生体内でPH、 pO₂、酸化還元状態とリン酸濃度および腫瘍微小環境におけるグルタチオンの EPR 評価

The overall goal of this in vivo electron paramagnetic resonance technology is to quantitatively and noninvasively assess the physiologically relevant parameters of local tissue microenvironment in preclinical settings in animal models of cancer. This technique is based on the combination of flourofield APR with special design functional paramagnetic probes to provide quantitative information on tissue oxygenation as CDT in dock status as well as interstitial organic phosphate and intercellular concentrations. This noninvasive technique provides unique insight into the world of tumor microenvironment and tumor regenesis and may contribute to the future rational design of tumor microenvironment targeted anticancer therapeutics.

The application of this technique extend toward brain's corelation between the parameters of application of multifunctional APR probes. Therefore providing additional insight into underlying biological mechanisms. In addition to me and Andrey, demonstrating the procedure will be Marieta Gencheva and Dr.Benoit Driesschaert and a graduate student, Kayla Steinberger from IMMR Center.

To prepare the particulate oxygen sensitive probe, first weigh 60 milligrams of the microcrystals. For oxygen sensitivity calibration, suspend microcrystals in three milliliters of dolbeckas modified eagle media. Sonicate for five minutes, on ice, with a probe sonicator at 20 kilohertz using seven watts of power in a five milliliter glass round bottom tube.

Next, place one milliliter of the sonicated microcrystals in a glass tube in the surface coil resignator of the L-band EPR spectrometer. Acquire continuous waves EPR spectra at the physiological temperature of 37 degrees Celsius and oxygen concentrations of zero, one, two, four, eight, and 20.9 percent. To prepare the dual function pH and redox probe, dissolve 6.34 milligrams of the probe in one milliliter of saline solution.

Adjust the pH to 7.2 with small allocates of hydrochloric acid or sodium hydroxide using a pH meter. To perform the pH calibration of the nitroxide probe, first add 0.1 milliliter of the probe stock solution to 0.9 milliliters of two millimolar sodium phosphate buffer, 150 millimolar sodium chloride. Then titrate the obtained one millimolar probe solution with allocates of hydrochloric acid or sodium hydroxide to the required pH using a pH meter.

Record the EPR spectra of the samples in 1.5 milliliter microcentrifuge tubes using the L-band EPR spectrometer. To prepare the GSH sensitive RSSR probe, dissolve 4.05 milligrams of the probe in one milliliter of DMSO solution. Next, determine the value of the rate constant of the reaction of RSSR with GSH.

To do so, first add 20 microliters of the RSSR stock solution to 0.98 milliliters of one millimolar phosphate buffer pH 7.2, 150 millimolar sodium chloride to obtain a 0.2 millimolar RSSR probe solution. Then mix equal volumes of 0.2 millimolar RSSR solution in one of the GSH solutions for a final concentration of the probe at 0.1 millimolar and of GSH at 0.5, one, or 2.5 millimolar. Immediately after RSSR and GSH solution mixing, place the sample in the EPR resignator and record the EPR spectra every 12 seconds for 10 minutes.

Then calculate the kinetics of the increase of the monoradical spectral amplitude for the multifunctional HOPE probe for oxygen pH and Pi assessment. Dissolve 10.7 milligrams of the probe in one milliliter of saline solution and adjust the pH to 7.4. Then add 20 microliters of the prepared stock solution to 0.98 milliliters of the saline solution to obtain a 0.2 millimolar probe solution.

For probe calibration of the pH, titrate 0.2 millimolar of the probe solution by the addition of a small volume of sodium hydroxide or hydrochloric acid so that the final dilution of sample less than 1%Acquire the EPR spectra at intermediate pH as described in the text protocol. For oxygen sensitivity probe calibration, acquire EPR spectra of the probe at various oxygen concentrations as described in the text protocol. For probe calibration of Pi, acquire EPR specra of the probe at various phosphate concentrations.

Use the radical solution with a pH value near the PKA and titrate it with various phosphate concentrations. Maintain the temperature and gas composition as before. In the case of the MET1 tumor model for internalization of the particulate probe into MET1 cells, add 100 microliters of the sonicated suspension to a T75 flask with 10 milliliters of culture media containing MET1 cells.

All procedures take place in a biosafety cabinet and the media contains penicillin and streptomycin to minimize potential infection. Incubate the cells at 37 degrees Celsius for 72 hours or until they reach approximately 80%confluency. Aspirate the media and wash the cells five times with 10 milliliters of PBS.

Detach the cells with five milliliters of tripson EDTA. After collecting the cells and centrifuging as before, stain the sample of cells of the exclusion dye to determine cell viability and quantity. Suspend the cells at a concentration of one million cells per 100 microliters in minimal DMEM.

Using an insulin syringe, slowly inject 100 microliters of the cell suspension containing the internalized microcrystals into the number four mammary fat pads of an eight week old, female, wild-type mouse as described in the text protocol. Monitor tumor initiation and growth. For EPR spectroscopic measurements, anesthetize the mice by inhalation of air isophlorane mixture using an anesthesia machine as described in the text protocol.

Perform functional measurements using an L-band EPR spectrometer by placing the surface coil resignator onto a normal mammary gland or a mammary tumor. Proceed to tune the spectrometer. In the case of soluble probes, acquire the EPR spectra immediately after probe injection for five to 10 minutes.

Acquire the EPR spectra from the implanted particulate probe for five to 10 minutes over several weeks after implantation. Finally, analyze the EPR spectra and the probes as described in the text protocol. The EPR spectral sensitivity of particulate LiNc-BuO probe to oxygen is shown here.

The EPR spectrum was measured at 0%of pO2 and 37 degrees Celsius. Representative dependents of the line width of the EPR spectrum of the probe suspension on oxygen partial pressure is shown. The line width increases linearly with pO2.

A spectrum measured in mammary tumor tissue of an anesthetized female mouse corresponds to tissue partial oxygen of 7.5 millimeters of mercury. L-band EPR spectra of the NR probe solution were acquired at 37 degrees Celsius at various pH levels. Dependents of the observed hyperfine splitting constant on pH is plotted and fitted with the standard titration curve.

Shown here is the multifunctional tumor microenvironment assessment combining the oxygen sensitive LiNc-BuO probe in the dual function pH in redox NR probe. The observed triplet spectrum of the NR is superimposed with the single EPR line of LiNc-BuO probe embedded within tumor. In vivo EPR assessment of intracellular tissue glutathione concentrations is shown here.

Kinetics of the monoradical spectral peek intensity change measured by L-band EPR in mammary tumor in normal mammary gland of FVBN mice immediately after intratissual injection of RSSR probe are shown. The faster kinetics reflects the higher GSH concentration in tumor tissue. The technique assists investigators in the field of cancer research with surpassed opportunity for in vivo performing of cellular physiological important parameters of tumor microenvironment and the transpotential for the translation.

The sensitives of the probes according to the previous procedures requires expertise in synthetic chemistry and is critical for presentation. Our center reported the difficult of this technology and to the sites on a collaborative basis.