant dependence, aN, of the NR radical on pH. The dependence is described by a standard titration curve with the pKa = 6.6 at 37 &#176;C. This dependence of aN(pH) serves as the calibration curve for corresponding in vivo measurements, allowing for pH measurements in the range from 5.6 to 7.6, with accuracy of 0.05 pH units.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56624/56624fig3.jpg" /> 
Figure 3. EPR spectral sensitivity of the soluble NR probe to pH. L-band EPR spectra of the NR probe solution were acquired at 37 &#176;C at various pH, and pH-dependence of the observed hyperfine splitting constant, aN, was plotted. The solid line is the fit of experimental data with the standard titration curve, <img alt="Equation 2" src="/files/ftp_upload/56624/56624eq2.jpg" />, yielding pKa = 6.6, aN(NR-H+) = 14.24 G, and aN(R) = 15.27 G. Insert: Exemplified EPR spectrum of 1 mM NR solution (pH 6.4) acquired using the following spectrometer settings: magnetic field sweep, 80 G; modulation amplitude, 2.5 G, acquisition time, 20 s. The measured hyperfine splitting constant is 14.63 G. Reproduced from reference23 with permission of John Wiley &#38; Sons, Inc. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56624/56624fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 4 exemplifies the pH and redox measurements in a breast tumor mouse model, performed as described under step 3.3 (i.t. probe injection) and section 4 (spectra acquisition and spectra analysis).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56624/56624fig4.jpg" /> 
Figure 4. EPR assessment of tissue reducing capacity in vivo. Insert: L-band EPR spectrum of the NR probe measured in vivo after i.t. injection (10 &#181;L, 10 mM) in mammary tumor tissue of anesthetized female FVB/N mice. The hyperfine splitting, aN, was found to be equal to 14.72 G, which corresponds to the value of pHe = 6.52 assuming tumor tissue temperature 34 &#176;C and pKa = 6.6. The NR reduction rate is assessed by following the decay of the central-field spectral component. The exemplified kinetics measured in mammary tumor (&#9632;) and mammary glands (o) demonstrate higher reducing capacity of the tumor tissue vs. normal mammary gland. The analysis of the initial part of the kinetics yields the rates of the EPR signal reduction, kred, in the extracellular media of the tissues as 2.5 x 10-3/s in tumor vs. 0.5 x 10-3/s in normal gland. Reproduced from reference11 with permission of John Wiley &#38; Sons, Inc. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56624/56624fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 5 summarizes the pHe and redox measurements performed for the group of female FVB/N MMTV-PyMT mice in tumors and normal mammary glands supporting tumor acidic extracellular microenvironment and high reducing capacity.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56624/56624fig5.jpg" /> 
Figure 5. In vivo EPR assessment of tissue acidity and reducing capacity. Extracellular tissue pH (filled bars) and the reduction rate (empty bars) values of NR nitroxide in normal mammary glands and mammary tumors of female FVB/N mice measured by in vivo EPR. Error bars denote SE. Reproduced from reference11 with permission of John Wiley &#38; Sons, Inc. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56624/56624fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Concurrent Tissue pHe, Redox, and pO2 Assessment by Combining NR and LiNc-BuO Probes:
Particulate LiNc-BuO is the probe of choice when repetitive longitudinal measurements of pO2 values are planned in a predetermined tissue of interest. Unfortunately, a major limitation of the particulate probe is that it only allows for the detection of oxygen and no other physiologically-relevant chemical parameters within the TME, and does not allow for tissue imaging. Thus, the use of soluble probes in EPR have the advantage of imaging and the detection of multiple physiologically-relevant TME parameters. In this respect, pO2 measurements using implanted LiNc-BuO particles can be combined with the dual function pH and redox NR probe because low- and high-field spectral components of the NR EPR spectrum do not overlap with the EPR line of the LiNc-BuO probe.
Figure 6 illustrates a typical EPR spectrum observed by the NR probe immediately after i.t. injection in breast tumor tissue of female C57Bl/6 mice. The observed triplet spectrum of the NR probe is overlaid with the single EPR line of LiNc-BuO microcrystals that have been embedded within the tumor (tumor growth was initiated by the injection of the cells with internalized particles as described in step 3.1. The pH value is calculated by measuring aN hyperfine splitting as a distance between low- and high-field components and using the calibration shown in Figure 3 (equal to 6.88) for the spectrum shown in Figure 6. Note that pO2 assessment is performed based on measurements of the linewidth of the LiNc-BuO signal before injection of the NR probe. The decay of the NR signal provides the value of tissue reducing capacity as illustrated in Figure 4.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56624/56624fig6.jpg" /> 
Figure 6. Multifunctional TME assessment combining LiNc-BuO and NR probes. EPR spectrum measured in vivo in breast tumor tissue of female C57Bl/6 mouse immediately after i.t. injection of the NR probe. The observed triplet spectrum of the NR is superimposed with a single EPR line of LiNc-BuO probes embedded within tumor. The pH value calculated from the splitting between low- and high-field components is 6.88. The decay of the NR signal provides the value of tissue reducing capacity. pO2 assessment is performed based on measurements of the linewidth of the LiNc-BuO before injection of the NR probe. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56624/56624fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In Vivo Assessment of Intracellular GSH:
The reaction of thiol-disulfide exchange between the RSSR probe and GSH splits the disulfide bond of the probe resulting in the formation of two monoradicals22 and cancelation of intramolecular spin exchange between the monoradical fragments. Figure 7A illustrates the effect of the reaction of the RSSR probe with GSH on the EPR spectra resulting in the disappearance of the &#34;biradical&#34; spectral components and corresponding increase of the intensity of the monoradical components. The observed rate constant of the reaction between GSH and RSSR is: kobs = 2.8 &#177; 0.2 M-1s-1 (T = 34 &#176;C, pH 7.2)11. Figure 7B exemplifies the kinetics of the monoradical spectral peak intensity change measured in mammary tumor (&#9679;) and normal mammary gland (o) using L-band EPR. The solid lines are the fits of the initial part of the kinetics by the monoexponent using kobs = 2.8 M-1s-1 and yielding [GSH] = 10.7 mM and 3.3 mM for the tumor and normal mammary gland, respectively.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56624/56624fig7.jpg" /> 
Figure 7. In vivo EPR assessment of intracellular tissue glutathione concentrations. (A) The X-band EPR spectra of 100 &#181;M RSSR measured at various time points after incubation with 2.5 mM GSH in 0.1 M Na-phosphate buffer, pH 7.2, and 1 mM DTPA at 34 &#176;C. The kinetics analysis provides the observed rate constant value of the reaction between GSH and RSSR, kobs (pH 7.2, 34 &#176;C) = (2.8 &#177; 0.2) M-1 s-1. (B) The kinetics of the monoradical spectral peak intensity change measured by L-band EPR in mammary tumor (&#9679;) and normal mammary gland (o) of FVB/N mice immediately after i.t. injection of RSSR probe (see step 3.3.2). The solid lines are the fits of the initial part of the kinetics by the monoexponent, supposing kobs (pH 7.2, 34 &#176;C) = 2.8 M-1s-1 and yielding [GSH] = 10.7 mM and 3.3 mM for the tumor and normal mammary gland, respectively. Reproduced from reference11 with permission of John Wiley &#38; Sons, Inc. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56624/56624fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Multifunctional Assessment of Tissue Extracellular pH, pO2, and Pi Using the Multifunctional HOPE Probe:
Figure 8 illustrates the sensitivity of the spectral parameters of the HOPE probe obtained using calibration procedures described under section 1.4.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56624/56624fig8.jpg" /> 
Figure 8. Multifunctional assessment of the chemical microenvironment using HOPE probe. (a) The scheme of pH-dependent equilibrium between two ionization states of the probe. (b) L-band EPR spectrum of HOPE. (c) The EPR linewidth of the HOPE is a pO2 marker (accuracy, &#8776; 1 mmHg; pO2 range, 1-100 mmHg). (d) The fraction of protonated HOPE is a pH marker in the range from 6 to 8.0 (accuracy, &#177; 0.05). (e) Dependence of proton exchange rate (expressed in mG) of the HOPE with inorganic phosphate (Pi) concentration extracted by spectra simulation (accuracy, &#177; 0.1 mM, range, 0.1-20 mM) 12,18. Reproduced from reference8 with permission of Nature Publishing Group. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56624/56624fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 9 illustrates multifunctional measurements performed in FVB/N wild type mammary glands and in the TME of MMTV-PyMT transgenic mice, which spontaneously develop breast cancer and emulate human tumor staging20. The mean values of pO2 (50 &#177; 3 mmHg in TME vs. 58 &#177; 3 mmHg in normal tissue) and pHe (6.99 &#177; 0.03 in TME vs. 7.1 &#177; 0.03 in normal tissue) support an appearance of hypoxic and acidic regions in the tumor. The most dramatic changes were observed in the concentration of interstitial Pi (1.8 &#177; 0.2 mM in TME vs. 0.84 &#177; 0.07 mM in normal tissue) indicating a potential role of interstitial Pi concentration as a TME marker of tumor progression. The individual measurements show significant variations around the mean values. An important advantage of a multifunctional probe is that all parameters are measured using a single probe, therefore allowing for correlation analyses independent of probe distribution and time of the measurements as illustrated in Figure 9b-e. The observed positive correlation between pO2 and pHe in normal mammary gland versus the absence of correlation in tumors supports tumor reliance on glycolysis independent of oxygen concentration; in our opinion this is an exemplified in vivo demonstration of the Warburg effect. In turn, the observed negative correlation between interstitial [Pi] and pO2 both in normal and tumor tissues is in agreement with the association of high [Pi] (and low ATP/Pi ratio) with changes in bioenergetics status upon lower oxygen supply.
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56624/56624fig9.jpg" /> 
Figure 9. In vivo tissue pO2, pHe, and Pi assessment using the HOPE probe and EPR. (a) Photograph of the set-up for in vivo L-band EPR measurements shows the anesthetized mouse between the magnets of the EPR spectrometer, with the insert on the right showing placement and positioning of the loop resonator on top of the measured tissue. Note the interstitial extracellular localization of the HOPE probe: it does not penetrate into the cells due to bulky charged structures and the HOPE signal from the blood is not detected by EPR due to signal broadening by HOPE complexation with plasma albumin24. (b-e) Correlation between interstitial pO2, pHe, and Pi values measured in normal mammary glands of FVB/N wild type mice and in the TME of breast cancer in MMTV-PyMT transgenic mice (n = 23). To extend the range of oxygen variations, anoxic conditions in interstitial space were established by i.t. injection of the oxygen-consuming enzymatic system of glucose/glucose oxidase (red symbols). Blue lines represent a linear fit for the total data sets. (b) A positive correlation between pO2 and pHe in normal tissue (r = 0.5, p = 0.014 for black symbols; r = 0.64, p = 1.8 &#215; 10-4 for total data set) vs. (c) no significant correlation between pO2 and pHe in TME (r = 0.01, p = 0.97 for black symbols; r = 0.23, p = 0.3 for total data set) were found. (d) A negative correlation between pO2 and Pi both in normal tissue (r = &#8722;0.51, p = 0.013 for black symbols; r = &#8722;0.7, p = 2.3 &#215; 10-5 for total data set) and (e) in TME (b, bottom: r = &#8722;0.4, p = 0.079 for black symbols; r = &#8722;0.62, p = 0.001 for total data set) were found. Adapted from reference8 with permission with of Nature Publishing Group. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56624/56624fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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Nanyang Technological University

Research

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Cancer Research

8.2K 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

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 ...

Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary sites. Recently, we established that cancer cell dissemination in primary breast cancer and at metastatic sites in the lung occurs only at doorways called Tumor MicroEnvironment of Metastasis (TMEM). TMEM doorway number is prognostic for distant recurrence of metastatic disease in breast cancer patients. TMEM doorways are composed of a cancer cell which over-expresses the actin regulatory protein Mena in direct contact with a perivascular, proangiogenic macrophage which expresses high levels of TIE2 and VEGF, where both of these cells are tightly bound to a blood vessel endothelial cell. Cancer cells can intravasate through TMEM doorways due to transient vascular permeability orchestrated by the joint activity of the TMEM-associated macrophage and the TMEM-associated Mena-expressing cancer cell. In this manuscript, we describe two methods for assessment of TMEM-mediated transient vascular permeability: intravital imaging and fixed tissue immunofluorescence. Although both methods have their advantages and disadvantages, combining the two may provide the most complete analyses of TMEM-mediated vascular permeability as well as microenvironmental prerequisites for TMEM function. Since the metastatic process in breast cancer, and possibly other types of cancer, involves cancer cell dissemination via TMEM doorways, it is essential to employ well established methods for the analysis of the TMEM doorway activity. The two methods described here provide a comprehensive approach to the analysis of TMEM doorway activity, either in na&#239;ve or pharmacologically treated animals, which is of paramount importance for pre-clinical trials of agents that prevent cancer cell dissemination via TMEM.

We describe two methods for assessing transient vascular permeability associated with tumor microenvironment of metastasis (TMEM) doorway function and cancer cell intravasation using intravenous injection of high-molecular weight (155 kDa) dextran in mice. The methods include intravital imaging in live animals and fixed tissue analysis using immunofluorescence.

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary ...

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 ...

Medicine

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Immunology and Infection

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

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 ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

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

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

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

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

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

Summary

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Chapters in this video

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

Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis

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

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

Real-time Imaging of Myeloid Cells Dynamics in Apc^Min/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

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

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

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